Ghrelin receptor antagonism and satiety attenuate Pavlovian-instrumental transfer.

Animals rely on learned cues to guide their behaviour for rewards such as food. The Pavlovian-instrumental transfer (PIT) task can be used to investigate the influence of Pavlovian stimuli on instrumental responding. Ghrelin, an orexigenic peptide, and its receptor, growth hormone secretagogue receptor 1A (GHS-R1A), has received growing interest for its role in reward-motivated learning and behaviours. A significant population of GHS-R1A have been identified within the ventral tegmental area (VTA), a critical node in the mesolimbic reward circuit that is necessary for the expression of PIT. As ghrelin has been found to increase dopaminergic activity in the VTA, we predicted that GHS-R1A antagonism with JMV-2959 would attenuate PIT. Further, given the relationship between hunger levels and changes in ghrelin signalling, we sought to compare the effects GHS-R1A antagonism with those of satiety, hypothesizing parallel effects, with each attenuating PIT. Rats received daily sessions of Pavlovian and then instrumental training over 3 weeks. Across three experiments, we examined the effects of a shift to satiety, or treatment with the GHS-R1A antagonist JMV-2959, either peripherally or directly into the VTA. We found that presentations of a stimulus paired with food reward enhanced responding for food across all conditions, thus demonstrating the expected PIT effect. Further, GHS-R1A antagonism, both peripherally and within the VTA, as well as satiety significantly reduced the magnitude of the PIT effect compared to control conditions. These results clarify our understanding of ghrelin signalling in PIT and begin to elucidate the role of feeding-related peptides in the modulation of reward-related responding.

Nucleus accumbens ghrelin signaling controls anxiety-like behavioral response to acute stress.

BACKGROUND: Anxiety disorders are one of the most common mental disorders. Ghrelin is a critical orexigenic brain-gut peptide that regulates food intake and metabolism. Recently, the ghrelin system has attracted more attention for its crucial roles in psychiatric disorders, including depression and anxiety. However, the underlying neural mechanisms involved have not been fully investigated. METHODS: In the present study, the effect and underlying mechanism of ghrelin signaling in the nucleus accumbens (NAc) core on anxiety-like behaviors were examined in normal and acute stress rats, by using immunofluorescence, qRT-PCR, neuropharmacology, molecular manipulation and behavioral tests. RESULTS: We reported that injection of ghrelin into the NAc core caused significant anxiolytic effects. Ghrelin receptor growth hormone secretagogue receptor (GHSR) is highly localized and expressed in the NAc core neurons. Antagonism of GHSR blocked the ghrelin-induced anxiolytic effects. Moreover, molecular knockdown of GHSR induced anxiogenic effects. Furthermore, injection of ghrelin or overexpression of GHSR in the NAc core reduced acute restraint stress-induced anxiogenic effects. CONCLUSIONS: This study demonstrates that ghrelin and its receptor GHSR in the NAc core are actively involved in modulating anxiety induced by acute stress, and raises an opportunity to treat anxiety disorders by targeting ghrelin signaling system.

A case control study investigating the methylation levels of GHRL and GHSR genes in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is a relapsing disease described as excessive use of alcohol. Evidence of the role of DNA methylation in addiction is accumulating. Ghrelin is an important peptide known as appetite hormone and its role in addictive behavior has been identified. Here we aimed to determine the methylation levels of two crucial genes (GHRL and GHSR) in ghrelin signaling and further investigate the association between methylation ratios and plasma ghrelin levels. METHODS: Individuals diagnosed with (n = 71) and without (n = 82) AUD were recruited in this study. DNA methylation levels were measured through methylation-sensitive high-resolution melting (MS-HRM). Acylated ghrelin levels were detected by ELISA. The GHRL rs696217 polymorphism was analyzed by the standard PCR-RFLP method. RESULTS: GHRL was significantly hypermethylated (P < 0.0022) in AUD between 25 and 50% methylation than in control subjects but no significant changes of GHSR methylation were observed. Moreover, GHRL showed significant positive correlation of methylation ratio between 25 and 50% with age. A significant positive correlation between GHSR methylation and ghrelin levels in the AUD group was determined (P = 0.037). The level of GHRL methylation and the ghrelin levels showed a significant association in the control subjects (P = 0.042). CONCLUSION: GHSR and GHRL methylation levels did not change significantly between control and AUD groups. However, GHRL and GHSR methylations seemed to have associations with plasma ghrelin levels in two groups. This is the first study investigating the DNA methylation of GHRL and GHSR genes in AUD.

