Identification, chemical synthesis, and receptor binding of a reptilian gecko ghrelin.

Ghrelin is known to be a gastrointestinal peptide hormone in vertebrates. It has a unique posttransrational modification, octanoylation, at the Ser side chain of the third position. In this study, we identified the genes encoding ghrelin and its receptor from the Schlegel's Japanese gecko Gekko japonicus. The C-terminal residue of gecko ghrelin was His, although the chemical synthesis method for the O-octanoyl peptide with a C-terminal His residue has not yet been well-established. Acyl-ghrelin has been synthesized using a Ser derivative without side chain protecting group in the solid-phase peptide synthesis, although this synthetic strategy has not yet been well-established. Here we show the efficient synthetic method with minimal side reactions, and G. japonicus ghrelin could be obtained in good yield. This would be useful and applicable to the synthesis of ghrelin from other animal species. The gecko ghrelin receptor was expressed in HEK 293 cells, which was fully responsive to the synthetic gecko ghrelin. These results indicate that the ghrelin system similar to mammals also exists in a reptilian gecko, G. japonicus.

Structure and dynamics of G protein-coupled receptor-bound ghrelin reveal the critical role of the octanoyl chain.

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

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.

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation.

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

Role of ghrelin isoforms in the mitigation of hepatic inflammation, mitochondrial dysfunction, and endoplasmic reticulum stress after bariatric surgery in rats.

BACKGROUND/OBJECTIVES: Bariatric surgery improves nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain elusive. We evaluated the potential role of ghrelin isoforms in the amelioration of hepatic inflammation after sleeve gastrectomy and Roux-en-Y gastric bypass (RYGB). SUBJECTS/METHODS: Plasma ghrelin isoforms were measured in male Wistar rats (n = 129) subjected to surgical (sham operation, sleeve gastrectomy, or RYGB) or dietary interventions [fed ad libitum a normal (ND) or a high-fat diet (HFD) or pair-fed diet]. The effect of acylated and desacyl ghrelin on markers of inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in primary rat hepatocytes under palmitate-induced lipotoxic conditions was assessed. RESULTS: Plasma desacyl ghrelin was decreased after sleeve gastrectomy and RYGB, whereas the acylated/desacyl ghrelin ratio was augmented. Both surgeries diminished obesity-associated hepatic steatosis, CD68+- and apoptotic cells, proinflammatory JNK activation, and Crp, Tnf, and Il6 transcripts. Moreover, a postsurgical amelioration in the mitochondrial DNA content, oxidative phosphorylation (OXPHOS) complexes I and II, and ER stress markers was observed. Specifically, following bariatric surgery GRP78, spliced XBP-1, ATF4, and CHOP levels were reduced, as were phosphorylated eIF2α. Interestingly, acylated and desacyl ghrelin inhibited steatosis and inflammation of palmitate-treated hepatocytes in parallel to an upregulation of OXPHOS complexes II, III, and V, and a downregulation of ER stress transducers IRE1α, PERK, ATF6, their downstream effectors, ATF4 and CHOP, as well as chaperone GRP78. CONCLUSIONS: Our data suggest that the increased relative acylated ghrelin levels after bariatric surgery might contribute to mitigate obesity-associated hepatic inflammation, mitochondrial dysfunction, and ER stress.

Unacylated Ghrelin Regulates Glucose-Sensitive Neurons Activity and Glycolipid Metabolism via Orexin-A Neurons in the Lateral Hypothalamic Area.

