Ghrelin Gene Research Articles

The role of ghrelin in patients with functional dyspepsia and its potential clinical relevance (Review)

Functional dyspepsia (FD) is a functional gastrointestinal disorder (FGID). According to the Rome III consensus, FD is divided into 2 subgroups: epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS). Although multiple mechanisms of FD pathogenesis have been suggested, its underlying etiology and pharmacological therapy remain unclear. Ghrelin is a gut-derived peptide found in the stomach. It plays a role in the regulation of gastric motility and appetite. The ghrelin gene encodes 3 molecular forms, acyl ghrelin, des-acyl ghrelin and obestatin. Acyl ghrelin acts as an endogenous ligand for growth hormone secretagogue receptor; furthermore, it is orexigenic, with effects on food intake, energy homeostasis and gastrointestinal motility. Des-acyl ghrelin exerts an opposite effect to acyl ghrelin. Obestatin exerts an inhibitory effect on the motor activity of the antrum and duodenum in fed animals. These peptides exert differential effects on gut motility and food intake. The therapeutic potential of ghrelin has attracted attention due to its varied bioactivities. Certain studies have shown that total ghrelin levels are significantly lower in patients with FD compared with healthy volunteers and that the acyl ghrelin levels of patients with FD are higher compared with healthy volunteers. However, a recent study demonstrated that acyl ghrelin levels in patients with PDS were lower compared with healthy volunteers; the association between FD and other ghrelin family gene products also remains unclear. Although certain studies have demonstrated the beneficial effects of acyl ghrelin administration and its agonist in patients with FD, only a few clinical reports exist. Further studies are required in order to examine the effects of ghrelin on FD.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Molecular characterization and expression profile of ghrelin gene during different reproductive phases in buffalo (Bubalus bubalis)

Ghrelin, a novel motilin-related endogenous ligand for growth hormone secretagouge receptor, is implicated in various biological functions, including regulation of female reproduction. But the presence of ghrelin and its role in reproductive functions in buffalo, a species with poor reproductive efficiency, is not known. In the present study full-length ghrelin cDNA was isolated from bubaline abomasum, which encodes the entire prepropeptide of 116 amino acids. The deduced amino acid sequence of ghrelin of buffalo showed >95% and 31% identity with that of ruminants (cattle, sheep, and goat) and humans, respectively. Analysis of synonymous and nonsynonymous nucleotide substitutions in the coding region of ghrelin indicated that these sequences of different species have been under purifying selection. The 3995-bp amplicon of ghrelin gene consisting of 4 exons and 3 introns was cloned with genomic DNA from buffalo. Further, ghrelin expression was determined by quantitative real-time PCR, in situ hybridization, and immunohistochemistry in bubaline endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated the persistent expression of ghrelin mRNA and protein in the endometrium during stage I (day 3–5), stage II (day 6–15), and stage III (day 16–21) of the estrous cycle and also during early (∼day 30–40) pregnancy. Immunohistochemistry and quantitative real-time PCR experiments indicated the relatively higher expression of ghrelin in the endometrium during stage II (day 6–15) of the estrous cycle and early pregnancy than during stage I (day 3–5) and stage III (day 16–21) of the estrous cycle, but no statistically significant difference in ghrelin expression was observed among stages. To conclude, the results of the present study indicate the persistent expression of ghrelin in the uterine endometrium throughout the estrous cycle and in early pregnancy which might be helpful in determining its role in buffalo reproduction.