Quinpirole ameliorates nigral dopaminergic neuron damage in Parkinson's disease mouse model through activating GHS-R1a/D2R heterodimers.

Growth hormone secretagogue receptor 1a (GHS-R1a) is an important G protein-coupled receptor (GPCR) that regulates a variety of functions by binding to ghrelin. It has been shown that the dimerization of GHS-R1a with other receptors also affects ingestion, energy metabolism, learning and memory. Dopamine type 2 receptor (D2R) is a GPCR mainly distributed in the ventral tegmental area (VTA), substantia nigra (SN), striatum and other brain regions. In this study we investigated the existence and function of GHS-R1a/D2R heterodimers in nigral dopaminergic neurons in Parkinson's disease (PD) models in vitro and in vivo. By conducting immunofluorescence staining, FRET and BRET analyses, we confirmed that GHS-R1a and D2R could form heterodimers in PC-12 cells and in the nigral dopaminergic neurons of wild-type mice. This process was inhibited by MPP+ or MPTP treatment. Application of QNP (10 µM) alone significantly increased the viability of MPP+-treated PC-12 cells, and administration of quinpirole (QNP, 1 mg/kg, i.p. once before and twice after MPTP injection) significantly alleviated motor deficits in MPTP-induced PD mice model; the beneficial effects of QNP were abolished by GHS-R1a knockdown. We revealed that the GHS-R1a/D2R heterodimers could increase the protein levels of tyrosine hydroxylase in the SN of MPTP-induced PD mice model through the cAMP response element binding protein (CREB) signaling pathway, ultimately promoting dopamine synthesis and release. These results demonstrate a protective role for GHS-R1a/D2R heterodimers in dopaminergic neurons, providing evidence for the involvement of GHS-R1a in PD pathogenesis independent of ghrelin.

"Sibling" battle or harmony: crosstalk between nesfatin-1 and ghrelin.

Ghrelin was first identified as an endogenous ligand of the growth hormone secretagogue receptor (GHSR) in 1999, with the function of stimulating the release of growth hormone (GH), while nesfatin-1 was identified in 2006. Both peptides are secreted by the same kind of endocrine cells, X/A-like cells in the stomach. Compared with ghrelin, nesfatin-1 exerts opposite effects on energy metabolism, glucose metabolism, gastrointestinal functions and regulation of blood pressure, but exerts similar effects on anti-inflammation and neuroprotection. Up to now, nesfatin-1 remains as an orphan ligand because its receptor has not been identified. Several studies have shown the effects of nesfatin-1 are dependent on the receptor of ghrelin. We herein compare the effects of nesfatin-1 and ghrelin in several aspects and explore the possibility of their interactions.

Effect of Growth Hormone Secretagogue Receptor Deletion on Growth, Pulsatile Growth Hormone Secretion, and Meal Pattern in Male and Female Mice.

INTRODUCTION: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. METHODS: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5-6 weeks old) or adult (10-19 weeks old) GHS-R KO (Ghsr-/-) and WT (Ghsr+/+) male and female mice. RESULTS: Adult Ghsr-/- male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr-/- mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr-/- females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. DISCUSSION/CONCLUSION: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging.

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.

The Role of Ghrelin/GHS-R1A Signaling in Nonalcohol Drug Addictions.

