Assessing the role of ghrelin and the enzyme ghrelin O-acyltransferase (GOAT) system in food reward, food motivation, and binge eating behavior.

The peripheral peptide hormone ghrelin is a powerful stimulator of food intake, which leads to body weight gain and adiposity in both rodents and humans. The hormone, thus, increases the vulnerability to obesity and binge eating behavior. Several studies have revealed that ghrelin's functions are due to its interaction with the growth hormone secretagogue receptor type 1a (GHSR1a) in the hypothalamic area; besides, ghrelin also promotes the reinforcing properties of hedonic food, acting at extra-hypothalamic sites and interacting with dopaminergic, cannabinoid, opioid, and orexin signaling. The hormone is primarily present in two forms in the plasma and the enzyme ghrelin O-acyltransferase (GOAT) allows the acylation reaction which causes the transformation of des-acyl-ghrelin (DAG) to the active form acyl-ghrelin (AG). DAG has been demonstrated to show antagonist properties; it is metabolically active, and counteracts the effects of AG on glucose metabolism and lipolysis, and reduces food consumption, body weight, and hedonic feeding response. Both peptides seem to influence the hypothalamic-pituitary-adrenal (HPA) axis and the corticosterone/cortisol level that drive the urge to eat under stressful conditions. These findings suggest that DAG and inhibition of GOAT may be targets for obesity and bingeing-related eating disorders and that AG/DAG ratio may be an important potential biomarker to assess the risk of developing maladaptive eating behaviors.

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.

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.

Ghrelin octanoylation by ghrelin O-acyltransferase: Unique protein biochemistry underlying metabolic signaling.

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Ghrelin signaling is implicated in a variety of neurological and physiological processes, but is most well known for its roles in controlling hunger and metabolic regulation. Ghrelin octanoylation is catalyzed by ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) enzyme family. From the status of ghrelin as the only substrate for GOAT in the human genome to the source and requirement for the octanoyl acyl donor, the ghrelin-GOAT system is defined by multiple unique aspects within both protein biochemistry and endocrinology. In this review, we examine recent advances in our understanding of the interactions and mechanisms leading to ghrelin modification by GOAT, discuss the potential sources for the octanoyl acyl donor required for ghrelin's activation, and summarize the current landscape of molecules targeting ghrelin octanoylation through GOAT inhibition.

Metabolic stress leads to divergent changes in the ghrelinergic system in goldfish (Carassius auratus) gonads.

Various endocrine factors that regulate energy homeostasis are also implicated in the reproductive physiology of mammals. However, the hormonal link between metabolism and reproduction in fish is poorly understood. Ghrelin is a multifunctional hormone with both metabolic and reproductive roles in vertebrates. Post-translational acylation by ghrelin-O-acyltransferase (GOAT) is critical for its biological actions. The expression of ghrelin, ghrelin or growth hormone secretagogue receptor (GHSR), and GOAT (which forms the ghrelinergic system) in fish under metabolic stress remains unclear. In this research, we used RT-qPCR and Western blot analysis to determine the expression of the ghrelinergic system in goldfish (during the reproductively active phase) hypothalamus and gonads under 7 and 28 days of fasting. We found a significant increase in preproghrelin mRNA expresson in the ovary, and GOAT mRNA expression in the testis of goldfish deprived of food for 7 days. In fish deprived of food for 28 days, preproghrelin, GHSR and GOAT mRNA expression was significantly increased in the hypothalamus of male goldfish. Such differences were not observed in the hypothalamus of female fish, and in the testis of 28 days fasted fish. Meanwhile, preproghrelin, GHSR, and GOAT expression (both mRNA and protein) was significantly increased in the ovary of female fish fasted for 28 days. Ghrelin has been shown to suppress oocyte maturation in fish. The upregulation of a system that has ovarian inbititory roles suggests a role for ghrelin in maintaining reduced reproductive capability during metabolically challenging periods.

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.

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.

Reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: reversal by growth hormone.