The objective of the study was to investigate the regulatory actions of unacylated ghrelin (UAG) on glucose-sensitive (GS) neurons and glycolipid metabolism in the lateral hypothalamus area (LHA) and its involvement with orexin-A-immunopositive neurons. The effects of UAG administered into the LHA on GS neurons discharges and glycolipid metabolism were detected by single neuron discharge recording, biochemical index analysis and quantitative real-time PCR; the level of c-fos protein in orexin-A-immunopositive neurons was observed using immunofluorescence staining. UAG microinjected into the LHA activated glucose-inhibited neurons, which were partially blocked by pre-administration of anti-orexin-A antibody in the LHA. Furthermore, UAG microinjected into the LHA significantly reduced serum triglycerides (TG), total cholesterol, low-density lipoprotein cholesterol, blood glucose, insulin and hepatic TG levels, while elevated serum high-density lipoprotein cholesterol levels. UAG elevated the mRNA expression of carnitine palmitoyltransferase-1 and reduced the mRNA expression of acetyl-CoA carboxylase-1 in the liver. The above-mentioned effects of UAG were partially blocked by pre-administration of anti-orexin-A antibody. The expressions of orexin-A and c-fos were observed in the LHA. After UAG injection into the LHA, some neurons showed double labeling, and the percentage of double-labeled orexin-A/c-fos neurons in orexin-A-immunopositive neurons increased significantly. UAG in the LHA regulates glycolipid metabolism by activating orexin-A-immunopositive neurons in the LHA.

Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract.

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.

Early Detection of Growth Hormone Secretagogue Receptor Antagonists Exploiting Their Atypical Behavior in Competitive Assays.

We report the proof-of-concept of a bioaffinity format designed for the early detection of growth hormone secretagogue receptor (GHS-R1a) antagonists in urine samples. We exploit here their atypical behavior in competitive experiments with labeled ghrelin (GHR), namely, the strong promoting effect on the GHR/GHS-R1a interaction at low molar ratios GHR/antagonist. The antagonists potentiate the GHR/GHS-R1a interaction, and they display the same effect on the interaction of GHS-R1a with other agonists listed as doping agents. The developed assay allows the estimation of affinity constants of ligand/receptor and antagonist/receptor binding and is amenable to optical, electrochemical, and mass-sensitive detection. The estimated affinity constants for GHR/GHS-R1a and antagonist/GHS-R1a in the absence of G proteins are in good agreement with recently reported data.

Physiological significance of ghrelin in the cardiovascular system.

Ghrelin, a growth hormone-releasing peptide first discovered in rat stomach in 1999, is a ligand for the growth hormone secretagogue receptor. It participates in the regulation of diverse processes, including energy balance and body weight maintenance, and appears to be beneficial for the treatment of cardiovascular diseases. In animal models of chronic heart failure, ghrelin improves cardiac function and remodeling; these findings have been recapitulated in human patients. In other animal models, ghrelin effectively diminishes pulmonary hypertension. Moreover, ghrelin administration early after myocardial infarction decreased the frequency of fatal arrhythmia and improved survival rate. In ghrelin-deficient mice, endogenous ghrelin protects against fatal arrhythmia and promotes remodeling after myocardial infarction. Although the mechanisms underlying the effects of ghrelin on the cardiovascular system have not been fully elucidated, its beneficial effects appear to be mediated through regulation of the autonomic nervous system. Ghrelin is a promising therapeutic agent for cardiac diseases.

Development and Characterization of an 18F-labeled Ghrelin Peptidomimetic for Imaging the Cardiac Growth Hormone Secretagogue Receptor.

One-third of patients with heart disease develop heart failure, which is diagnosed through imaging and detection of circulating biomarkers. Imaging strategies reveal morphologic and functional changes but fall short of detecting molecular abnormalities that can lead to heart failure, and circulating biomarkers are not cardiac specific. Thus, there is critical need for biomarkers that are endogenous to myocardial tissues. The cardiac growth hormone secretagogue receptor 1a (GHSR1a), which binds the hormone ghrelin, is a potential biomarker for heart failure. We have synthesized and characterized a novel ghrelin peptidomimetic tracer, an 18F-labeled analogue of G-7039, for positron emission tomography (PET) imaging of cardiac GHSR1a. In vitro analysis showed enhanced serum stability compared to natural ghrelin and significantly increased cellular uptake in GHSR1a-expressing OVCAR cells. Biodistribution studies in mice showed that tissue uptake of the tracer was independent of circulating ghrelin levels, and there was negligible cardiac uptake and high uptake in the liver, intestines, and kidneys. Specificity of tracer uptake was assessed using ghsr -/- mice; both static and dynamic PET imaging revealed no difference in cardiac uptake, and there was no significant correlation between cardiac standardized uptake values and GHSR1a expression. Our study lays the groundwork for further refinement of peptidomimetic PET tracers targeting cardiac GHSR1a.