Drug addiction causes constant serious health, social, and economic burden within the human society. The current drug dependence pharmacotherapies, particularly relapse prevention, remain limited, unsatisfactory, unreliable for opioids and tobacco, and even symptomatic for stimulants and cannabinoids, thus, new more effective treatment strategies are researched. The antagonism of the growth hormone secretagogue receptor type A (GHS-R1A) has been recently proposed as a novel alcohol addiction treatment strategy, and it has been intensively studied in experimental models of other addictive drugs, such as nicotine, stimulants, opioids and cannabinoids. The role of ghrelin signaling in these drugs effects has also been investigated. The present review aims to provide a comprehensive overview of preclinical and clinical studies focused on ghrelin's/GHS-R1A possible involvement in these nonalcohol addictive drugs reinforcing effects and addiction. Although the investigation is still in its early stage, majority of the existing reviewed experimental results from rodents with the addition of few human studies, that searched correlations between the genetic variations of the ghrelin signaling or the ghrelin blood content with the addictive drugs effects, have indicated the importance of the ghrelin's/GHS-R1As involvement in the nonalcohol abused drugs pro-addictive effects. Further research is necessary to elucidate the exact involved mechanisms and to verify the future potential utilization and safety of the GHS-R1A antagonism use for these drug addiction therapies, particularly for reducing the risk of relapse.

H. pylori effects on ghrelin axis: Preliminary change in gastric pathogenesis.

Ghrelin and its receptors are present in the stomach, suggesting that the ghrelin axis plays an essential role in gastrointestinal complications. This investigation aimed to explore the effects of H. pylori infection and gastritis on serum ghrelin and ghrelin axis gene expression. In this study, we enrolled 68 adult ambulatory people referred for upper gastrointestinal endoscopy. The individuals were classified into three groups based on H. pylori infection and gastritis. Total serum ghrelin and tissue gene expression were tested with ELISA and quantitative RT-PCR, respectively. Serum ghrelin and mRNA expression were significantly lower in H. pylori-positive with gastritis subjects compared with both H. pylori-negative with and without gastritis. Growth hormone secretagogue receptor1a mRNA expression was not different between groups while GHSR1b expression was significantly higher in patients with H. pylori infection and gastritis. We propose the ghrelin axis intermediaries, such as GHSR1b, as a potential clinical target for gastric disorders.

Relationship between Orexigenic Peptide Ghrelin Signal, Gender Difference and Disease.

Growth hormone secretagogue receptor 1a (GHS-R1a), which is one of the G protein-coupled receptors (GPCRs), is involved in various physiological actions such as energy consumption, growth hormone secretion promoting action, and cardiovascular protective action. The ligand was searched for as an orphan receptor for a while, but the ligand was found to be acylated ghrelin (ghrelin) discovered by Kangawa and Kojima et al. in 1999. Recently, it has also been reported that dysregulation of GHS-R1a mediates reduced feeding in various diseases. On the other hand, since the physiological effects of ghrelin have been studied exclusively in male mice, few studies have been conducted on gender differences in ghrelin reactivity. In this review, we describe (1) the characteristics of GHS-R1a, (2) the role of ghrelin in hypophagia due to stress or anticancer drugs, and (3) the gender differences in the physiological effects of GHS-R1a and the influence of stress on it.

ß Cell GHS-R Regulates Insulin Secretion and Sensitivity.

