Guest guest Posted November 11, 2005 Report Share Posted November 11, 2005 Hi All, Discovery of a hormone related to hunger are in the pdf-available below from Science, not yet in Medline. There appears to be amply explanation of the articles in the below popular press report, reviews and article (1-5). Two-day fasting is a long time for rats, is it not? 1. Appetite-suppressing hormone identified Last Updated Thu, 10 Nov 2005 19:16:44 EST CBC News Scientists used clues from the Human Genome Project to find a hormone that seems to suppress appetite in rats. Three hormones, leptin, melanocortin and ghrelin, are known to act on appetite and weight. The discovery of each raised interest in weight-control strategies, but scientists say dozens of hormones are likely involved. Little is understood about the stomach and brain's complex system of regulating weight. Researchers at Stanford University School of Medicine identified the latest hormone, obestatin, by studying human genome data for hormone receptors. The hunger hormone ghrelin was found to have obestatin attached to it. The team then found obestatin was present in rat stomach tissues and brains. When obestatin was injected into rats, it suppressed food intake in the animals by about half compared to those not given the hormone, the team reports in the Nov. 11 issue of the journal Science. Obestatin seems to act by putting a brake on ghrelin, say the researchers, who found it also slowed the movement of digested food from the stomach into the intestines. The normal rats lost 20 per cent of their weight in eight days. Researchers plan to test its effect on obese rats next. They also need to test if obestatin made the rats eat less by suppressing appetite directly or by making the rodents feel ill. & Pharmaceutical Research & Development, LLC sponsored the research into the newly identified hormone. The company has licence rights on the discovery. http://www.cbc.ca/story/science/national/2005/11/10/appetite-hormone051110.html 2. Dueling Hunger Hormones? Science 11 November 2005:? Perspectives BIOMEDICINE: Separation of Conjoined Hormones Yields Appetite Rivals Nogueiras and Matthias Tschöp In their Perspective, Nogueiras and Tschöp discuss the study of Zhang et al. that has identified obestatin, a new hormone regulator of energy balance. Obestatin suppresses food intake, body weight gain, and gastrointestinal motility in rodents. The paradox is that obestatin is derived from a precursor peptide that also yields ghrelin, a hormone that increases appetite and body weight. 3. This Week in Science Science 11 November 2005:939 Dueling Hunger Hormones? Ghrelin, a circulating peptide hormone produced in the stomach, has attracted much attention because of its stimulatory effect on food intake, but the effect of ghrelin may represent only half of the story. Using a bioinformatics approach, Zhang et al. (p. 996; see the Perspective by Nogueiras and Tschöp) show that ghrelin encodes a second peptide hormone that is processed from the same protein precursor as ghrelin. In rodents, a synthetic version of this hormone, obestatin, has the opposite physiological effect as ghrelin--it suppresses food intake. Obestatin mediates its actions through an orphan G protein-coupled receptor, GPR39, which shares sequences with, but is distinct from, the receptor targeted by ghrelin. 4. Science 11 November 2005: 985-986 Perspectives BIOMEDICINE: Separation of Conjoined Hormones Yields Appetite Rivals Nogueiras and Matthias Tschöp When we refer to our " gut feelings, " not many of us actually visualize how the gastrointestinal tract spills myriads of small peptide hormones into our bloodstream to activate defined circuits of the central nervous system. Nevertheless, that picture does reflect a current scientific concept called the " gutbrain axis. " This model consists of a complex network of hormonal and neuronal signaling pathways that is believed to balance numerous homeostatic and behavioral processes (1, 2). In this context, our stomach does not just collect, process, and transport ingested food, but it also represents a multileveled conversational partner of the central nervous system. A key element of this communication process is the hunger-inducing hormone ghrelin, which is believed to convey information about nutrient availability from the stomach to the brain (3, 4). Zhang and colleagues (5) now report on page 996 of this issue that ghrelin not only has a sibling derived from the same peptide precursor (preproghrelin), but also that this new ghrelin-associated peptide behaves as a physiological opponent of ghrelin. Guided by bioinformatics-based predictions for typical enzymatic cleavage sites, they identified a 23-amino acid region of preproghrelin that is highly conserved across species, suggesting a relevant biological function. The authors purified