Molecular characterization and expression patterns of ghrelin in the reindeer (Rangifer tarandus).

Ghrelin is a novel growth hormone (GH)-releasing peptide, which has been identified as an endogenous ligand for the GH-sretagogue receptor. The sequence and expression of ghrelin has been determined in many species. In this study, to reveal the molecular characterization and expression patterns of ghrelin in the reindeer (Rangifer tarandus), the full-length DNA and cDNA encoding ghrelin were cloned from reindeer stomach using genome walking and rapid amplification of complementary deoxyribonucleic acid ends (RACE). The expression of ghrelin in almost all tissues was examined by real-time quantitative PCR (RT-qPCR). The 4076 bp amplicon of the ghrelin gene consisting of 4 exons and 3 introns was cloned from reindeer. Results of cDNA cloning and sequence analysis revealed that the full-length ghrelin cDNA was composed of 539 bp that included a 5'-untranslated region (46 bp), an open reading frame (ORF) (351 bp), and a 3'-untranslated region (142 bp). In addition, ghrelin was expressed in the all tissues examined, with the expression in the abomasum significantly higher than that in other tissues (p<0.05), followed by the pancreas, duodenum, testis and oesophagus. The results show that the expression of ghrelin in the reindeer gastrointestinal tract is extensive, suggesting its may have a role in regulating the digestive function.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation.

The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small molecule therapeutics for treatment of obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence of the involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine-modifying electrophiles, revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness.

BACKGROUND: The Ghrelin-system is a complex, pleiotropic family composed of several peptides, including native-ghrelin and its In1-ghrelin splicing variant, and receptors (GHSR 1a/b), which are dysregulated in various endocrine-related tumors, where they associate to pathophysiological features, but the presence, functional role, and mechanisms of actions of In1-ghrelin splicing variant in prostate-cancer (PCa), is completely unexplored. Herein, we aimed to determine the presence of key ghrelin-system components (native-ghrelin, In1-ghrelin, GHSR1a/1b) and their potential pathophysiological role in prostate cancer (PCa). METHODS: In1-ghrelin and native-ghrelin expression was evaluated by qPCR in prostate tissues from patients with high PCa-risk (n = 52; fresh-tumoral biopsies), and healthy-prostates (n = 12; from cystoprostatectomies) and correlated with clinical parameters using Spearman-test. In addition, In1-ghrelin and native-ghrelin was measured in plasma from an additional cohort of PCa-patients with different risk levels (n = 30) and control-healthy patients (n = 20). In vivo functional (proliferation/migration) and mechanistic (gene expression/signaling-pathways) assays were performed in PCa-cell lines in response to In1-ghrelin and native-ghrelin treatment, overexpression and/or silencing. Finally, tumor progression was monitored in nude-mice injected with PCa-cells overexpressing In1-ghrelin, native-ghrelin and empty vector (control). RESULTS: In1-ghrelin, but not native-ghrelin, was overexpressed in high-risk PCa-samples compared to normal-prostate (NP), and this expression correlated with that of PSA. Conversely, GHSR1a/1b expression was virtually absent. Remarkably, plasmatic In1-ghrelin, but not native-ghrelin, levels were also higher in PCa-patients compared to healthy-controls. Furthermore, In1-ghrelin treatment/overexpression, and to a much lesser extent native-ghrelin, increased aggressiveness features (cell-proliferation, migration and PSA secretion) of NP and PCa cells. Consistently, nude-mice injected with PC-3-cells stably-transfected with In1-ghrelin, but not native-ghrelin, presented larger tumors. These effects were likely mediated by ERK1/2-signaling activation and involved altered expression of key oncogenes/tumor suppressor genes. Finally, In1-ghrelin silencing reduced cell-proliferation and PSA secretion from PCa cells. CONCLUSIONS: Altogether, our results indicate that In1-ghrelin levels (in tissue) and circulating levels (in plasma) are increased in PCa where it can regulate key pathophysiological processes, thus suggesting that In1-ghrelin may represent a novel biomarker and a new therapeutic target in PCa.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts.

