Growth Hormone Secretagogue Receptor Expression Research Articles

Intestinal expression profiles and hepatic expression of LEAP2, ghrelin and their common receptor, GHSR, in humans

Liver-expressed antimicrobial peptide 2 (LEAP2) and ghrelin have reciprocal effects on their common receptor, the growth hormone secretagogue receptor (GHSR). Ghrelin is considered a gastric hormone and LEAP2 a liver-derived hormone and both have been proposed to be involved in the pathophysiology of obesity and type 2 diabetes (T2D). We investigated the mRNA expression of LEAP2, ghrelin and GHSR along the intestinal tract of individuals with and without TD2, and in the liver of men with and without obesity. Mucosal biopsies retrieved with 30-cm intervals throughout the small intestine and from 7 well-defined locations along the large intestine from 12 individuals with T2D and 12 healthy controls together with liver biopsies from 15 men with obesity and 15 lean men were subjected to bulk transcriptomics analysis. Both in individuals with and without T2D, mRNA expression of LEAP2 increased through the small intestine until dropping at the ileocecal valve, with little LEAP2 mRNA expression in the large intestine. Pronounced LEAP2 expression was observed in the liver of men with and without obesity. Robust ghrelin mRNA expression was observed in the duodenum of individuals with and without T2D, gradually decreasing along the small intestine with little expression in the large intestine. Ghrelin mRNA expression was not detected in the liver biopsies, and GHSR mRNA expression was not. In conclusion, we provide unique mRNA expression profiles of LEAP2, ghrelin and GHSR along the human intestinal tract showing no T2D-associated changes, and in the liver showing no differences between men with and without obesity.

Growth Hormone Secretagogue Receptor (GHSR) Signaling in the Ventral Tegmental Area (VTA) Mediates Feeding Produced by Chronic Social Defeat Stress in Male Mice

Ghrelin, a hormone secreted by the stomach, binds to the growth hormone secretagogue receptor (GHSR) in various brain regions to produce a number of behavioral effects that include increased feeding motivation. During social defeat stress, ghrelin levels rise in correlation with increased feeding and potentially play a role in attenuating the anxiogenic effects of social defeat. One region implicated in the feeding effects of ghrelin is the ventral tegmental area (VTA), a region implicated in reward seeking behaviors, and linked to social defeat in mice. Here we examined the role of GHSR signaling in the VTA in feeding behavior in mice exposed to social defeat stress. Male C57BL/J6 mice that were socially defeated once daily for 3 weeks ate more, had higher plasma ghrelin level and increased GHSR expression in the VTA compared to non-stressed mice. Socially defeated GHSR KO mice failed to increase their caloric intake in response to this stressor but rescue of GHSR expression in the VTA restored feeding responses. Finally, we pharmacologically blocked VTA GHSR signalling with JMV2959 infused via an indwelling VTA cannula connected to a minipump. Vehicle-treated mice increased their caloric intake during social defeat, but JMV2959-infusions attenuated feeding responses and increased anxiety-like behaviors. The data suggest that GHSR signalling in the VTA is critical for the increases in appetite observed during chronic social defeat stress. Furthermore, these data support the idea that GHSR signaling in the VTA may also have anxiolytic effects, and blocking GHSR in this region may result in an anxiety-like phenotype.

PS-B11-6: ENDOTHELIAL GHRELIN RECEPTOR INCREASE WHITE FAT WEIGHT THROUGH ACTIVATION OF MTOR-PPARGAMMA PATHWAY

Ghrelin secretion from the stomach acts on peripheral tissues to cause weight gain and fat accumulation in white adipose tissue (WAT). The role of endothelial growth hormone secretagogue-receptor (GHSR) in lipid accumulation was evaluated by restoring the expression of GHSR only in the endothelial cells of GHSR-deficient mice using the Cre-LoxP system (GHSR-EC). Radioisotope (RI) -labeled H-triolein was orally administered to evaluate lipid uptake in WAT. GHSR-deficient mice had reduced RI uptake in WAT compared to control mice, and GHSR-EC mice recovered the reduced RI uptake in GHSR-deficient mice. Furthermore, the body weight and WAT weight of GHSR-deficient mice after high-fat diet loading were significantly decreased compared with control mice, but increased in GHSR-EC mice. In vitro studies, ghrelin stimulation increased the uptake of free fatty acids by GHSR-mediated activation of the mTOR pathway and increased expression of PPARgamma. From these results, ghrelin takes up lipids via endothelial GHSR and contributes to fat accumulation in white adipose tissue.

