Ghrelin octanoylation by ghrelin O-acyltransferase: protein acylation impacting metabolic and neuroendocrine signalling

The acylated peptide hormone ghrelin impacts a wide range of physiological processes but is most well known for controlling hunger and metabolic regulation. Ghrelin requires a unique posttranslational modification, serine octanoylation, to bind and activate signalling through its cognate GHS-R1a receptor. Ghrelin acylation is catalysed by ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) enzyme family. The ghrelin/GOAT/GHS-R1a system is defined by multiple unique aspects within both protein biochemistry and endocrinology. Ghrelin serves as the only substrate for GOAT within the human proteome and, among the multiple hormones involved in energy homeostasis and metabolism such as insulin and leptin, acts as the only known hormone in circulation that directly stimulates appetite and hunger signalling. Advances in GOAT enzymology, structural modelling and inhibitor development have revolutionized our understanding of this enzyme and offered new tools for investigating ghrelin signalling at the molecular and organismal levels. In this review, we briefly summarize the current state of knowledge regarding ghrelin signalling and ghrelin/GOAT enzymology, discuss the GOAT structural model in the context of recently reported MBOAT enzyme superfamily member structures, and highlight the growing complement of GOAT inhibitors that offer options for both ghrelin signalling studies and therapeutic applications.     

Altered Lipid and Salt Taste Responsivity in Ghrelin and GOAT Null Mice

Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin^−/−), and GOAT knockout (GOAT^−/−) mice. Ghrelin^−/− mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT^−/− mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin^−/− and GOAT^−/− mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin^−/− mice, yet potentiated in GOAT^−/− mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT^−/− mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin^−/− and GOAT^−/− mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities.     

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.     

Functional group and stereochemical requirements for substrate binding by ghrelin O-acyltransferase revealed by unnatural amino acid incorporation

Ghrelin is a small peptide hormone that undergoes a unique posttranslational modification, serine octanoylation, to play its physiological roles in processes including hunger signaling and glucose metabolism. Ghrelin O-acyltransferase (GOAT) catalyzes this posttranslational modification, which is essential for ghrelin to bind and activate its cognate GHS-R1a receptor. Inhibition of GOAT offers a potential avenue for modulating ghrelin signaling for therapeutic effect. Defining the molecular characteristics of ghrelin that lead to binding and recognition by GOAT will facilitate the development and optimization of GOAT inhibitors. We show that small peptide mimics of ghrelin substituted with 2,3-diaminopropanoic acid in place of the serine at the site of octanoylation act as submicromolar inhibitors of GOAT. Using these chemically modified analogs of desacyl ghrelin, we define key functional groups within the N-terminal sequence of ghrelin essential for binding to GOAT and determine GOAT’s tolerance to backbone methylations and altered amino acid stereochemistry within ghrelin. Our study provides a structure-activity analysis of ghrelin binding to GOAT that expands upon activity-based investigations of ghrelin recognition and establishes a new class of potent substrate-mimetic GOAT inhibitors for further investigation and therapeutic interventions targeting ghrelin signaling.     

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

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

     

Knockdown of ghrelin‐O‐acyltransferase attenuates colitis through the modulation of inflammatory factors and tight junction proteins in the intestinal epithelium

Ghrelin‐O‐acyltransferase (GOAT) is a membrane‐bound enzyme that attaches eight‐carbon octanoate to a serine residue in ghrelin and thereby acylates inactive ghrelin to produce active ghrelin. In this study, we investigated the function of GOAT in the intestinal mucosal barrier. The intestinal mucosal barrier prevents harmful substances such as bacteria and endotoxin from entering the other tissues, organs, and blood circulation through the intestinal mucosa. Here, we established 5% dextran sodium sulfate (DSS)‐induced colitis in mice and found that the body weight and colon weight were significantly decreased in these mice. Furthermore, increased inflammation and apoptosis were observed in the tissues of DSS‐induced colitis mice, with increased expression of tumor necrosis factor‐α, interleukin‐6, phosphorylation of nuclear factor kappa B‐p65 (p‐NF‐κB‐p65), and cleaved caspase‐3, and decreased expression of tight junction (TJ) proteins such as zonula occluden‐1 and occludin. The knockdown of GOAT significantly attenuated colitis‐induced inflammation responses and apoptosis, while GOAT overexpression significantly enhanced the induction of colitis. These results suggest that knockdown of GOAT may attenuate colitis‐induced inflammation, ulcers, and fecal occult blood by decreasing the intestinal mucosal permeability via the modulation of inflammatory factors and TJ proteins.     

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of K_m for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0–9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.     

Ghrelin O-acyltransferase (GOAT) has a preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor

Ghrelin is a peptide hormone in which serine 3 is modified by n-octanoic acid through GOAT (ghrelin O-acyltransferase). However, the enzymological properties of GOAT remain to be elucidated. We analyzed the in vitro activity of GOAT using the recombinant enzyme. Unexpectedly, although the main active form of ghrelin is modified by n-octanoic acid, GOAT had a strong preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor. Moreover, a four-amino acid peptide derived from the N-terminal sequence of ghrelin can be modified by GOAT, indicating that these four amino acids constitute the core motif for substrate recognition by the enzyme.     