Metabolic responses to exogenous ghrelin in obesity and early after Roux-en-Y gastric bypass in humans.

AIMS: Ghrelin is a gastric-derived hormone that stimulates growth hormone (GH) secretion and has a multi-faceted role in the regulation of energy homeostasis, including glucose metabolism. Circulating ghrelin concentrations are modulated in response to nutritional status, but responses to ghrelin in altered metabolic states are poorly understood. We investigated the metabolic effects of ghrelin in obesity and early after Roux-en-Y gastric bypass (RYGB). MATERIALS AND METHODS: We assessed central and peripheral metabolic responses to acyl ghrelin infusion (1 pmol kg-1 min-1 ) in healthy, lean subjects (n = 9) and non-diabetic, obese subjects (n = 9) before and 2 weeks after RYGB. Central responses were assessed by GH and pancreatic polypeptide (surrogate for vagal activity) secretion. Peripheral responses were assessed by hepatic and skeletal muscle insulin sensitivity during a hyperinsulinaemic-euglycaemic clamp. RESULTS: Ghrelin-stimulated GH secretion was attenuated in obese subjects, but was restored by RYGB to a response similar to that of lean subjects. The heightened pancreatic polypeptide response to ghrelin infusion in the obese was attenuated after RYGB. Hepatic glucose production and hepatic insulin sensitivity were not altered by ghrelin infusion in RYGB subjects. Skeletal muscle insulin sensitivity was impaired to a similar degree in lean, obese and post-RYGB individuals in response to ghrelin infusion. CONCLUSIONS: These data suggest that obesity is characterized by abnormal central, but not peripheral, responsiveness to ghrelin that can be restored early after RYGB before significant weight loss. Further work is necessary to fully elucidate the role of ghrelin in the metabolic changes that occur in obesity and following RYGB.

Structure-activity analysis of human ghrelin O-acyltransferase reveals chemical determinants of ghrelin selectivity and acyl group recognition.

Ghrelin O-acyltransferase (GOAT) is an integral membrane acyltransferase responsible for catalyzing a serine-octanoylation posttranslational modification within the peptide hormone ghrelin. Ghrelin requires this octanoylation for its biological activity in stimulating appetite and in regulating other physiological pathways involved in energy balance. Blocking ghrelin acylation using GOAT inhibitors is a new potential avenue to treat health conditions impacted by ghrelin signaling, such as obesity and diabetes. Designing novel and potent GOAT inhibitors as potential therapeutics requires insight into the interactions between the ghrelin and octanoyl coenzyme A substrates and the GOAT active site. Through structure-activity investigation of ghrelin-mimetic peptide substrates and inhibitors, we have analyzed the amino acid selectivity of the enzyme as well as the functional groups involved in substrate recognition by human GOAT (hGOAT). This analysis reveals that hGOAT both prefers and tolerates a distinct set of chemical properties at each position within the N-terminal sequence of ghrelin and that sequence elements downstream of the ghrelin N-terminal sequence contribute to ghrelin binding to hGOAT. We also found that the hGOAT active site exhibits a marked preference for binding an eight-carbon acyl chain, which potentially explains the biological observation of ghrelin octanoylation in light of the acyl donor promiscuity reported for GOAT. Bioinformatics analysis, guided by our reactivity data, supports the conclusion that ghrelin is a unique substrate for hGOAT within the human proteome, providing further justification for the ghrelin-hGOAT system as a desirable drug target. By defining an array of substrate-enzyme interactions used by hGOAT to bind, recognize, and acylate ghrelin, this study yields novel insight into the character of the hGOAT active site that can serve as a guide toward the rational design of hGOAT inhibitors.