Growth hormone secretagogue receptor (GHS-R) is widely known to regulate food intake and adiposity, but its role in glucose homeostasis is unclear. In this study, we investigated the expression of GHS-R in mouse pancreatic islets and its role in glycemic regulation. We used Ghsr-IRES-tauGFP mice, with Green Fluorescent Protein (GFP) as a surrogate for GHS-R, to demonstrate the GFP co-localization with insulin and glucagon expression in pancreatic islets, confirming GHS-R expression in ß and α cells. We then generated ß-cell-specific GHSR-deleted mice with MIP-Cre/ERT and validated that GHS-R suppression was restricted to the pancreatic islets. MIP-Cre/ERT;Ghsrf/f mice showed normal energy homeostasis with similar body weight, body composition, and indirect calorimetry profile. Interestingly, MIP-Cre/ERT;Ghsrf/f mice exhibited an impressive phenotype in glucose homeostasis. Compared to controls, MIP-Cre/ERT;Ghsrf/f mice showed lower fasting blood glucose and insulin; reduced first-phase insulin secretion during a glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test in vivo. The isolated pancreatic islets of MIP-Cre/ERT;Ghsrf/f mice also showed reduced insulin secretion during GSIS ex vivo. Further, MIP-Cre/ERT;Ghsrf/f mice exhibited improved insulin sensitivity during insulin tolerance tests (ITT). Overall, our results confirmed GHS-R expression in pancreatic ß and α cells; GHS-R cell-autonomously regulated GSIS and modulated systemic insulin sensitivity. In conclusion, ß cell GHS-R was an important regulator of glucose homeostasis, and GHS-R antagonists may have therapeutic potential for Type 2 Diabetes.

17ß-Estradiol Increases Arcuate KNDy Neuronal Sensitivity to Ghrelin Inhibition of the M-Current in Female Mice.

Obesity and anorexia result in dysregulation of the hypothalamic-pituitary-gonadal axis, negatively impacting reproduction. Ghrelin, secreted from the stomach, potentially mediates negative energy states and neuroendocrine control of reproduction by acting through the growth hormone secretagogue receptor (GHSR). GHSR is expressed in hypothalamic arcuate (ARC) Kisspeptin/Neurokinin B (Tac2)/Dynorphin (KNDy) neurons. Ghrelin is known to inhibit the M-current produced by KCNQ channels in other ARC neurons. In addition, we have shown 17ß-estradiol (E2) increases Ghsr expression in KNDy neurons 6-fold and increases the M-current in NPY neurons. We hypothesize that E2 increases GHSR expression in KNDy neurons to increase ghrelin sensitivity during negative energy states. Furthermore, we suspect ghrelin targets the M-current in KNDy neurons to control reproduction and energy homeostasis. We utilized ovariectomized Tac2-EGFP adult female mice, pretreated with estradiol benzoate (EB) or oil vehicle and performed whole-cell-patch-clamp recordings to elicit the M-current in KNDy neurons using standard activation protocols in voltage-clamp. Using the selective KCNQ channel blocker XE-991 (40 µM) to target the M-current, oil- and EB-treated mice showed a decrease in the maximum peak current by 75.7 ± 13.8 pA (n = 10) and 68.0 ± 14.7 pA (n = 11), respectively. To determine the actions of ghrelin on the M-current, ghrelin was perfused (100 nM) in oil- and EB-treated mice resulting in the suppression of the maximum peak current by 58.5 ± 15.8 pA (n = 9) and 59.2 ± 11.9 pA (n = 9), respectively. KNDy neurons appeared more sensitive to ghrelin when pretreated with EB, revealing that ARC KNDy neurons are more sensitive to ghrelin during states of high E2.

Expression of the ghrelin/growth hormone secretagogue receptor axis and its functional role in promoting tumor growth in primary central nervous system lymphomas.

Ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), have been found in a variety of malignant tumor tissues, suggesting a biological function of the ghrelin/GHS-R axis in tumor growth and progression. Among central nervous system tumors, primary central nervous system lymphomas (PCNSLs) are relatively rare and characterized by a rapid progression and poor prognosis. In order to clarify ghrelin expression and its functional role in promoting tumor growth and progression in PCNSLs, we undertook an immunohistochemical investigation for ghrelin and GHS-R expression in 43 patients and tested the effect of ghrelin inhibition on lymphoma cells. Furthermore, we investigated the expression of CD105, a marker for tumor angiogenesis, to explore its association with the ghrelin/GHS-R axis. The Kaplan-Meier method and Cox's proportional hazards regression model were used to determine the association of ghrelin/GHS-R expression with overall survival rate. The immunohistochemical study showed moderate/strong immunostaining of cells for ghrelin and GHS-R in 40 patients (93.0%) and 39 patients (90.7%), respectively. A ghrelin inhibitor did not affect tumor cell proliferation in vitro. Expression levels of ghrelin and GHS-R were divided into high and low groups by the rate of moderate-strong staining cells to tumor cells. The survival rate was significantly lower in patients with high GHS-R expression (P = 0.0368 by log-rank test; P = 0.0219 by Wilcoxon test). In addition, multivariate analysis of overall survival using Cox's proportional hazards regression model indicated that GHS-R was a significant independent prognostic factor (P = 0.0426). CD105 expression on tumor vessels was positive in 33 patients (33/37, 89.2%). There was a positive correlation between the moderate-strong staining rate of ghrelin and CD105-positive vessel count. These results indicated that the ghrelin/GHS-R axis plays a potential role in promoting tumor growth and progression through neoangiogenesis, rather than the proliferation of tumor cells.