Correlation of ghrelin concentration and ghrelin,ghrelin-O-acetyltransferase (GOAT) and growth hormone secretagogue receptor 1a mRNAs expression in the proventriculus and brain of the growing chicken

To determine mechanisms for age-related decrease of GHS-R1a expression in the chicken proventriculus, changes in mRNA expression of ghrelin and ghrelin-O-acetyltransferase (GOAT) as well as ghrelin concentrations in the proventriculus and plasma were examined in growing chickens. Changes in expression levels of ghrelin, GOAT and GHS-R1a mRNAs were also examined in different brain regions (pituitary, hypothalamus, thalamus, cerebellum, cerebral cortex, olfactory bulb, midbrain and medulla oblongata). Ghrelin concentrations in the proventriculus and plasma increased with aging and reached plateaus at 30–50 days after hatching. High level of ghrelin mRNA decreased at 3 days after hatching, and it became stable at half of the initial level. Expression levels of GHS-R1a and GOAT decreased 3 or 5 days after hatching and became stable at low levels. Significant negative correlations were found between plasma ghrelin and mRNA levels of GOAT and GHS-R1a. Expression levels of ghrelin mRNA were different in the brain regions, but a significant change was not seen with aging. GHS-R1a expression was detected in all brain regions, and age-dependent changes were observed in the pituitary and cerebellum. Different from the proventriculus, the expression of GOAT in the brain increased or did not change with aging. These results suggest that decreased GHS-R1a and GOAT mRNA expression in the proventriculus is due to endogenous ghrelin-induced down-regulation. Expression levels of ghrelin, GOAT and GHS-R1a in the brain were independently regulated from that in the proventriculus, and age-related and region-dependent regulation pattern suggests a local effect of ghrelin system in chicken brain.     

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—a novel generation of anti‐obesity drug: design,pharmacomodulation and biological activity of ghrelin analogues

Ghrelin is a unique bioactive peptide with respect to both the structure and its biological function. This 28‐amino acid peptide is modified with an n‐octanoyl group at serine‐3, and accordingly is the only lipidated biologically active peptide hormone known so far. Ghrelin binds to the so‐called ghrelin or GHS receptor, a member of the class A of G‐protein coupled receptors, which leads to Ca²⁺ release intracellularly due to the activation of the Gq‐system. Interestingly, the ghrelin receptor shows a significant constitutive activity which means that in addition to agonists and antagonists, inverse agonists play an important role in receptor modulation. In this review, the major activities of ghrelin are summarized with a strong focus on the regulation of food intake. So far reported agonists, antagonists and inverse agonists are shown and structure activitiy relationships are discussed. Furthermore, the application of ghrelin ligands as novel anti‐obesity drugs is outlined and the state of the art in this field is summarized. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone

Ghrelin is a 28 amino acid, appetite-stimulating peptide hormone secreted by the food-deprived stomach. Serine-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is required for its endocrine actions. Here, we identify GOAT (Ghrelin O-Acyltransferase), a polytopic membrane-bound enzyme that attaches octanoate to serine-3 of ghrelin. Analysis of the mouse genome revealed that GOAT belongs to a family of 16 hydrophobic membrane-bound acyltransferases that includes Porcupine, which attaches long-chain fatty acids to Wnt proteins. GOAT is the only member of this family that octanoylates ghrelin when coexpressed in cultured endocrine cell lines with prepro-ghrelin. GOAT activity requires catalytic asparagine and histidine residues that are conserved in this family. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues. Identification of GOAT will facilitate the search for inhibitors that reduce appetite and diminish obesity in humans.     

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..     

Ghrelin acylation and metabolic control

Since its discovery, many physiologic functions have been ascribed to ghrelin, a gut derived hormone. The presence of a median fatty acid side chain on the ghrelin peptide is required for the binding and activation of the classical ghrelin receptor, the growth hormone secretagogue receptor (GHSR)-1a. Ghrelin O-acyl transferase (GOAT) was recently discovered as the enzyme responsible for this acylation process. GOAT is expressed in all tissues that have been found to express ghrelin and has demonstrated actions on several complex endocrine organ systems such as the hypothalamus-pituitary-gonadal, insular and adrenal axis as well as the gastrointestinal (GI) tract, bone and gustatory system. Ghrelin acylation is dependent on the function of GOAT and the availability of substrates such as proghrelin and short- to medium-chain fatty acids (MCFAs). This process is governed by GOAT activity and has been shown to be modified by dietary lipids. In this review, we provided evidence that support an important role of GOAT in the regulation of energy homeostasis and glucose metabolism by modulating acyl ghrelin (AG) production. The relevance of GOAT and AG during periods of starvation remains to be defined. In addition, we summarized the recent literature on the metabolic effects of GOAT specific inhibitors and shared our view on the potential of targeting GOAT for the treatment of metabolic disorders such as obesity and type 2 diabetes.     