a secreted peptide of the predicted size and sequence from rat stomach tissue and also detected it in rat blood. Similar to ghrelin, which requires posttranslational modification close to its amino terminus by acylation (6), the biological activity of the ghrelin- associated peptide also depends on modification, but by much more common amidation at its carboxyl terminus. The ghrelin-motilin receptor family and their ligands. Each of these gastrointestinal hormones acts on a specific G protein-coupled receptor from the same family to affect food intake and gastrointestinal motility (9-11). Similar dual effects on satiety and gastrointestinal motility are known for glucagon-like peptide 1, cholecystokinine, or peptide YY. Collectively, these peptides may serve to couple meal termination with inhibition of upper gastrointestinal function to prevent malabsorption and postprandial metabolic disturbances (1, 2, 8). The surprising finding is the pharmacological effects of the newly identified peptide in comparison with the known actions of ghrelin. Whereas ghrelin increases food intake and body weight (7), the ghrelin-associated peptide decreases food intake and body weight gain in rodents. Moreover, Zhang et al. observed that the new peptide decelerates gastric emptying and decreases intestinal contractility in mice, both of which counteract the well-defined effects of ghrelin (8). Through a targeted screen of mammalian orphan receptors and subsequent analyses in cultured mammalian cells, Zhang et al. show that the ghrelin-associated peptide binds to and activates the orphan receptor GPR39 (9). This G protein-coupled receptor has been mapped to human chromosome 2 and is expressed in multiple tissues, including the stomach, intestine, and hypothalamus. This localization is consistent with a role in energy balance regulation (10). GPR39 is a member of a family that includes the receptors for ghrelin and motilin, another gastrointestinal hormone that stimulates food intake, gastric emptying, and gut motility (9, 11). These facts support a somewhat counterintuitive, but nevertheless intriguing, relationship between ghrelin and the ghrelin-associated peptide. To denote its anorexigenic actions, Zhang and colleagues named this new gastric hormone obestatin (from the Latin term obedere, meaning to " devour " ). Inevitably, the terms " obesity " and " statins, " a class of lipid-lowering drugs, come to mind. However, obestatin has not been tested in animal models of obesity and there is no evidence for a lipid-lowering effect. Furthermore, even its effect on body weight appears to be very subtle. The failure of obestatin treatment to decrease leptin levels in mice may indicate lack of lipolytic potency. Effects of obestatin on food intake regulation following administration to peripheral circulation or directly into the brain of mice suggest the typical action profile of a gastrointestinal satiety hormone. However, it is possible that obestatin may simply suppress appetite by triggering nausea or visceral illness. Recent examples have emphasized the importance of excluding nonspecific appetite suppression when examining anti-obesity drug candidates (12). Furthermore, despite sequence homologies between rodent and human obestatin (87%) and GPR39 (93%) sequences (5, 9), data from rodents cannot always be translated to humans, where the effects of obestatin have yet to be determined. Another concern regarding a role for obestatin in energy balance regulation arises from its quantification in blood. Although Zhang et al. confirmed earlier findings that the level of plasma ghrelin increases upon fasting and decreases following nutrient ingestion (5, 11), they did not observe any changes in circulating obestatin upon fasting or feeding in rodents. Detection methods for differentiating between circulating amidated and nonamidated obestatin are not yet available, but could still reveal an association with nutrient availability. Nevertheless, total plasma obestatin generally appears to be a fraction of the level of plasma ghrelin. Should hormones derived from the same prepropeptide not circulate in an equimolar ratio? The Yin and Yang personalities of ghrelin and obestatin. Both hormones derive from the same precursor protein and are predominantly secreted by the stomach and released into the blood. Each acts on a different receptor (GPR39 and GHS-R, as shown) and has an opposite effect on food intake, body weight, and gastrointestinal motility. CREDIT: K. SUTLIFF/SCIENCE Another peptide precursor that gives birth to antipodal regulators of food intake may provide some answers. The neuropeptide proopiomelanocortin is cleaved into several active fragments that include the appetite-suppressing - and melanocyte-stimulating hormones (-, -MSH) and the appetite-stimulating hormone -endorphin (13). Tissue-specific