There is growing evidence that the ghrelin axis, including ghrelin (GHRL) and its receptor, the growth hormone secretagogue receptor (GHSR), play a role in cancer progression. Ghrelin gene and ghrelin receptor gene polymorphisms have been reported to have a range of effects in cancer, from increased risk, to protection from cancer, or having no association. In this study we aimed to clarify the role of ghrelin and ghrelin receptor polymorphisms in cancer by performing a meta-analysis of published case-control studies.

Ghrelin is a multifunctional gut peptide with a unique structure, which is modified by a medium chain fatty acid at the third serine by ghrelin O-acyl transferase (GOAT). It is well known that the major source of plasma ghrelin is the stomach, but the transcriptional regulation of gastric ghrelin and GOAT is incompletely understood. Here, we studied the involvement of the nuclear receptors REV-ERBα and REV-ERBβ on ghrelin and GOAT gene expression in vivo and in vitro. Reverse-transcriptase polymerase chain reaction analysis showed that REV-ERBα and REV-ERBβ mRNAs were expressed in the stomach and a stomach-derived ghrelin cell line (SG-1 cells). In vivo experiments with mice revealed the circadian rhythm of ghrelin, GOAT, and REV-ERBs. The peak expression of ghrelin and GOAT mRNAs occurred at Zeitgeber time (ZT) 4, whereas that of REV-ERBα and REV-ERBβ was observed at ZT8 and ZT12, respectively. Treatment of SG-1 cells with SR9009, a REV-ERB agonist, led to a significant reduction in ghrelin and GOAT mRNA levels. Overexpression of REV-ERBα and REV-ERBβ decreased ghrelin and GOAT mRNA levels in SG-1 cells. In contrast, small-interfering RNA (siRNA)-mediated double-knockdown of REV-ERBα and REV-ERBβ in SG-1 cells led to the upregulation in the expression of ghrelin and GOAT mRNAs. These results suggest that REV-ERBs suppress ghrelin and GOAT mRNA expression.

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.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild-type rats, WAS-induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex.

Obesity is a hallmark of aging in many Western societies, and is a precursor to numerous serious age-related diseases. Ghrelin (Ghrl), via its receptor (growth hormone secretagogue receptor, GHS-R), is shown to stimulate GH secretion and appetite. Surprisingly, our previous studies showed that Ghrl(-/-) mice have impaired thermoregulatory responses to cold and fasting stresses, while Ghsr(-/-) mice are adaptive.

Adipose tissue (AT) atrophy is a hallmark of cancer cachexia contributing to increased morbidity/mortality. Ghrelin has been proposed as a treatment for cancer cachexia partly by preventing AT atrophy. However, the mechanisms mediating ghrelin's effects are incompletely understood, including the extent to which its only known receptor, GHSR-1a, is required for these effects. This study characterizes the pathways involved in AT atrophy in the Lewis Lung Carcinoma (LLC)-induced cachexia model and those mediating the effects of ghrelin in Ghsr +/+ and Ghsr -/- mice. We show that LLC causes AT atrophy by inducing anorexia, and increasing lipolysis, AT inflammation, thermogenesis and energy expenditure. These changes were greater in Ghsr -/-. Ghrelin administration prevented LLC-induced anorexia only in Ghsr +/+, but prevented WAT lipolysis, inflammation and atrophy in both genotypes, although its effects were greater in Ghsr +/+. LLC-induced increases in BAT inflammation, WAT and BAT thermogenesis, and energy expenditure were not affected by ghrelin. In conclusion, ghrelin ameliorates WAT inflammation, fat atrophy and anorexia in LLC-induced cachexia. GHSR-1a is required for ghrelin's orexigenic effect but not for its anti-inflammatory or fat-sparing effects.

Cancer chemotherapy is commonly accompanied by mucositis, anorexia, weight loss, and anxiety independently from cancer-induced anorexia-cachexia, further aggravating clinical outcome. Ghrelin is a peptide hormone produced in gastric mucosa that reaches the brain to stimulate appetite. In plasma, ghrelin is protected from degradation by ghrelin-reactive immunoglobulins (Ig). To analyze possible involvement of ghrelin in the chemotherapy-induced anorexia and anxiety, gastric ghrelin expression, plasma levels of ghrelin, and ghrelin-reactive IgG were studied in rats treated with methotrexate (MTX).

Ghrelin, the endogenous growth hormone secretagogue, has an important role in metabolic homeostasis. It exists in two major molecular forms: acylated (AG) and unacylated (UAG). Many studies suggest different roles for these two forms of ghrelin in energy balance regulation. In the present study, we compared the effects of acute intracerebroventricular administration of AG, UAG and their combination (AG+UAG) to young adult Wistar rats on food intake and central melanocortin system modulation. Although UAG did not affect food intake it significantly increased the number of c-Fos positive neurons in the arcuate (ARC), paraventricular (PVN) and solitary tract (NTS) nuclei. In contrast, UAG suppressed AG-induced neuronal activity in PVN and NTS. Central UAG also modulated hypothalamic expression of Mc4r and Bmp8b, which were increased and Mc3r, Pomc, Agrp and Ucp2, which were decreased. Finally, UAG, AG and combination treatments caused activation of c-Fos in POMC expressing neurons in the arcuate, substantiating a physiologic effect of these peptides on the central melanocortin system. Together, these results demonstrate that UAG can act directly to increase neuronal activity in the hypothalamus and is able to counteract AG-induced neuronal activity in the PVN and NTS. UAG also modulates expression of members of the melanocortin signaling system in the hypothalamus. In the absence of an effect on energy intake, these findings indicate that UAG could affect energy homeostasis by modulation of the central melanocortin system.