Differential distribution of ghrelin-O-acyltransferase (GOAT) immunoreactive cells in the mouse and rat gastric oxyntic mucosa

The enzyme that acylates ghrelin was recently identified in mice as the fourth member of the membrane-bound O-acyltransferases superfamily (MBOAT4) and named ghrelin-O-acyltransferase (GOAT). Only one report showed GOAT mRNA expression in ghrelin-expressing cells of the mouse stomach. We investigated the distribution of GOAT protein in peripheral tissues and co-expression with endocrine markers in the gastric mucosa using a custom-made anti-GOAT antibody. Tissues were collected from male Sprague-Dawley rats and C57BL/6 mice. Western blot revealed two immunoreactive bands in rat and mouse gastric corpus mucosal proteins, a 50 kDa band corresponding to the GOAT protein and a 100 kDa band likely corresponding to a dimer. Western blot also detected GOAT in the plasma and levels were strongly increased after 24-h fasting in mice and slightly in rats. GOAT-immunoreactive cells were located in the gastric corpus mucosa and the anterior pituitary gland, whereas other peripheral tissues of rats and mice examined were negative. In mice, GOAT-immunoreactive cells were mainly distributed throughout the middle portion of the oxyntic glands, whereas in rats they were localized mainly in the lower portion of the glands. Double labeling showed that 95 ± 1% of GOAT-immunoreactive cells in mice co-labeled with ghrelin, whereas in rats only 56 ± 4% of GOAT-positive cells showed co-expression of ghrelin. The remainder of the GOAT-immunopositive cells in rats co-expressed histidine decarboxylase (44 ± 3%). No co-localization was observed with somatostatin in rats or mice. These data suggest species differences between rats and mice in gastric GOAT expression perhaps resulting in a different role of the MBOAT4 enzyme in the rat stomach. Detection of GOAT in the plasma raises the possibility that ghrelin octanoylation may occur in the circulation and the fasting-induced increase in GOAT may contribute to the increase of acylated ghrelin after fasting.     

GOAT induced ghrelin acylation regulates hedonic feeding

Ghrelin is an orexigenic hormone that regulates homeostatic and reward-related feeding behavior. Recent evidence indicates that acylation of ghrelin by the gut enzyme ghrelin O-acyl transferase (GOAT) is necessary to render ghrelin maximally active within its target tissues. Here we tested the hypothesis that GOAT activity modulates food motivation and food hedonics using behavioral pharmacology and mutant mice deficient for GOAT and the ghrelin receptor (GHSR). We evaluated operant responding following pharmacological administration of acyl-ghrelin and assessed the necessity of endogenous GOAT activity for operant responding in GOAT and GHSR-null mice. Hedonic-based feeding behavior also was examined in GOAT-KO and GHSR-null mice using a “Dessert Effect” protocol in which the intake of a palatable high fat diet “dessert” was assessed in calorically-sated mice. Pharmacological administration of acyl-ghrelin augmented operant responding; notably, this effect was dependent on intact GHSR signaling. GOAT-KO mice displayed attenuated operant responding and decreased hedonic feeding relative to controls. These behavioral results correlated with decreased expression of the orexin-1 receptor in reward-related brain regions in GOAT-KO mice. In summary, the ability of ghrelin to stimulate food motivation is dependent on intact GHSR signaling and modified by endogenous GOAT activity. Furthermore, GOAT activity is required for hedonic feeding behavior, an effect potentially mediated by forebrain orexin signaling. These data highlight the significance of the GOAT–ghrelin system for the mediation of food motivation and hedonic feeding.     

The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index

Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30–40, 40–50 and >50 were recruited (n = 9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (−42%, p < 0.01) whereas obese patients with BMI > 50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62–78%, p < 0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r = 0.71, p < 0.001) and a negative correlation with ghrelin (r = −0.60, p < 0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.     

Stress-related alterations of acyl and desacyl ghrelin circulating levels: mechanisms and functional implications

Ghrelin is the only known peripherally produced and centrally acting peptide hormone that stimulates food intake and digestive functions. Ghrelin circulates as acylated and desacylated forms and recently the acylating enzyme, ghrelin-O-acyltransferase (GOAT) and the de-acylating enzyme, thioesterase 1/lysophospholipase 1 have been identified adding new layers of complexity to the regulation of ghrelin. Stress is known to alter gastrointestinal motility and food intake and was recently shown to modify circulating ghrelin and GOAT levels with differential responses related to the type of stressors including a reduction induced by physical stressors (abdominal surgery and immunological/endotoxin injection, exercise) and elevation by metabolic (cold exposure, acute fasting and caloric restriction) and psychological stressors. However, the pathways underlying the alterations of ghrelin under these various stress conditions are still largely to be defined and may relate to stress-associated autonomic changes. There is evidence that alterations of circulating ghrelin may contribute to the neuroendocrine and behavioral responses along with sustaining the energetic requirement needed upon repeated exposure to stressors. A better understanding of these mechanisms will allow targeting components of ghrelin signaling that may improve food intake and gastric motility alterations induced by stress.