Plasma growth hormone (GH) and hepatic autophagy each have been reported to protect against hypoglycemia in the fasted state, but previous data have not linked the two. Here we demonstrate a connection using a mouse model of fasting in a fat-depleted state. Mice were subjected to 1 wk of 60% calorie restriction, causing them to lose nearly all body fat. They were then fasted for 23 h. During fasting, WT mice developed massive increases in plasma GH and a concomitant increase in hepatic autophagy, allowing them to maintain viable levels of blood glucose. In contrast, lethal hypoglycemia occurred in mice deficient in the GH secretagogue ghrelin as a result of knockout of the gene encoding ghrelin O-acyltransferase (GOAT), which catalyzes a required acylation of the peptide. Fasting fat-depleted Goat(-/-) mice showed a blunted increase in GH and a marked decrease in hepatic autophagy. Restoration of GH by infusion during the week of calorie restriction maintained autophagy in the Goat(-/-) mice and prevented lethal hypoglycemia. Acute injections of GH after 7 d of calorie restriction also restored hepatic autophagy, but failed to increase blood glucose, perhaps owing to ATP deficiency in the liver. These data indicate that GH stimulation of autophagy is necessary over the long term, but not sufficient over the short term to maintain blood glucose levels in fasted, fat-depleted mice.

Effect of preduodenal lipase inhibition in suckling rats on dietary octanoic acid (C8:0) gastric absorption and plasma octanoylated ghrelin concentration.

Part of medium chain fatty acids (MCFAs) coming from dietary triglycerides (TGs) can be directly absorbed through the gastric mucosa after the action of preduodenal lipase (lingual lipase in the rat). MCFA gastric absorption, particularly that of octanoic acid (C8:0), may have a physiological importance in the octanoylation of ghrelin, the orexigenic gastric peptide acting as an endogenous ligand of the hypothalamic growth hormone secretagogue receptor 1a (GHSR-1a). However, the amount of C8:0 absorbed in the stomach and its metabolic fate still haven't been clearly characterized. The purpose of the present study was to further characterize and quantify the importance of preduodenal lipase activity on the release and gastric absorption of dietary C8:0 and on the subsequent ghrelin octanoylation in the stomach mucosa. Fifteen days old rats received fat emulsions containing triolein or [1,1,1-(13)C]-Tri-C8:0 and a specific inhibitor of preduodenal lipase, 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one or BemPPOX. The fate of the (13)C-C8:0 was followed in rat tissues after 30 and 120min of digestion and octanoylated ghrelin was measured in the plasma. This work (1) demonstrates that part of C8:0 coming from Tri-C8:0 is directly absorbed at the gastric level, (2) allows the estimation of C8:0 gastric absorption level (1.3% of the (13)C-C8:0 in sn-3 position after 30min of digestion), as well as (3) the contribution of rat lingual lipase to total lipolysis and to duodenal absorption of dietary FAs (at least 30%), (4) shows no short-term effect of dietary Tri-C8:0 consumption and subsequent increase of C8:0 gastric tissue content on plasma octanoylated ghrelin concentration.

The role of acylated-ghrelin in the regulation of sucrose intake.

The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin's biological interactions and avenues for controlling ghrelin signaling.

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Initially demonstrated to stimulate hunger and appetite, ghrelin-dependent signaling is implicated in a variety of neurological and physiological processes influencing diseases such as diabetes, obesity, and Prader-Willi syndrome. In addition to its cognate receptor, recent studies have revealed ghrelin interacts with a range of binding partners within the bloodstream. Defining the scope of ghrelin's interactions within the body, understanding how these interactions work in concert to modulate ghrelin signaling, and developing molecular tools for controlling ghrelin signaling are essential for exploiting ghrelin for therapeutic effect. In this review, we discuss recent findings regarding the biological effects of ghrelin signaling, outline binding partners that control ghrelin trafficking and stability in circulation, and summarize the current landscape of inhibitors targeting ghrelin octanoylation.

A new class of ghrelin O-acyltransferase inhibitors incorporating triazole-linked lipid mimetic groups.