The expression and function of growth hormone secretagogue receptor in immune cells: A current perspective.

The orexigenic hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), have been extensively studied in the last two decades, revealing that ghrelin signaling has important implications in health and disease. Metabolic diseases, such as obesity and diabetes, are often accompanied by low-grade chronic inflammation, that has been coined as "meta-inflammation." Immune cells are key cellular mediators of meta-inflammation, controlling both initiation and resolution of inflammation. Immune cells exhibit dynamic changes in cellular characteristics and functional output in response to the stimuli/insults from their surrounding microenvironment. Emerging evidence shows that ghrelin has an important effect on inflammation, in addition to its well-known effects on metabolism. However, the cellular/molecular mechanism of ghrelin signaling in immunity is largely unknown because the knowledge in regard to the expression and function of GHS-R in immune cells is currently sparse. In this review, we have accumulated the recent findings related to the expression and functions of GHS-R in various immune cells under different physiological and pathological states. This review aims to inspire further investigation of the immunological roles of ghrelin signaling and advance the therapeutic applications of ghrelin signaling in meta-inflammation.

Ablation of Ghrelin Receptor Mitigates the Metabolic Decline of Aging Skeletal Muscle.

The orexigenic hormone ghrelin has multifaceted roles in health and disease. We have reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), protects against metabolic dysfunction of adipose tissues in aging. Our further observation interestingly revealed that GHS-R deficiency phenocopies the effects of myokine irisin. In this study, we aim to determine whether GHS-R affects the metabolic functions of aging skeletal muscle and whether GHS-R regulates the muscular functions via irisin. We first studied the expression of metabolic signature genes in gastrocnemius muscle of young, middle-aged and old mice. Then, old GHS-R knockout (Ghsr−/−) mice and their wild type counterparts were used to assess the impact of GHS-R ablation on the metabolic characteristics of gastrocnemius and soleus muscle. There was an increase of GHS-R expression in skeletal muscle during aging, inversely correlated with the decline of metabolic functions. Remarkedly the muscle of old GHS-R knockout (Ghsr−/−) mice exhibited a youthful metabolic profile and better maintenance of oxidative type 2 muscle fibers. Furthermore, old Ghsr−/− mice showed improved treadmill performance, supporting better functionality. Also intriguing to note was the fact that old GHS-R-ablated mice showed increased expression of the irisin precursor FNDC5 in the muscle and elevated plasma irisin levels in circulation, which supports a potential interrelationship between GHS-R and irisin. Overall, our work suggests that GHS-R has deleterious effects on the metabolism of aging muscle, which may be at least partially mediated by myokine irisin.

The controversial role of the vagus nerve in mediating ghrelin's actions: gut feelings and beyond.

Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells.

Expression of ghrelin or growth hormone secretagogue receptor in the brain of postpartum stress mice.

Postpartum depression is one of the most common mental diseases that occur in women after childbirth; this disorder is extremely painful for women and represents a major burden on the society. Therefore, we designed this study to explore the possible material basis of the disease, and provide potential novel antidepressants therapy using a mouse model. We established a postpartum immobilization stress model. Maternal body weight changes and food intake were recorded for half a month after delivery, and levels of ghrelin and its receptor, growth hormone secretagogue receptor (GHSR) were measured. The mice in the immobilization stress group showed stress activity as well as low body weight and low feeding status. Ghrelin expression was elevated in blood whereas ghrelin or GHSR expression decreased in the hippocampus and prefrontal cortex of the immobilization stress mice, and the number of ghrelin-active and GHSR cells reduced.

Synergistic Control of KNDy Neuronal Influence on Energy Balance by Ghrelin and Estradiol