ASSOCIATION OF GHRELIN RECEPTOR AND INFLAMMATION IN PERI-ATRIAL ADIPOSE TISSUE FROM OBESE PATIENTS WITH POSTOPERATIVE ATRIAL FIBRILLATION.

CONTEXT: Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice. The increasing evidence supports links between inflammation and AF. There is evidence showing that obesity is a major cause of adipose tissue (AT) inflammation. Ghrelin (GHRL), through its growth hormone secretagogue receptor (GHS-R) present on adipose tissue macrophages (ATMs), could modulate AT inflammation. OBJECTIVE: Our study aimed to evaluate the role of adipose tissue macrophages (ATMs) and their GHS-R in adipose tissue samples of right atrial appendages (RAA) biopsies. SUBJECTS AND METHOD: We obtained RAA biopsies from 10 obese patients, undergoing cardiac surgery for coronary artery bypass graft (CABG) and developing postoperative atrial fibrillation (POAF). The epicardial tissue samples were examined using immunohistochemistry to visualize and quantify CD68 and GSH-R expression of the ATMs. RESULTS: Histologically, the mean adipocyte diameter (MAD) of epicardial adipose tissue (EAT) was larger in EAT samples with inflammation as compared to EAT without inflammation (84.2 µm vs. 79.6 µm). The expression of CD68 was lower in EAT without inflammation as compared to EAT with inflammation in adipose tissue samples. Similarly, the expression of GSH-R was lower in EAT samples without inflammation as compared to EAT samples with inflammation in adipose tissue. CONCLUSIONS: Increased epicardial fat area, macrophage infiltration, and GHS-R expression in epicardial ATMs appeared to be associated with postoperative atrial fibrillation in obese patients.

Regulation of compensatory growth by molecular mechanism in Labeo rohita juveniles under different feeding regimes.

A study was carried out to assess the regulation of compensatory growth under different restriction feeding regimes in Labeo rohita juveniles by the interaction of various feed intake and growth regulating genes. A 60 day feeding trial was conducted with five treatment groups, Control (3% body weight, bw), T1 (alternate days), T2 (0.5% bw), T3 (1% bw) and T4 (2% bw) and feeding was done for first 30 days of the trial. For next 30 days, all the treatment groups were fed at a rate of 3% bw as in the control group. There was significant (p < 0.05) difference in the weight gain among the treatment groups with lowest FCR and highest PER was found in T2 group. Ghrelin gene mRNA levels were upregulated during first 30th days of the trial with highest expression levels in the T2 group. The expression levels of leptin gene mRNA were found significantly different (p < 0.05) among the treatments, which was down-regulated during initial 30 days and upregulated as the experiment progress towards 60th day. The IGF-1 mRNA expression levels were upregulated more in liver compared to the muscle tissue. The results of the study suggest that increased ghrelin levels and decreased leptin levels lead to hyperphagia during the onset of refeeding, which further triggers the compensatory growth in L. rohita. The present study describes the molecular mechanism behind the compensatory growth following a different feed restriction regime in L. rohita which is regulated due to the interaction of different energy homeostasis and growth regulating genes.