The dimerization properties of the ghrelin receptor (GRLN-R) and its non-signalling, naturally occurring, truncated splice variant (GHS-R1b) have been investigated in human embryonic kidney 293 cells heterologously expressing these proteins. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the formation of GRLN-R homodimers and GRLN-R/GHS-R1b heterodimers, but ghrelin-induced conformational changes were only detected in the GRLN-R homodimers. When the expression of GHS-R1b exceeded that of GRLN-R, there was a decrease in the cell surface expression of GRLN-R with a consequent decrease in constitutive activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Furthermore, there was no change in ghrelin affinity, and the efficacy of cell signalling as measured by stimulation of PI-PLC and extracellular signal-regulated kinase 1/2 was unchanged. Cellular localization studies suggest that GRLN-R is normally distributed between the plasma membrane and cytosolic fractions, but in the presence of GHS-R1b, GRLN-R is localized to the nucleus. Therefore, we propose that the decrease in GRLN-R constitutive signalling was due to translocation of GRLN-R to the nucleus due to the formation of GRLN-R/GHS-R1b heterodimers. Therefore, GHS-R1b appears to act as a dominant-negative mutant of the full-length GRLN-R.

Ghrelin is a stomach-derived hormone that regulates several metabolic functions including growth hormone release, appetite, adiposity, and gastric motility. Nutrients, the autonomic nervous system, and other metabolic hormones have all been implicated in the regulation of ghrelin secretion. Despite this, ongoing efforts to develop modulators of ghrelin secretion in human diseases are still underway. Hydrogen sulfide (H2 S) is a gaseous signaling molecule that is produced both endogenously in many tissues and by the gut microbiome. H2 S has established roles in cardiovascular and immune health, however, more recently H2 S has been implicated in the regulation of metabolic hormone secretion. We hypothesized that H2 S is able to directly regulate ghrelin secretion and in turn, regulate appetite. We first demonstrated that GYY4137 (an H2 S donor molecule) directly suppresses ghrelin secretion in rat primary gastric culture, in part through the activation of the protein kinase B (AKT) pathway. We then demonstrated the colocalization of ghrelin-positive gastric cells with the H2 S producing enzyme cystathionine-γ-lyase (CSE). While GYY4137 suppressed ghrelin secretion, inhibition of CSE caused a stimulation in ghrelin secretion in primary gastric culture. In mice, GYY4137 treatment prolonged the postprandial drop of circulating ghrelin and caused reduced food consumption up to 4 h after treatment. These results demonstrate for the first time a role for H2 S in the regulation of ghrelin and appetite. Modulating H2 S levels may be a novel approach to regulate ghrelin secretion in the treatment of metabolic diseases.

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates.

Growth hormone secretagogue receptors (GHSRs) in the central nervous system (CNS) mediate hyperphagia and adiposity induced by acyl ghrelin (AG). Evidence suggests that des-AG (dAG) has biological activity through GHSR-independent mechanisms. We combined in vitro and in vivo approaches to test possible GHSR-mediated biological activity of dAG. Both AG (100 nmol/L) and dAG (100 nmol/L) significantly increased inositol triphosphate formation in human embryonic kidney-293 cells transfected with human GHSR. As expected, intracerebroventricular infusion of AG in mice increased fat mass (FM), in comparison with the saline-infused controls. Intracerebroventricular dAG also increased FM at the highest dose tested (5 nmol/day). Chronic intracerebroventricular infusion of AG or dAG increased glucose-stimulated insulin secretion (GSIS). Subcutaneously infused AG regulated FM and GSIS in comparison with saline-infused control mice, whereas dAG failed to regulate these parameters even with doses that were efficacious when delivered intracerebroventricularly. Furthermore, intracerebroventricular dAG failed to regulate FM and induce hyperinsulinemia in GHSR-deficient (Ghsr(-/-)) mice. In addition, a hyperinsulinemic-euglycemic clamp suggests that intracerebroventricular dAG impairs glucose clearance without affecting endogenous glucose production. Together, these data demonstrate that dAG is an agonist of GHSR and regulates body adiposity and peripheral glucose metabolism through a CNS GHSR-dependent mechanism.