The gut peptide, ghrelin, mediates negative energy homeostasis and the neuroendocrine control of energy homeostasis by acting through its receptor, growth hormone secretagogue receptor (GHSR). GHSR, expressed in hypothalamic Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons in the arcuate (ARC), is well known to regulate energy balance. We have previously shown 17-beta-estradiol (E2) robustly increases Ghsr expression in KNDy neurons, enhancing their sensitivity to ghrelin. We hypothesize that E2-induced increase in GHSR expression augments KNDy sensitivity in a fasting state by elevating ghrelin to reduce energy expenditure in females. We developed a Kiss1-specific GHSR knockout to determine the role of GHSR in ARC KNDy neurons and fed them either a low-fat diet (LFD) or a high-fat diet (HFD). Knockout (experimental) females were resistant to HFD in terms of body weight gain, adiposity, and food intake compared to HFD-fed controls. HFD-fed experimental females also exhibited slower glucose clearance compared to HFD-fed controls. Experimental females, regardless of diet, exhibited elevated fasting (5h) glucose. Metabolic rates (V.O2, V.CO2) and energy expenditure (heat) were not different. Respiratory Exchange Ratio (RER) was elevated in LFD-fed females, indicating the utilization of carbohydrates over fat for energy. Further meal pattern analysis revealed a reduction in meal duration in HFD-fed females, but elevated meal frequency, while HFD-fed experimental females exhibited a reduced meal size. In two separate meal pattern experiments, experimental and control females were fasted for 24h and refed or injected with ghrelin (I.P. 1mg/kg) or saline. We observed a striking delay in refeeding behavior in experimental females compared to controls during the refeeding period after fasting. After injection, control females responded to ghrelin with a rapid and sustained increase in food intake which was blunted in experimentals. Collectively, these data suggest that GHSR activation in KNDy neurons modulates metabolism, glucose homeostasis, and feeding behavior, illustrating a novel mechanism for E2 and ghrelin to synergistically control KNDy neuronal output and their subsequent behavioral and physiological outcomes.

Contribution of growth hormone secretagogue receptor (GHSR) signaling in the ventral tegmental area (VTA) to the regulation of social motivation in male mice

Most psychiatric disorders are characterized by deficits in the ability to interact socially with others. Ghrelin, a hormone normally associated with the regulation of glucose utilization and appetite, is also implicated in the modulation of motivated behaviors including those associated with food and sex rewards. Here we hypothesized that deficits in ghrelin receptor (growth hormone secretagogue receptor; GHSR) signaling are also associated with deficits in social motivation in male mice. To test this hypothesis, we compared social motivation in male mice lacking GHSR or mice treated with the GHSR antagonist JMV2959 with that of WT or vehicle-treated mice. GHSR signaling in dopamine cells of the ventral tegmental area (VTA) has been implicated in the control of sexual behavior, thus we further hypothesized that GHSR signaling in the VTA is important for social motivation. Thus, we conducted studies where we delivered JMV2959 to block GHSR in the VTA of mice, and studies where we rescued the expression of GHSR in the VTA of GHSR knockout (KO) mice. Mice lacking GHSR or injected with JMV2959 peripherally for 3 consecutive days displayed lower social motivation as reflected by a longer latency to approach a novel conspecific and shorter interaction time compared to WT or vehicle-treated controls. Furthermore, intra-VTA infusion of JMV2959 resulted in longer latencies to approach a novel conspecific, whereas GHSR KO mice with partial rescue of the GHSR showed decreased latencies to begin a novel social interaction. Together, these data suggest that GHSR in the VTA facilitate social approach in male mice, and GHSR-signaling deficits within the VTA result in reduced motivation to interact socially.

β 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 β and α cells. We then generated β-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 β and α cells; GHS-R cell-autonomously regulated GSIS and modulated systemic insulin sensitivity. In conclusion, β cell GHS-R was an important regulator of glucose homeostasis, and GHS-R antagonists may have therapeutic potential for Type 2 Diabetes.

Genome-Wide Identification of RNA Editing Sites Affecting Intramuscular Fat in Pigs.