Inhibitors of ghrelin O-acyltransferase (GOAT) have untapped potential as therapeutics targeting obesity and diabetes. We report the first examples of GOAT inhibitors incorporating a triazole linkage as a biostable isosteric replacement for the ester bond in ghrelin and amide bonds in previously reported GOAT inhibitors. These triazole-containing inhibitors exhibit sub-micromolar inhibition of the human isoform of GOAT (hGOAT), and provide a foundation for rapid future chemical diversification and optimization of hGOAT inhibitors.

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 Km 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.

Efficacy and safety of the ghrelin-O-acyltransferase inhibitor BI 1356225 in overweight/obesity: Data from two Phase I, randomised, placebo-controlled studies.

BACKGROUND: Obesity is a complex disease associated with multiple concurrent complications, and the coordinated targeting of multiple pathways in pharmacological treatment may improve weight loss outcomes. During synthesis, ghrelin is converted from the 'inactive' unacylated ghrelin (UAG) to the active acylated ghrelin (AG) by the enzyme ghrelin-O-acyltransferase (GOAT), stimulating appetite and food intake. AIMS: To report the results of two Phase I studies investigating single rising doses (SRDs) or multiple rising doses (MRDs) of the novel oral GOAT inhibitor BI 1356225 versus placebo in male and postmenopausal/sterilised female subjects with overweight or obesity. METHODS: The SRD study investigated single doses of BI 1356225 (0.1-20.0 mg) in healthy male subjects with a BMI of 18.5-29.9 kg/m2 (SRD cohort) and assessed doses of 2.5 mg BI 1356225 under fed and fasted conditions (bioavailability [BA] cohort). The MRD study investigated multiple doses of BI 1356225 (0.2, 1.0, 2.5 or 10.0 mg) or 5.0 mg BI 1356225 with a single dose of midazolam and celecoxib (drug-drug interaction part) over 28 days in adults with a BMI of 27.0-39.9 kg/m2. RESULTS: Sixty-five subjects were treated in the SRD study. Drug-related adverse events (AEs) were reported for five subjects (9.1 %) in the SRD cohort and two subjects (20.0 %) in the BA cohort, with the most frequent being headache (SRD: n = 4, 9.8 %; BA: n = 1, 10.0 %). In the MRD study, two (2.3 %) of the 87 subjects treated discontinued treatment because of AEs. Drug-related AEs were reported for 18 subjects (20.7 %), did not increase with dose and were most frequently reported as headache (n = 5, 5.7 %) and gastrointestinal disorders (n = 5, 5.7 %). In both studies, exposure parameters (area under the concentration-time curve [AUC] and maximum plasma concentration [Cmax]) of BI 1356225 increased across dose groups, although this was less than dose-proportional across the entire dose range. In the BA cohort of the SRD study, AUC0-∞ was slightly increased and Cmax slightly decreased in fed versus fasted conditions, with fed/fasted ratios (90 % CI) of 101.10 % (92.42, 110.60) and 91.67 % (78.50, 107.05), respectively. In both studies, AG concentrations and the AG/UAG ratio were dose-dependently decreased after BI 1356225 treatment from baseline versus placebo. In the MRD study, UAG concentrations were increased from baseline, but not dose-dependently. No differences were observed in bodyweight, appetite, food cravings, ad libitum food uptake or obesity-related biomarkers after 28 days of treatment with BI 1356225. CONCLUSIONS: Treatment with SRDs and MRDs of BI 1356225 was well tolerated by healthy males and subjects with overweight/obesity. BI 1356225 treatment over 28 days reduced AG concentrations and the AG/UAG ratio by >80 %, but no effect was seen on bodyweight, hunger/satiety, control of eating or energy intake. Although, at 4 weeks, the MRD study was fairly short, a reduction in bodyweight would be expected to be evident by this time, suggesting that a reduction of AG via a GOAT inhibitor is not sufficient to induce clinically relevant bodyweight loss.

Discovery of new oxadiazolo pyridine derivatives as potent ghrelin O-acyltransferase inhibitors using molecular modeling techniques.