Effects of Cigarette Smoking on Plasma Concentration of the Appetite-Regulating Peptide Ghrelin.

BACKGROUND: Weight gain is a common but only a partially understood consequence of smoking cessation. Existing data suggest modulating effects of the orexigenic peptide ghrelin on food intake. The aim of the present study was to investigate the effect of tobacco withdrawal on plasma concentration of acetylated and total ghrelin. METHODS: Fifty four normal-weighted smokers and 30 non-smoking healthy controls were enrolled in our study. Concentrations of acetylated and total ghrelin were measured in blood plasma drawn two hours after a standardized meal and three hours after the smokers smoked their last cigarette. The severity of tobacco addiction was assessed based on cotinine plasma concentration, the Fagerström Test for Nicotine Dependence (FTND) and the number of cigarettes smoked per day. RESULTS: The plasma concentration of acetylated ghrelin, but not total ghrelin, was significantly higher in smokers than in non-smokers. Moreover, we found significant negative correlations between acetylated ghrelin and all measures of the severity of nicotine dependence. CONCLUSIONS: Early abstinence from tobacco smoking seems to be associated with increased plasma concentration of the orexigenic peptide acetylated ghrelin. This could be one reason for increased food craving during nicotine withdrawal and subsequent weight gain. Smokers might compensate these effects by increasing tobacco intake.

Cloning and tissue distribution of appetite-regulating peptides in pirapitinga (Piaractus brachypomus).

Pirapitinga (or red-bellied pacu, Piaractus brachypomus, Characiforme, Serrasalmidae) is an economically important South American fish for which the endocrine mechanism of the regulation of feeding has never been examined. To better understand these mechanisms, cDNAs encoding the appetite-regulating peptides orexin, cocaine- and amphetamine-regulated transcript (CART), apelin, cholecystokinin (CCK), peptide YY (PYY), leptin and ghrelin were isolated in pirapitinga and their mRNA distributions examined in peripheral tissues and brain. When compared to other fish, the sequences obtained for all peptides were most similar to those of other Characiforme fish (i.e. Mexican cavefish) and Siluriformes (catfish) as well as Cypriniformes (i.e. goldfish, zebrafish). All peptides were widely expressed within the brain. With the exception of CART, which was only expressed in brain, the mRNAs of all peptides were present in several peripheral tissues, including gastrointestinal tract, kidneys and gills. The widespread and peptide-specific distributions suggest that each peptide might have distinct physiological actions in the brain and on peripheral tissues, in particular on the gastrointestinal tract, which include feeding regulation. This preliminary study opens new avenues for further functional studies on the endocrine regulation of feeding in Serrasalmidae fish, including pirapitinga.

Ghrelin and lipid levels in panic disorder before and after treatment and their relationship with agoraphobia.

BACKGROUND: We aimed to evaluate serum ghrelin (GHR) levels and lipid profile in panic disorder (PD), with and without agoraphobia, and to compare these parameters before and after treatment. SUBJECTS AND METHODS: The GHR and lipid profiles were measured in blood samples taken from 31 PD patients with agoraphobia, 22 PD patients without agoraphobia, and 53 control group subjects. 23 of the 53 patients who were prescribed 20 to 40 mg/day paroxetine had continued treatment. The 23 patients who had continued treatment were measured again at the end of twelve weeks. RESULTS: The GHR and triglyceride (TRG), total cholesterol (Total-C), low-density lipoproteins (LDL-C), and very low-density lipoproteins (VLDL-C) levels were higher in the PD with agoraphobia group than the PD without agoraphobia and control groups. The 23 patients that had continued their treatment were re-evaluated, and the serum GHR, Total-C levels, and BMI after treatment were significantly decreased, compared to the values before treatment. CONCLUSIONS: There may be a pathophysiological relationship between the GHR and lipid profiles that interact with each other in PD. In fact, this relationship was more marked in PD with agoraphobia than in PD without agoraphobia.

Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase.

Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes.