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.     

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.

     

Role of ghrelin system in neuroprotection and cognitive functions: implications in Alzheimer's disease

Alzheimer's disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by loss of memory and cognitive deficits, strongly influenced by the metabolic status, in which the impairment of neuropeptides/neurotransmitters systems has been previously observed. Ghrelin is a multifunctional hormone produced in a wide variety of tissues, which has been associated with the progression of obesity and metabolic syndrome, but has been also linked to neuromodulation, neuroprotection and memory and learning processes. In addition, ghrelin system also acts in an autocrine/paracrine fashion where the majority of its components [ghrelin variants (native ghrelin, In1-ghrelin), acylation enzyme (GOAT) and receptors (GHS-Rs)] are expressed in the different regions of central nervous system. In spite of all these pieces of information strongly suggesting a close association between ghrelin system and AD, which could be of pathophysiological relevance, few studies have been addressed to clarify this relationship. In this work, the role of ghrelin system in neuroprotection, memory consolidation and learning is reviewed, and its influence in AD, as well as the regulation of its expression in the brain of AD patients, is discussed.     

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.     

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.     

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..     

Reduced intake of carbohydrate prevents the development of obesity and impaired glucose metabolism in ghrelin O-acyltransferase knockout mice

A close relationship between acylated-ghrelin and sucrose intake has been reported. However, little has been examined about the physiological action of ghrelin on preference for different types of carbohydrate such as glucose, fructose, and starch. The current study was aimed to investigate the role of acylated-ghrelin in the determinants of the choice of carbohydrates, and pathogenesis of chronic disorders, including obesity and insulin resistance. In a two-bottle-drinking test, ghrelin O-acyltransferase (GOAT) knockout (KO) mice consumed a less amount of glucose and maltodextrin, and almost the same amount of fructose and saccharin solution compared to WT littermates. The increased consumption of glucose and maltodextrin was observed when acylated-ghrelin, but not unacylated-ghrelin, was exogeneously administered in normal C57BL/6J mice, suggesting an association of acylated-ghrelin with glucose-containing carbohydrate intake. When fed a diet rich in maltodextrin, starch and fat for 12 weeks, GOAT KO mice showed less food intake and weight gain, as well as improved glucose tolerance and insulin sensitivity than WT mice. Our data suggests that blockade of GOAT activity may offer a therapeutic option for treatment of obesity and its associated metabolic syndrome by preventing from overconsumption of carbohydrate-rich food.     

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.     

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.     

Genetic variants of ghrelin in metabolic disorders

An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual's risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In cross-sectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity.     

Ultrastructure of islet ghrelin cells in the human fetus

Ghrelin is a peptide hormone predominantly produced in the stomach. Ghrelin expression has also been reported in other tissues including the pancreas. We have reported that ghrelin cells constitute a novel endocrine cell type in the human and the developing rat islets. The cells are most numerous pre- and neonatally and, in humans, constitute 10% of all islet cells from mid-gestation to birth. Since gastric ghrelin expression is low before birth, the islets may be the main source of circulating ghrelin during this time. In the present investigation, we have performed an ultrastructural analysis of pancreatic ghrelin cells in human fetuses by using transmission electron microscopy and immunogold labelling. In addition, morphometrical analysis of secretory granules size was performed. Our data provide evidence for the unique ultrastructural features of ghrelin cells versus other islet cells. Notably, the secretory granules of ghrelin cells were of small size with a mean dense-core diameter of 110 nm. We conclude that ghrelin cells constitute a novel islet cell type, distinct from the previously hormonally characterised islet cell types.This work was supported by grants from the Swedish Medical Research Council (Project No. 4499), the Royal Physiographic Society and the Novo Nordic, Påhlsson and Gyllenstiernska Krapperup Foundations     

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.     

Physiological roles revealed by ghrelin and ghrelin receptor deficient mice

Ghrelin is a hormone made in the stomach and known primarily for its growth hormone releasing and orexigenic properties. Nevertheless, ghrelin through its receptor, the GHS-R1a, has been shown to exert many roles including regulation of glucose homeostasis, memory & learning, food addiction and neuroprotection. Furthermore, ghrelin could promote overall health and longevity by acting directly in the immune system and promoting an extended antigen repertoire. The development of mice lacking either ghrelin (ghrelin−/−) or its receptor (ghsr−/−) have provided a valuable tool for determining the relevance of ghrelin and its receptor in these multiple and diverse roles. In this review, we summarize the most important findings and lessons learned from the ghrelin−/− and ghsr−/− mice.     

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.     

A novel human ghrelin variant (In1-ghrelin) and ghrelin-O-acyltransferase are overexpressed in breast cancer: potential pathophysiological relevance

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.     

Voluntary exercise attenuates obesity-associated inflammation through ghrelin expressed in macrophages

Chronic low-level inflammation is associated with obesity and a sedentary lifestyle, causing metabolic disturbances such as insulin resistance. Exercise training has been shown to decrease chronic low-level systemic inflammation in high-fat diet (HFD)-induced obesity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Ghrelin is a peptide hormone predominantly produced in the stomach that stimulates appetite and induces growth hormone release. In addition to these well-known functions, recent studies suggest that ghrelin localizes to immune cells and exerts an anti-inflammatory effect. The purpose of the current study was to investigate the role of ghrelin expressed in macrophages in the anti-inflammatory effects of voluntary exercise training. Expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1 and F4/80 was increased in adipose tissue from mice fed a HFD (HFD mice) compared with mice fed a standard diet (SD mice), whereas the expression of these inflammatory cytokines was markedly decreased in mice performing voluntary wheel running during the feeding of a HFD (HFEx mice). The expression of TNF-α was also increased in peritoneal macrophages by a HFD and exercise training inhibited the increase of TNF-α expression. Interestingly, expression of ghrelin in peritoneal macrophages was decreased by a HFD and recovered by exercise training. Suppression of ghrelin expression by siRNA increased TNF-α expression and LPS-stimulated NF-κB activation in RAW264 cells, which is a macrophage cell line. TNF-α expression by stimulation with LPS was significantly suppressed in RAW264 cells cultured in the presence of ghrelin. These results suggest that ghrelin exerts potent anti-inflammatory effects in macrophages and functions as a mediator of the beneficial effects of exercise training.     

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.     

Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes

Ghrelin is a 28 amino acid peptide hormone that is produced both centrally and peripherally. Regulated by the ghrelin O-acyl transferase enzyme, ghrelin exerts its action through the growth hormone secretagogue receptor, and is implicated in a diverse range of physiological processes. These implications have placed the ghrelin signaling pathway at the center of a large number of candidate gene and genome-wide studies which aim to identify the genetic basis of human heterogeneity. In this review we summarize the available data on the genetic variability of ghrelin, its receptor and its regulatory enzyme, and their association with obesity, stature, type 2 diabetes, cardiovascular disease, eating disorders, and reward seeking behavior.