Simple SummaryIn order to investigate RNA editing sites affecting IMF (which is associated with pork quality and human insulin resistance.), we analyzed the transcriptome and genome sequencing data of a high- and low- groups composed of full-sib pairs pig with opposite IMF phenotypes. Finally, a total of 36 nonredundant RNA editing sites in the longissimus dorsi muscle, which may reveal the potential importance of RNA editing in IMF were identified. Four were selected as candidate sites associated with IMF. Our findings provide some new insights of RNA editing function in pig longissimus dorsi muscle.Intramuscular fat (IMF) is essential for improving the palatability and flavor of meat, and it is strongly associated with human insulin resistance. RNA editing is a widespread regulating event in different tissues. Here, we investigated the global RNA editing difference of two groups of pig with different IMF contents to find the potential editing sites affecting IMF. In this research, RES-Scanner and REDItools were used to identify RNA editing sites based on the whole genome and transcriptome sequencing data of the high and low groups composed of three full-sib pairs with opposite IMF phenotypes. A total of 295 RNA editing sites were investigated in the longissimus dorsi muscle, and 90.17% of these sites caused A to G conversion. After annotation, most editing sites were located in noncoding regions (including five sites located on the 3′ UTR regions). Five editing sites (including two sites that could lead to nonsynonymous amino acid changes) were located in the exons of genes. A total of 36 intergroup (high and low IMF) differential RNA editing sites were found in 33 genes. Some candidate editing sites, such as sites in acyl-coenzymeA: cholesterol acyltransferase 1 (ACAT1), coatomer protein, subunit alpha (COPA), and nuclear receptor coactivator 3 (NCOA3), were selected as candidate RNA editing sites associated with IMF. One site located on the 3′ UTR region of growth hormone secretagogue receptor (GHSR) may regulate GHSR expression by affecting the interaction of miRNA and mRNA. In conclusion, we identified a total of 36 nonredundant RNA editing sites in the longissimus dorsi muscle, which may reveal the potential importance of RNA editing in IMF. Four were selected as candidate sites associated with IMF. Our findings provide some new insights of RNA editing function in pig longissimus dorsi muscle which useful for pig IMF breeding or human insulin resistances research.

254-LB: Characterization of Ghrelin Receptor Expression in Mouse Islets Reveals Pancreatic Polypeptide Cells as a Key Ghrelin Target

Objective: While expression of the ghrelin receptor, GHSR (growth hormone secretagogue receptor), by pancreatic islets has been investigated, data regarding the specific islet cell types that express GHSR are inconsistent and incomplete. For instance, transcriptomic studies indicate marked GHSR expression by somatostatin (SST)-secreting delta cells vs. little to no GHSR expression by alpha cells and beta cells, which contrasts studies suggesting direct actions of ghrelin on GHSRs expressed by alpha cells and beta cells. Also, to our knowledge, GHSR expression by pancreatic polypeptide (PP) cells has not previously been directly investigated. The aims of this study were to determine GHSR expression within the four principal islet endocrine cell types and the effects of ghrelin on circulating PP. Methods: Ghsr-IRES-Cre mice, which express Cre recombinase directed by the GHSR promoter, crossed to Cre-dependent ROSA26-YFP reporter mice were used to histologically identify GHSR expression in islets. Double and triple labeling for YFP-immunoreactivity (IR) with insulin-IR, glucagon-IR, SST-IR, and/or PP-IR was performed to determine GHSR co-expression with islet hormones. Pharmacological manipulations were used to examine in vivo effects of acyl-ghrelin on plasma PP. Results: Eighty-five % of YFP-IR cells expressed PP-IR, 50% expressed SST-IR, and none expressed insulin-IR or glucagon-IR. GHSR antagonist raised plasma PP in overnight-fasted C57BL/6N mice. However, acyl-ghrelin administration had no effect on plasma PP in C57BL/6N mice. Conclusions: These results suggest that within mouse pancreatic islets, GHSR expression occurs most abundantly in PP cells and that pharmacologic blockade of ghrelin action raises plasma PP. Disclosure D. Gupta: None. B.K. Mani: None. K. Shankar: None. S. Osborne-Lawrence: None. N. Metzger: None. J.M. Zigman: Stock/Shareholder; Self; Medtronic. Funding Diana and Richard C. Strauss Professorship in Biomedical Research; Mr. and Mrs. Bruce G. Brookshire Professorship in Medicine; Kent and Jodi Foster Distinguished Chair in Endocrinology; University of Texas Southwestern Medical Center (to J.M.Z.)

Growth hormone secretagogue receptor in dopamine neurons controls appetitive and consummatory behaviors towards high-fat diet in ad-libitum fed mice

Growth hormone secretagogue receptor (GHSR), the receptor for ghrelin, is expressed in key brain nuclei that regulate food intake. The dopamine (DA) pathways have long been recognized to play key roles mediating GHSR effects on feeding behaviors. Here, we aimed to determine the role of GHSR in DA neurons controlling appetitive and consummatory behaviors towards high fat (HF) diet. For this purpose, we crossed reactivable GHSR-deficient mice with DA transporter (DAT)-Cre mice, which express Cre recombinase under the DAT promoter that is active exclusively in DA neurons, to generate mice with GHSR expression limited to DA neurons (DAT-GHSR mice). We found that DAT-GHSR mice show an increase of c-Fos levels in brain areas containing DA neurons after ghrelin treatment, in a similar fashion as seen in wild-type mice; however, they did not increase food intake or locomotor activity in response to systemically- or centrally-administered ghrelin. In addition, we found that satiated DAT-GHSR mice displayed both anticipatory activity to scheduled HF diet exposure and HF intake in a binge-like eating protocol similar to those in wild-type mice, whereas GHSR-deficient mice displayed impaired responses. We conclude that GHSR expression in DA neurons is sufficient to both mediate increased anticipatory activity to a scheduled HF diet exposure and fully orchestrate binge-like HF intake, but it is insufficient to restore the acute orexigenic or locomotor effects of ghrelin treatment. Thus, GHSR in DA neurons affects appetitive and consummatory behaviors towards HF diet that take place in the absence of caloric needs.