enzymes determine which of these are generated. A similar scenario could determine how and where preproghrelin is fragmented into bioactive peptides. An earlier study postulated one other circulating preproghrelin fragment, a 13-amino acid peptide called C-ghrelin (14). In addition, turnover rates of ghrelin and obestatin may differ appreciably, according to their acylation or amidation rates, which again would be a parallel to the acetylation of the proopiomelanocortin derivative -MSH (15). Dissecting the posttranslational cleavage, activation, or degradation processes of peptide hormones may reveal elegant enzymatic drug targets: Simultaneous activation of an agonist and deactivation of its endogenous functional antagonist could provide a powerful strategy for homeostatic control. If obestatin lives up to its name as a circulating hormone with a physiologically relevant anorectic as well as an obesity-preventing function, the puzzling discrepancy between the very mild phenotype of mice lacking ghrelin (16, 17) and the unsurpassed pharmacological effects of ghrelin on energy balance would receive an unexpected--but logical--explanation. The absence of an orexigenic hormone may be counterbalanced by the simultaneous deletion of an equally potent satiety factor. Targeted mouse mutagenesis is widely used as a strategy to unmask or validate the biological function of a gene product. An obvious abnormality of such a knockout mouse is usually interpreted as a reliable indicator of the target's physiological role. However, subtle or absent differences between gene-disrupted mice and their wild-type littermates are often regarded as evidence of negligible biological relevance. Such conclusions should be regarded with caution because developmental compensation may mask loss of function. However, rarely has such compensation been defined on a molecular level. The Zhang et al. findings caution against the interpretation of results based exclusively on gene disruption or messenger RNA quantification due to an additional level of complexity represented by posttranslational processing of proteins. The discovery of obestatin leaves several questions unanswered. Why does a mouse that is deficient for the ghrelin receptor not exhibit an impressive phenotype? Should the absence of ghrelin action in the presence of an intact obestatin signaling pathway not generate a robust negative energy balance? Why does obestatin, unlike ghrelin, not affect growth hormone secretion from the pituitary gland, despite the presence of the obestatin receptor in this organ? Although the adversarial relationship between ghrelin and obestatin certainly is an important contribution to our understanding of body weight regulation, the search for a magic bullet against obesity is likely to continue--admittedly, a gut feeling. 5. Obestatin, a Peptide Encoded by the Ghrelin Gene, Opposes Ghrelin's Effects on Food Intake Jian V. Zhang, Pei-Gen Ren, Orna Avsian-Kretchmer, Ching-Wei Luo, Rami Rauch, Klein, and J. W. Hsueh Science 11 November 2005: 996-999. Ghrelin, a circulating appetite-inducing hormone, is derived from a prohormone by posttranslational processing. On the basis of the bioinformatic prediction that another peptide also derived from proghrelin exists, we isolated a hormone from rat stomach and named it obestatin—a contraction of obese, from the Latin " obedere, " meaning to devour, and " statin, " denoting suppression. Contrary to the appetite-stimulating effects of ghrelin, treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain. Obestatin bound to the orphan G protein–coupled receptor GPR39. Thus, two peptide hormones with opposing action in weight regulation are derived from the same ghrelin gene. After differential modification, these hormones activate distinct receptors.The increasing prevalence of obesity is a global problem. Body weight is regulated in part by peptide hormones produced in the brain or gut or both (1). Earlier studies on synthetic and peptidyl growth hormone (GH) secretagogues (2–4) led to the identification of a specific G protein–coupled receptor (GPCR), the GH secretagogue receptor (GHSR) (5, 6), and subsequently to the discovery of its endogenous ligand, ghrelin (7), a gut-derived circulating hormone that stimulates food intake (4, 8). Human ghrelin, a 28–amino acid peptide, is derived by posttranslational cleavage from a prepropeptide of 117 residues. On the basis of bioinformatic searches of putative hormones derived from the prepropeptides of known peptide hormones, we identified a ghrelin-associated peptide. We searched GenBank for orthologs of the human ghrelin gene and compared preproghrelin sequences from 11 mammalian species. In addition to the known ghrelin mature peptide, which immediately follows the signal peptide, we identified another conserved region that was flanked by