Obestatin Enhances In Vitro Generation of Pancreatic Islets through Regulation of Developmental Pathways

Availability of large amounts of in vitro generated β-cells may support replacement therapy in diabetes. However, methods to obtain β-cells from stem/progenitor cells are limited by inefficient endocrine differentiation. We have recently shown that the ghrelin gene product obestatin displays beneficial effects on pancreatic β-cell survival and function. Obestatin prevents β-cell apoptosis, preserves β-cell mass and stimulates insulin secretion in vitro and in vivo, in both normal and diabetic conditions. In the present study, we investigated whether obestatin may promote in vitro β-cell generation from mouse pancreatic islet-derived precursor cells. Treatment of cultured islets of Langerhans with obestatin (i) enriched cells expressing the mesenchymal/neuronal marker nestin, which is associated with pancreatic precursors; (ii) increased cell survival and reduced apoptosis during precursor selection; (iii) promoted the generation of islet-like cell clusters (ICCs) with increased insulin gene expression and C-peptide secretion. Furthermore, obestatin modulated the expression of fibroblast growth factor receptors (FGFRs), Notch receptors and neurogenin 3 (Ngn3) during islet-derived precursor cell selection and endocrine differentiation. These results indicate that obestatin improves the generation of functional β-cells/ICCs in vitro, suggesting implications for cell-based replacement therapy in diabetes. Moreover, obestatin may play a role in regulating pathways involved in pancreas development and regeneration.

Ontogeny expression of ghrelin, neuropeptide Y and cholecystokinin in blunt snout bream, Megalobrama amblycephala

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding in fish and mammals. To better understand the role of the three peptides in appetite regulation in the early developmental stages of blunt snout bream (Megalobrama amblycephala), partial cDNA sequences of ghrelin, NPY and CCK genes were cloned. And then, real-time quantitative PCR and RT-PCR were used to detect and quantify the mRNA expressions of these genes from zygotes to larvae of 50 days after hatching (DAH). Ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. Ghrelin and NPY mRNA expressions were upregulated at 1, 3, 5 DAH, while CCK mRNA expression was reduced significantly at 3 DAH. The mRNA expression levels of three genes in larvae varied significantly until 30 DAH. In adult fish, all three peptides were detected to be expressed in brain and several peripheral tissues. Ghrelin mRNA was mainly expressed in the intestine, whereas NPY and CCK mRNAs were mainly expressed in the brain. Taken together, these results indicate that ghrelin, NPY and CCK may have roles in early development and participate in the regulation of feeding of larvae in blunt snout bream and will be helpful for further investigation into feed intake regulation in adults of this species.

Abstract 583: Ghrelingene expression avoiding the D-ribose caused HepG2 cell death.

Abstract Liver cancer patients could suffer from high saccharide environment due to the malfunction of liver. To mimic this high saccharide environment, HepG2 cells were culture with 30 mM D-riboses. We found that this high concentration of saccharide cause cell death. Cinnamon was report to reduce glucose in model rats. We hypothesized that the administration of cinnamon extracts may benefit liver cancer patients whose metabolism is defected. To test the hypothesis, three different chemical subtypes of cinnamon extracts were added into the culture medium. The adding of all three cinnamon extracts protected HepG2 cells from D-ribose administration and increased around 20% of surviving cells. These protections were even better than the addition of aminoguanidine hemisulfate salt, a chemical used in the treatment of diabetes. The expressions of ghrelin gene and two ghrelin-related genes was monitored by RT-PCR. Ghrelin gene expression was up-regulated in cinnamon treated cells. Also, the ghrelin modify gene, MBOAT4, and ghrelin process enzyme, furin, gene were all detected in HepG2 cells and cinnnamon treated cells. These results demonstrated that if ghrelin expressed in HepG2 cell and maybe liver as well, it can be processed properly. Both ghrelin transcript variants 1 and 3 recombinant plasmid were transfected into HepG2 cells and both transcript variants protected HepG2 cells from the addition of D-ribose without the administration of cinnamon extracts. We conclude that the cinnamon avoids HepG2 cell damage from D-ribose induced oxidative stress by inducing ghrelin gene expression, and through the ghrelin signaling increases cell surviving. Since ghrelin level is abnormally regulated in patients with hepatocellular carcinoma and post-hepatitic liver cirrhosis, the understanding of how ghrelin been regulated in liver may benefit liver disease patients. Citation Format: Jia Huei Shiu, Chin Hao Huang, Tai Lin Lee, Shih Chieh Lee, Shu Ying Liu, Meng Feng Tsai. Ghrelingene expression avoiding the D-ribose caused HepG2 cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2013-583

Fermented barley averts diet induced obesity via modulating the lipid metabolic gene expression in Sprague dawley rats.