Recognition of Invasive Prostate Cancer Using a GHRL Polypeptide Probe Targeting GHSR in a Mouse Model In Vivo.

Ghrelin (GHRL) is a polypeptide that can specifically bind to the growth hormone secretagogue receptor (GHSR). The expression of GHSR is significantly different in normal and prostate cancer (PC) tissues in humans. It is important to find an effective diagnostic method for the diagnosis and prognosis of invasive PC/neuroendocrine prostate cancer (NEPC). GHRL and GHSR mRNA levels were determined by a quantitative real-time polymerase chain reaction in PC tissues. The expression of GHRL and GHSR proteins was assessed by Western blot assay and immunohistochemistry. A GHRL polypeptide probe was synthesized by standard solid-phase polypeptide synthesis, and labeled with Alexa Fluor 660. Confocal microscopy was used to capture fluorescence images. Living imaging analysis showed tumor areas of different invasiveness in mice models. The levels of GHRL and GHSR copy number amplification and mRNA expression were increased in invasive PC/NEPC, and the protein expression levels of GHRL and GHSR were similarly increased in NEPC. The GHRL polypeptide probe could effectively bind to GHSR. In PC3 cells, it was found that the GHRL probe specifically binds to GHSR on the cell membrane and accumulates in the cells through internalization after binding. Live imaging in mice models showed that there were different signal intensities in tumor areas with different invasiveness. GHSR and GHRL might be used in molecular imaging diagnosis for invasive PC/NEPC in the future.

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.

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. 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. 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. 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. Increased epicardial fat area, macrophage infiltration, and GHS-R expression in epicardial ATMs appeared to be associated with postoperative atrial fibrillation in obese patients.

Expression of Growth and Hunger Related Genes and Physio-Biochemical Responses in Labeo rohita (Hamilton, 1822) Fed with Lysine and Betaine.

The growth promoting effect of lysine and betaine as well as the expression of candidate genes reflecting their efficacy, such as ghrelin, leptin, Growth Hormone Secretagogue Receptor (GHS-R), Insulin like Growth Factor (IGF- 1) and Growth Hormone Releasing Hormone (GHRH) was examined in Labeo rohita fingerlings. One hundred eighty healthy juveniles from a homologous population were randomly distributed to 15 rectangular tanks of 150 litres capacity. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. At the end of trial, the growth parameters such as weight gain, SGR, PER were estimated from the weight of the triplicate groups. The digestive, metabolic and antioxidant enzymes were analysed using spectrophotometric methods. The intestine, brain and liver were sampled from the treatments and expression of different genes ghrelin, leptin, GHSR, IGF-1 and GHRH was also performed by realtime PCR. A significant (P<0.05) increase in weight gain, SGR, PER and lowest FCR was found in T4 group which was significantly (p < 0.05) different from other experimental groups. The highest mRNA expression levels of expression were found in T4 group which was similar to that of ghrelin gene mRNA of T2 group. The significantly (p<0.05) highest GHSR, GHRH and IGF-1 gene expression levels were found in T4 treatment group compared to other groups. The present study reveals that the lysine and betaine stimulate growth and expression of ghrelin GHRH, GHS-R and IGF-1 genes. The increase of IGF-I mRNA expression with lysine and betaine supplementation revealed that these compounds act as growth modulators. However, lysine was found to be a more potent modulator of growth compared to betaine.

Effects of β‑hydroxybutyric acid and ghrelin on the motility and inflammation of gastric antral smooth muscle cells involving the regulation of growth hormone secretagogue receptor.