potential convertase cleavage sites (fig. S1, underlined). This region encodes a putative 23–amino acid peptide, with a flanking conserved glycine residue at the C terminus, suggesting that it might be amidated (9). We named this ghrelin-associated peptide obestatin. Characterization of endogenous obestatin. To detect endogenous obestatin, we prepared a synthetic obestatin peptide and performed radioimmunoassays on rat-tissue extracts with obestatin-specific antibodies. As shown in Fig. 1A, the stomach extract displaced I125-obestatin binding to the obestatin antibodies. Obestatin-like activities from stomach extracts were purified. Immunoreactive (ir) obestatin was eluted in a Sephadex G-50 gel permeation column (Amersham Biosciences, Piscataway, NJ) with estimated sizes of 2.6 and 1.5 kilo-daltons (kD), distinct from the elution position of mature ghrelin (Fig. 1B). We subjected peak 1 (2.6 kD) fractions to ion-exchange fast protein liquid chromatography (FPLC). A single peak of ir obestatin was eluted (Fig. 1C) and shown by mass spectrometry and Edman sequencing to contain a peptide with a molecular mass of 2516.3 (Fig. 1D) and with a sequence of FNAPFDVGIKLSGAQYQQHG-XX (10). Combined with molecular-weight determination, the full sequence of the purified peptide was predicted to be FNAPFDVGIKLSGAQYQQHGRALNH2, consistent with the obestatin sequence deduced from rat ghrelin cDNA. In addition, mass spectrometric analyses suggested that peak 2 (1.5 kD) represented the last 13 residues of amidated obestatin, indicating further processing. To investigate differential secretion of ghrelin and obestatin in vivo, we fasted adult male rats for 48 hours before refeeding. Consistent with earlier findings (11), fasting led to a major increase in serum ghrelin levels, whereas subsequent refeeding for 2 hours by allowing animals free access to food or drinking water containing dextrose decreased circulating ghrelin (Fig. 1E). In contrast, serum levels of obestatin determined by a radioimmunoassay were constant in the different treatment groups. Obestatin suppression of food intake and gastrointestinal functions. We next synthesized amidated human obestatin and tested its effect on food intake in adult male mice. Intraperitoneal injection of obestatin suppressed food intake in a time- and dose-dependent manner (Fig. 2A). Intracerebroventricular treatment with obestatin also decreased food intake (Fig. 2B), similar to the anorexigenic effect of the synthetic melanocortin agonists MTII (12). In contrast, treatment with the nonamidated obestatin (NA-obestatin) was less effective. We also investigated the effect of obestatin, ghrelin, or vehicle alone on body weight in adult male rats. Treatment with ghrelin (1 µmol per kg body weight, three times daily) increased body weight, whereas the same dose of obestatin suppressed body-weight gain (Fig. 2C). Serum leptin levels were not affected after treatment with either obestatin or ghrelin (fig. S2), suggesting minimal modulation of body-fat content. Furthermore, treatment with obestatin led to a sustained suppression of gastric emptying activity (Fig. 2D). In vitro, isometric force measurement demonstrated that obestatin treatment decreased the contractile activity of jejunum muscle strips and antagonized the stimulatory effect of ghrelin (Fig. 2E) (13). The observed inhibition of jejunal contraction may trigger an afferent vagus signal to induce a central satiety response. Unlike ghrelin, obestatin did not increase GH secretion by cultured rat pituitary cells (fig. S3). Obestatin is the cognate ligand for GPR39. Experiments with crude plasma-membrane preparation of rat jejunum revealed that I125-obestatin bound to jejunal preparations with a high affinity (dissociation constant Kd = 4 nM), and this binding was not competed by ghrelin, motilin, neurotensin, or neuromedin U (fig. S4). Furthermore, NA-obestatin and truncated (des1-10)obestatin showed a lower binding affinity than did obestatin. I125-obestatin also bound to the pituitary, stomach, ileum, and hypothalamus, but less so to other tissues (fig. S4). We hypothesized that obestatin interacts with an orphan GPCR, and we tested obestatin binding to Chinese hamster ovary (CHO) cells transfected with 30 individual orphan receptor cDNAs. I125-obestatin interacted with high affinity (Kd = 1 nM) to the orphan receptor GPR39, which belongs to the ghrelin receptor subfamily (Fig. 3A) (14, 15). I125-obestatin binding to GPR39 was competed by obestatin but not by ghrelin or several other brain/gut hormones including motilin, neurotensin, or neuromedin U (Fig. 3B). In addition, NA-obestatin and truncated (des1-10)obestatin had a lower affinity for GPR39 than did obestatin. In CHO cells overexpressing GPR39, treatment with obestatin stimulated cyclic adenosine monophosphate (cAMP) production, whereas treatment with