Ghrelin promotes hepatic lipogenesis by activation of mTOR-PPARγ signaling pathway.

Although ghrelin has been demonstrated to stimulate energy intake and storage through a central mechanism, its effect on hepatic lipid metabolism remains largely uncharacterized. Ghrelin receptor antagonism or gene deletion significantly decreased obesity-associated hepatic steatosis by suppression of de novo lipogenesis, whereas exogenous ghrelin stimulated lipogenesis, leading to hepatic lipid accumulation in mice. The effects of ghrelin were mediated by direct activation of its receptor on hepatocytes. Cultured hepatocytes responded to ghrelin with increased lipid content and expression of lipogenesis-related genes. Ghrelin increased phosphorylation of S6, the downstream target of mammalian target of rapamycin (mTOR) signaling in cultured hepatocytes, whereas ghrelin receptor antagonism reduced hepatic phosphorylation of S6 in db/db mice. Inhibition of mTOR signaling by rapamycin markedly attenuated ghrelin-induced up-regulation of lipogenesis in hepatocytes, whereas activation of hepatic mTOR signaling by deletion of TSC1 increased hepatic lipogenesis. By interacting with peroxisome proliferator-activated receptor-γ (PPARγ), mTOR mediates the ghrelin-induced up-regulation of lipogenesis in hepatocytes. The stimulatory effect of ghrelin on hepatic lipogenesis was significantly attenuated by PPARγ antagonism in cultured hepatocytes and in PPARγ gene-deficient mice. Our study indicates that ghrelin activates its receptor on hepatocytes to promote lipogenesis via a mechanism involving the mTOR-PPARγ signaling pathway.

Suppression of GHS-R in AgRP Neurons Mitigates Diet-Induced Obesity by Activating Thermogenesis.

Ghrelin, an orexigenic hormone released primarily from the gut, signals the hypothalamus to stimulate growth hormone release, enhance appetite and promote weight gain. The ghrelin receptor, aka Growth Hormone Secretagogue Receptor (GHS-R), is highly expressed in the brain, with highest expression in Agouti-Related Peptide (AgRP) neurons of the hypothalamus. We recently reported that neuron-specific deletion of GHS-R completely prevents diet-induced obesity (DIO) in mice by activating non-shivering thermogenesis. To further decipher the specific neuronal circuits mediating the metabolic effects of GHS-R, we generated AgRP neuron-specific GHS-R knockout mice (AgRP-Cre;Ghsrf/f). Our data showed that GHS-R in AgRP neurons is required for ghrelin's stimulatory effects on growth hormone secretion, acute food intake and adiposity, but not for long-term total food intake. Importantly, deletion of GHS-R in AgRP neurons attenuated diet-induced obesity (DIO) and enhanced cold-resistance in mice fed high fat diet (HFD). The HFD-fed knockout mice showed increased energy expenditure, and exhibited enhanced thermogenic activation in both brown and subcutaneous fat; this implies that GHS-R suppression in AgRP neurons enhances sympathetic outflow. In summary, our results suggest that AgRP neurons are key site for GHS-R mediated thermogenesis, and demonstrate that GHS-R in AgRP neurons plays crucial roles in governing energy utilization and pathogenesis of DIO.

GHSR deficiency suppresses neointimal formation in injured mouse arteries.

Growth hormone secretagogue receptor (GHSR) is involved in appetite regulation and energy homeostasis. In the present study, we examined the role of GHSR in neointimal formation following vascular injury. In the mouse model of femoral artery wire injury, we found that vessel intima-to-media ratio was significantly reduced in GHSR deficiency (GHSR-/-) mice compared with that in wild-type mice. Immunohistochemical staining showed that the smooth muscle cell (SMCs) in the neointima were significantly decreased in the injured arteries of GHSR-/- mice which was associated with decreased SMC proliferation and migration. Furthermore, immunoblotting demonstrated that, in cultured rat aortic SMCs, small interfering RNA-mediated GHSR knockdown suppressed the activation of Akt and ERK1/2 signaling pathway. These findings suggested a novel role of GHSR in neointimal formation likely via promoting the proliferation and migration of SMCs involving Akt and ERK1/2 signaling. Therefore, GHSR may be a potential therapeutic target in restenosis and vascular remodeling.