Ghrelin is an orexigenic hormone that is produced by gastric cells. Ghrelin stimulates food intake and increases gastric movement. In rat model, injected β-hydroxybutyric acid (β-HB) leads to a decrease in body weight. It has been reported that patients with gastric erosions are slower to evacuate the stomach. The aim of the present study was to investigate the effects of ghrelin and β-HB on motility and inflammation in rat gastric antral smooth muscle cells (GASMCs). GASMCs were extracted from rat gastric antrum. Cell viability was determined using the Cell Counting Kit-8 assay. A reactive oxygen species (ROS) assay kit was used to analyze the levels of ROS using flow cytometry. Protein levels were determined using western blotting, and the expression levels of mRNAs were evaluated using reverse transcription-quantitative PCR. β-HB inhibited the expression of myosin regulatory light polypeptide 9 (MYL9), myosin light chain kinase (MLCK), transforming protein RhoA (RhoA), Rho-associated protein kinase-1 (ROCK-1) and growth hormone secretagogue receptor (GHS-R). By contrast, ghrelin increased the expression of MYL9, MLCK, RhoA, ROCK-1 and GHS-R in β-HB-treated GASMCs. β-HB increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and ROS, and decreased the levels of manganese (Mn) superoxide dismutase (SOD), copper/zinc (Cu/Zn)SOD and catalase. Ghrelin decreased the expression of TNF-α, IL-6, ROS and catalase, whereas ghrelin promoted the expression of MnSOD and Cu/ZnSOD in β-HB-treated GASMCs. Short interfering RNA targeting GHS-R inhibited the expression of MYL9, MLCK, RhoA and ROCK-1, and increased the levels of TNF-α, IL-6 and ROS in β-HB-treated or ghrelin-treated GASMCs. The present study provided preliminary evidence that β-HB inhibits the motility of GASMCs and promotes inflammation in GASMCs, whereas ghrelin decreases these effects. GHS-R acted as a primary regulator of motility and inflammation in GASMCs treated with β-HB and ghrelin.

THE GROWTH HORMONE SECRETAGOGUE RECEPTOR, GHRELIN, AND BNP IN HUMAN HEART DISEASE WITH PRESERVED EJECTION FRACTION

Heart disease (HD) diagnosis is based on clinical features, imaging, and circulating biomarkers, notably B-type natriuretic peptide (BNP), the gold standard marker of heart failure. However, a cardiac-specific biomarker detecting contractile dysfunction at early stages is lacking. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin may be cardiac-specific biomarkers. We have characterized a fluorescent analog of ghrelin, Cy5-ghrelin (1-19) that detects changes in cardiac GHSR in situ in diabetic cardiomyopathy and heart failure. We are now using this tool to map changes in regional and cellular distribution of GHSR in HD. We obtained tissue from the left ventricle (LV) and right atrium (RA) from 10 cardiac surgery patients with preserved ejection fraction (pEF) and from 10 deceased donors with no history of HD. GHSR levels were measured by quantitative fluorescence microscopy using Cy5-ghrelin(1-19). Ghrelin and BNP were measured by immunofluorescence. High-resolution images were acquired using a Nikon A1R confocal microscope and fluorescence intensities were quantified using ImageJ FIJI's built-in algorithms (Shanbhag for GHSR or RenyiEntropy for ghrelin and BNP). Values of fluorescence intensities were compared based on individual algorithms and colocalization analysis was conducted with the NIS-Elements software. Data were analysed by t-test, one-way ANOVA, and Pearson correlation coefficient (PCC) with significance set at p < 0.05. There were significantly lower levels of GHSR in RA tissue samples from cardiac surgery patients (p=0.041) and no significant differences in ghrelin or BNP expression (Figure 1). There was strong colocalization of ghrelin and BNP in both healthy (PCC=0.935) and diseased (PCC=0.856) tissue samples. In healthy tissue, colocalization appeared as large, discretely defined punctate areas near the nucleus, whereas in the tissue from cardiac surgery patients, ghrelin and BNP signals were more diffuse and contained in smaller areas surrounding the nucleus (Figure 2, arrows). We have shown expression of GHSR, ghrelin and BNP in both healthy and diseased cardiac tissue with only GHSR significantly changing in HD. This may indicate GHSR expression is decreased in HD with pEF whereas BNP (elevated in end stage HF) and ghrelin are not changed. The strong correlation between ghrelin and BNP indicates that these markers may be stored in the same intracellular compartment near the nucleus where they are released upon cellular stress induced by HD. This is the first study showing colocalization between ghrelin and BNP in human cardiac tissue. We are currently stratifying these results based on sex, type of HD, and region of the heart.View Large Image Figure ViewerDownload Hi-res image Download (PPT)