Diabesity is a major global health concern, and ghrelin O-acyltransferase (GOAT) acts as an important target for the development of new inhibitors of this disease. The present work highlights a detailed QSAR study using QSARINS software, which provides an excellent model equation using descriptors. Here, the best model equation developed has two variables, namely MLFER_E and XlogP, with statistical parameters R2 = 0.8433, LOF = 0.0793, CCCtr = 0.915, Q2LOO = 0.8303, Q2LMO = 0.8275, CCCcv = 0.9081, R2ext = 0.7712, and CCCext = 0.8668. A higher correlation of the key structural fragments with activity is validated by the developed QSAR model. Furthermore, molecular docking helped us identify the binding interactions. Thirty four new molecules with better predicted biological activity (pIC50) were designed. The binding energy of four compounds have shown higher binding activity into the membrane protein (PDB Id: 6BUG). Molecular dynamics simulation has established the stability of the protein-ligand complex over 100 ns. DFT and ADME-toxicity analyses also confirmed their drug-like properties. Based on our findings, we report that these new oxadiazolo pyridine derivatives lead to the development of potent candidates for further development. Graphical abstract: METTL3-mediated HOTAIRM1 promotes vasculogenic mimicry in glioma via regulating IGFBP2 expression. METTL3 expression is high in glioma cells and tissues that stabilize and enhance HOTAIRM1 expression. This HOTAIRM1 then interacts with IGFBP2 which in turn promotes glioma cell malignancy and vasculogenic mimicry (VM) formation, thus providing a new direction for glioma therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00167-z.

Effects of a 12-Week Diet versus Diet plus Aerobic and Resistance Exercise Program on Acylated and Desacylated Ghrelin, and Ghrelin O-Acyltransferase in Adolescent Girls with Obesity.

This study investigated the effects of a 12-week diet versus diet plus aerobic and resistance exercise programme on acylated ghrelin (AG), desacylated ghrelin (DAG), and ghrelin O-acyltransferase (GOAT) concentrations in girls with obesity. We randomised 30 adolescents with obesity to a 12-week aerobic and resistance exercise group (EG) or a control group (CG). At baseline and at 4, 8, and 12 weeks, we measured their body composition, lipid profile, glucose, AG, DAG, and GOAT concentrations. In the EG, the body fat percentage decreased by 2.37% and was significantly lower than that in the CG. The DAG concentrations significantly increased by 48.3% and 27.4% in the EG and CG, respectively. At 4, 8, and 12 weeks, DAG concentrations were significantly higher in the EG than in the CG. AG concentrations were higher at week 12 than at baseline in both groups. In both groups, the GOAT concentrations increased at weeks 8 and 12; however, no between-group differences were observed in the changes in GOAT concentrations. This study showed increased DAG concentrations and non-significant changes in AG and GOAT concentrations after a 12-week aerobic and resistance exercise programme in girls with obesity. These findings suggest that an aerobic and resistance exercise programme influences appetite-regulating hormones, mainly through changes in DAG concentrations.

Discovery of novel inhibitors of ghrelin O-acyltransferase enzyme: an in-silico approach.

Background and purpose: Ghrelin is known as a hunger hormone and plays a pivotal role in appetite, food intake, energy balance, glucose metabolism, and insulin secretion, making it a potential target for the treatment of obesity and type 2 diabetes. The essential maturation step of ghrelin to activate the GHS-R1a is the octanoylation of the Ser3, which is catalyzed by the ghrelin O-acyltransferase enzyme (GOAT) enzyme. Therefore, the inhibition of GOAT may be useful for treating ghrelin-related diseases. Experimental approach: To discover the novel inhibitors against GOAT enzyme by a fast and accurate computational method, here, we tried to develop the homology model of GOAT. Subsequently, the generated model was stabilized by molecular dynamics simulation. The consecutive process of docking, pharmacophore mapping, and large-scale virtual screening were performed to find the potential hit compounds. Findings / Results: The homology model of the GOAT enzyme was generated and the quality of 3D structures was increased to the highest level of > 99.8% of residue in allowed regions. The model was inserted into the lipid bilayer and was stabilized by molecular dynamics simulation in 200 ns. The sequential process of pharmacophore-based virtual screening led to the introduction of three compounds including ethaverine, kaempferitrin, and reglitazar as optimal candidates for GOAT inhibition. Conclusion and implications: The results of this study may provide a starting point for further investigation for drug design in the case of GOAT inhibitors and help pave the way for clinical targeting of obesity and type 2 diabetes.