Orexigenic peptide ghrelin and its receptor have been extensively investigated as potential therapeutic targets, primarily because of their role in feeding initiation and growth hormone (GH) release. However, no specific ghrelin targeting anti-obesity or cachexia therapeutics are available for clinical use thus far and further efforts in this direction are warranted. The present study aimed to find new peptide drug leads modulating ghrelin signal transduction. By targeting neutralising antibodies against ghrelin with phage display libraries, we aimed to identify peptides binding to the cognate receptor. Four synthetic peptides were selected and tested using calcium screening assays. The most effective competitive antagonist FSFLPPE was further tested in vivo. Administration of the peptide produced no significant effect on either food intake or GH release. Surprisingly, when co-administered with ghrelin, the peptide significantly enhanced GH secretion and c-Fos expression. The evidence obtained in the present study indicates that FSFLPPE might act as an ago-allosteric modulator.

Focusing on the caprylic acid (C8:0), this study aimed at investigating the discrepancy between the formerly described beneficial effects of dietary medium chain fatty acids on body weight loss and the C8:0 newly reported effect on food intake via ghrelin octanoylation. During 6 weeks, Sprague-Dawley male rats were fed with three dietary C8:0 levels (0, 8 and 21% of fatty acids) in three experimental conditions (moderate fat, caloric restriction and high fat). A specific dose-response enrichment of the stomach tissue C8:0 was observed as a function of dietary C8:0, supporting the hypothesis of an early preduodenal hydrolysis of medium chain triglycerides and a direct absorption at the gastric level. However, the octanoylated ghrelin concentration in the plasma was unchanged in spite of the increased C8:0 availability. A reproducible decrease in the plasma concentration of unacylated ghrelin was observed, which was consistent with a decrease in the stomach preproghrelin mRNA and stomach ghrelin expression. The concomitant decrease of the plasma unacylated ghrelin and the stability of its acylated form resulted in a significant increase in the acylated/total ghrelin ratio which had no effect on body weight gain or total dietary consumption. This enhanced ratio measured in rats consuming C8:0 was however suspected to increase (i) growth hormone (GH) secretion as an increase in the GH-dependent mRNA expression of the insulin like growth Factor 1 (IGF-1) was measured (ii) adipocyte diameters in subcutaneous adipose tissue without an increase in the fat pad mass. Altogether, these results show that daily feeding with diets containing C8:0 increased the C8:0 level in the stomach more than all the other tissues, affecting the acylated/total ghrelin plasma ratio by decreasing the concentration of circulating unacylated ghrelin. However, these modifications were not associated with increased body weight or food consumption.

Sarcopenia, the progressive loss of muscle mass and dysfunction, universally affects the elderly and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight upon acylation, which activates its receptor GHSR1a. Recent studies have demonstrated that acyl and unacylated ghrelin are protective against acute pathological conditions of skeletal muscle. We hypothesized that both acyl ghrelin receptor agonist (HM01) and unacylated ghrelin ameliorate muscle atrophy and contractile dysfunction in oxidative stress-induced sarcopenia. HM01, unacylated ghrelin, or saline was delivered via osmotic pump. HM01 increased food consumption transiently, while the body weight remained elevated. It also decreased lean body mass and muscle mass of wildtype and Sod1KO. In contrast, unacylated ghrelin ameliorated loss of muscle mass by 15-30% in Sod1KO mice without changes in food consumption or body weights. Contractile force was decreased by ~30% in Sod1KO mice, but unacylated ghrelin prevented the force deficit by ~80%. We identified downregulation of transcription factor FoxO3a and its downstream E3 ligase MuRF1 by unacylated ghrelin. Our data show a direct role of unacylated ghrelin in redox-dependent sarcopenia independent of changes of food consumption or body weight.

Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.

Ghrelin (GHRL) and its receptor (GHSR) are involved in various bioactivities. In this study, the complete cDNA and 5' flanking region of the duck GHRL (dGHRL) gene and a 3717 bp fragment of the duck GHSR (dGHSR) gene were obtained. A total of 19, 8, 43, and 48 SNPs identified in 2751, 1358, 3671, and 3567 bp of the chicken GHRL (cGHRL), chicken GHSR (cGHSR), dGHRL, and dGHSR genes, respectively. Both cGHRL and dGHRL were expressed predominantly in the proventriculus, whereas the highest mRNA levels of cGHSR and dGHSR were detected in the breast muscle and pituitary. Association analysis showed that C-2047G, A-2355C, and A-2220C of the cGHRL gene were significantly associated with abdominal fat weight (AFW; P = .01), crude protein content of leg muscle (CPCLM; P = .02), and CPCLM (P = .0009), respectively. C-1459T of the cGHSR gene was also significantly associated with CPCLM (P = .0004). C-729T of dGHRL and A3427T of dGHSR were both significantly associated with subcutaneous fat thickness (SFT; P = .04). It was indicated by this study that the GHRL and GHSR genes were related to fat deposition in both chicken and duck.

Desacyl ghrelin is acylated by ghrelin O-acyltransferase (GOAT) and converted to acyl ghrelin. To date, little is known about the relationship among the levels of these two forms of ghrelin, GOAT level, and insulin resistance in Asian individuals. The purpose of this study was to determine the relationship between insulin resistance and the levels of plasma acyl ghrelin, desacyl ghrelin, and GOAT in asymptomatic middle-aged Korean men.