Effects of ghrelin and motilin on smooth muscle contractility of the isolated gastrointestinal tract from the bullfrog and Japanese fire belly newt.

Ghrelin has been identified in some amphibians and is known to stimulate growth hormone release and food intake as seen in mammals. Ghrelin regulates gastrointestinal motility in mammals and birds. The aim of this study was to determine whether ghrelin affects gastrointestinal smooth muscle contractility in bullfrogs (anuran) and Japanese fire belly newts (urodelian) in vitro. Neither bullfrog ghrelin nor rat ghrelin affected longitudinal smooth muscle contractility of gastrointestinal strips from the bullfrog. Expression of growth hormone secretagogue receptor 1a (GHS-R1a) mRNA was confirmed in the bullfrog gastrointestinal tract, and the expression level in the gastric mucosa was lower than that in the intestinal mucosa. In contrast, some gastrointestinal peptides, including substance P, neurotensin and motilin, and the muscarinic receptor agonist carbachol showed marked contraction, indicating normality of the smooth muscle preparations. Similar results were obtained in another amphibian, the Japanese fire belly newt. Newt ghrelin and rat ghrelin did not cause any contraction in gastrointestinal longitudinal muscle, whereas substance P and carbachol were effective causing contraction. In conclusion, ghrelin does not affect contractility of the gastrointestinal smooth muscle in anuran and urodelian amphibians, similar to results for rainbow trout and goldfish (fish) but different from results for rats and chickens. The results suggest diversity of ghrelin actions on the gastrointestinal tract across animals. This study also showed for the first time that motilin induces gastrointestinal contraction in amphibians.

Signaling of ghrelin and its functional receptor, the growth hormone secretagogue receptor, promote tumor growth in glioblastomas.

Ghrelin is a 28-amino-acid peptide that is the endogenous ligand for the pituitary growth hormone secretagogue receptor (GHS-R). Ghrelin is mainly produced from the stomach, but it is also expressed by various other tissues, including the CNS under normal conditions. Physiologically, ghrelin regulates appetite, gut motility, and GH release from the anterior pituitary, as well as cardiovascular and immune systems. Recent studies also indicate that ghrelin and GHS-R may play an important autocrine/paracrine role in neoplastic conditions. In order to clarify the role of ghrelin/GHS-R in gliomas, the present study assessed the expression of ghrelin and its functional receptor, GHS-R1a, in 39 glioblastomas (GBs), 13 anaplastic astrocytomas (AAs) and 11 diffuse astrocytomas (DAs) using immunohistochemical analyses. Immunohistochemical staining was evaluated as follows: no staining; 1+, 0-10% positive cells; 2+, 10-50% positive cells; 3+, >50% positive cells. Ghrelin expression was detected in 52 of 63 cases of which 38, 13 and one were scored as 3+, 2+ and 1+, respectively. GHS-R1a expression was detected in 45 of 63 cases of which 29, 15 and one were scored as 3+, 2+ and 1+, respectively. Ghrelin immunoreactivity was observed in 38 of 39 GBs, 12 of 13 AAs and two of 11 DAs. GHS-R1a immunoreactivity was observed in 39 of 39 GBs, five of 13 AAs, and one of 11 DAs. AAs and GBs showed moderate or strong immunostaining of ghrelin/GHS-R1a in the tumor cells and in proliferating microvessels. Patients were classified into lower to moderate-score, and high-score ghrelin/GHS-R categories according to the principal component and cluster analyses. Multivariate analysis of overall survival indicated that there was a significant difference (P = 0.0001) in the survival rate between these two groups. The combined results indicated that expression of the ghrelin/GHS-R1a axis increases the growth of AAs and GBs through an autocrine/paracrine mechanism.