The Efficacy, Safety, and Pharmacology of a Ghrelin O-Acyltransferase Inhibitor for the Treatment of Prader-Willi Syndrome.

CONTEXT: Acylated ghrelin (AG) stimulates appetite and is elevated compared to its unacylated (UAG) counterpart in Prader-Willi syndrome (PWS). GLWL-01 is a selective, reversible inhibitor of ghrelin O-acyltransferase (GOAT), the enzyme that converts UAG into AG. OBJECTIVE: This work aimed to assess the efficacy, pharmacokinetics, pharmacodynamics, and safety of GLWL-01 in the treatment of PWS patients. METHODS: A double-blind, placebo-controlled, phase 2 crossover study was conducted with 2 active treatment periods of 28 days in 19 patients (aged 16-65 years; body mass index (BMI) ≥ 28) with genetically confirmed PWS. The study took place in 7 hospital-based study centers in the United States and Canada. Patients received placebo or GLWL-01 (450 mg twice daily) orally after lead-in placebo and washout periods. The Hyperphagia Questionnaire for Clinical Trials and Caregiver Global Impression of Change were used to measure reductions in hyperphagia. Plasma concentrations of AG and UAG were evaluated as correlates. RESULTS: Treatment resulted in statistically significant differences compared to placebo in plasma AG (P = .0002), UAG (P = .0488), and AG/UAG (P = .0003). GLWL-01 did not statistically significantly reduce hyperphagia-related behavior or bring about changes in global clinical end points, as assessed by caregivers. Anthropometric and clinical parameters correlated with obesity did not statistically significantly change in response to treatment. Less than half of patients reported a treatment-emergent adverse event (TEAE). No deaths, serious adverse events, or severe TEAEs were reported. CONCLUSION: GLWL-01 is safe and well tolerated. Pharmacological parameters confirmed the inhibition of GOAT following administration of GLWL-01. Patients' eating behaviors, BMI, blood glucose, and total cholesterol, among other similar measures, were not modified.

The ghrelin system follows a precise post-natal development in mini-pigs that is not impacted by dietary medium chain fatty-acids.

The ghrelin-ghrelin receptor (GHSR1) system is one of the most important mechanisms regulating food intake and energy balance. To be fully active, ghrelin is acylated with medium-chain fatty acids (MCFA) through the ghrelin-O-acetyl transferase (GOAT). Several studies reported an impact of dietary MCFA on ghrelin acylation in adults. Our study aimed at describing early post-natal development of the ghrelin system in mini-pigs as a model of human neonates and evaluating the impact of dietary MCFA. Suckled mini-pigs were sacrificed at post-natal day (PND) 0, 2, 5, and 10 or at adult stage. In parallel, other mini-pigs were fed from birth to PND10 a standard or a dairy lipid-enriched formula with increased MCFA concentration (DL-IF). Plasma ghrelin transiently peaked at PND2, with no variation of the acylated fraction except in adults where it was greater than during the neonatal period. Levels of mRNA coding pre-proghrelin (GHRL) and GOAT in the antrum did not vary during the post-natal period but dropped in adults. Levels of antral pcsk1/3 (cleaving GHRL into ghrelin) mRNA decreased significantly with age and was negatively correlated with plasma acylated, but not total, ghrelin. Hypothalamic ghsr1 mRNA did not vary in neonates but increased in adults. The DL-IF formula enriched antral tissue with MCFA but did not impact the ghrelin system. In conclusion, the ghrelin maturation enzyme PCSK1/3 gene expression exhibited post-natal modifications parallel to transient variations in circulating plasma ghrelin level in suckling piglets but dietary MCFA did not impact this post-natal development.

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.