ghrelin or motilin was ineffective (Fig. 3C). Consistent with the reported activation of the serum response element (SRE) by constitutive active GPR39 (14), hormonal treatment of CHO cells co-transfected with GPR39 and a SRE promoter-luciferase construct led to obestatin but not ghrelin or motilin signaling (Fig. 3D). Similar stimulation of cAMP production and the SRE promoter by obestatin was found when GPR39 was overexpressed in HEK293T cells (fig. S5). Although CHO cells expressing GHSR did not respond to treatment with obestatin or ghrelin, cotransfection with a chimeric Gsq protein, which is capable of switching Gq-mediated signaling to Gs proteins (16), led to cAMP increases induced by ghrelin but not obestatin (Fig. 3E). Likewise, cells expressing the Gsq protein and the motilin receptor responded to treatment with motilin but not obestatin (Fig. 3F). Cross-linking studies further demonstrated that I125-obestatin bound to recombinant GPR39, forming a high–molecular-weight complex (fig. S6). Real-time reverse-transcription polymerase chain reaction (RT-PCR) analyses indicated that GPR39 is expressed in the jejunum, duodenum, stomach, pituitary, ileum, liver, hypothalamus, and other tissues (Fig. 3G), consistent with obestatin binding studies. Discussion. Ghrelin is implicated in meal initiation and body-weight regulation. Chronic ghrelin administration increases food intake and decreases energy expenditure, thus causing weight gain. In contrast to ghrelin, which causes hyperphagia and obesity in rats (17), obestatin appears to act as an anorexic hormone by decreasing food intake, gastric emptying activities, jejunal motility, and body-weight gain. Mutant mice with a deletion of the ghrelin gene did not show impaired growth or appetite (6, 18), most likely because these animals lacked both orexigenic ghrelin and anorexic obestatin. Indeed, transgenic mice bearing the preproghrelin gene under the control of the chicken ß-actin promoter produced high levels of inactive des-acyl ghrelin but exhibited lower body weights (19), most likely due to excessive obestatin biosynthesis. The discovery of amidated obestatin and its cognate receptor underscores the power of comparative genomic analyses in the postgenomic era. A peptide derived from the 66 C-terminal amino acids of proghrelin, named C-ghrelin, was detected in human circulation, and its serum levels were elevated in patients with heart failure (20). Although the antibodies used to detect C-ghrelin overlap with obestatin by 13 residues, the exact chemical nature and function of the circulating C-ghrelin remain unclear. Our finding that two peptide hormones derived from the same proprotein act through distinct receptors and exert opposing physiological actions highlights the importance of posttranslational regulatory mechanisms. Thus, monitoring of ghrelin transcript levels does not accurately reflect the secretion of these two polypeptides. After removal of the signal peptides from prepropeptides, convertases cleave prohormones at mono- or dibasic residues (21). In processed peptides with a C-terminal glycine, the residue is further amidated (9). Similar to the importance of posttranslational amidation for obestatin bioactivity, ghrelin also requires acylation on its serine-3 residue for bioactivity (7). Ghrelin binds to GHSR, which belongs to the subgroup of type A GPCRs consisting of GPR39 and receptors for ghrelin and motilin (22). Our discovery that obestatin is the cognate ligand for GPR39 suggests that GHSR and GPR39 could have evolved from a common ancestor but diverged in their functions, thus maintaining a delicate balance of body-weight regulation. This scenario is similar to the divergent and sometimes opposing actions of two paralogous corticotropin-releasing hormone receptors and their ligands in the regulation of adaptive stress responses (23–25). In addition to roles in meal initiation, weight regulation, and gastrointestinal activity, ghrelin also regulates the pituitary hormone axis, carbohydrate metabolism, and various functions of the heart, kidney, pancreas, adipose tissues, and gonads (26). Because ghrelin mRNA was found in almost all human tissues analyzed (27), the identification of obestatin derived from the same gene product as ghrelin provides a basis for future elucidation of the differential posttranslational processing and modification of these two peptides. A better understanding of the roles of ghrelin and obestatin in the intricate balance of energy homeostasis and body-weight control may be essential for the successful treatment of obesity. Al Pater, PhD; email: old542000@... __________________________________ FareChase: Search multiple travel sites in one click. http://farechase. Quote Link to comment Share on other sites More sharing options...
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