This study investigated the anti‐obesity effect of fermented barley on diet induced obesity in Sprague dawley (SD) rats. Animals were randomly divided into 6 groups (n=6); ND‐C, ND‐SH, normal diet plus guar gum or 1500 mg/kg bodyweight (BW) fermented barley group respectively; HD‐C, high fat diet plus guar gum; HD‐SL, HD‐SM and HD‐SH, high fat diet plus 500, 1000 and 1500 mg/kg BW fermented barley respectively. Guar gum supplementation was equivalent to the amount of soluble fiber in the fermented barley. After 19 weeks animals were sacrificed, serum lipid profile, leptin, ghrelin and hepatic lipid metabolic gene expression level were examined. A significant reduction in bodyweight, bodyweight gain, BMI, obesity index, epididymal adipose tissue weight, feed and energy intake was observed in HD‐SH group compared to HD‐C and HD‐SL group. Serum leptin decreased significantly in a dose dependent manner. Serum triglyceride and ghrelin level did not show significant change compared to the control group. Total cholesterol and LDL‐cholesterol tended to decrease; HDL‐cholesterol did not show significant changes. The mRNA expression of FAS was significantly decreased; ACO, PPARγ and PPARα increased dose dependently. CPT1α level was at similar level in all fermented barley group. These data indicated that fermented barley exerts an anti‐obesity effect against diet induced obesity conditions in rats.

Polymorphisms in the ghrelin gene and their associations with milk yield and quality in water buffaloes

Ghrelin is a gastrointestinal hormone that acts in releasing growth hormone and influences the body general metabolism. It has been proposed as a candidate gene for traits such as growth, carcass quality, and milk production of livestock because it influences feed intake. In this context, the aim of this study was to verify the existence of polymorphisms in the ghrelin gene and their associations with milk, fat and protein yield, and percentage in water buffaloes (Bubalus bubalis). A group of 240 animals was studied. Five primer pairs were used and 11 single nucleotide polymorphisms (SNP) were found in the ghrelin gene by sequencing. The animals were genotyped for 8 SNP by PCR-RFLP. The SNP g.960G>A and g.778C>T were associated with fat yield and the SNP g.905T>C was associated with fat yield and percentage and protein percentage. These SNP are located in intronic regions of DNA and may be in noncoding RNA sites or affect transcriptional efciency. The ghrelin gene in buffaloes influences milk fat and protein synthesis. The polymorphisms observed can be used as molecular markers to assist selection.

Molecular Cloning, Tissue Distribution, and Ontogenetic Expression of Ghrelin and Regulation of Expression by Fasting and Refeeding in the Grass Carp (Ctenopharyngodon idellus)

Ghrelin is a recently discovered brain-gut peptide in fish and mammals. It has two main physiological functions-stimulating growth hormone secretion and regulating appetite. To investigate the biological function of ghrelin in appetite regulation in grass carp Ctenopharyngodon idellus, the full-length cDNA sequence of the ghrelin gene was obtained by RT-PCR and rapid amplification of cDNA ends methods. Homology analysis indicated that ghrelin cDNA sequence is conserved in teleosts. The grass carp ghrelin gene consists of four exons and three introns, which is similar to that of common carp, zebrafish and humans. Real-time quantitative PCR was used to detect ghrelin mRNA expression in the study. In adult tissues, high levels of gene expression were found in the foregut; moderate levels in the muscle, liver, hypothalamus, white adipose tissue, midgut, heart, and pituitary; and lower levels in the three other examined tissues. During embryonic development, ghrelin mRNA expression could be detected as early as fertilized egg stage and displayed an increase in expression until heart appearance stage. After hatching, the level of ghrelin mRNA expression decreased sharply to the lowest level at 1 day post-hatching (dph), then increased to a higher level at 7 dph and subsequently decreased to a relative stabilized level from 15 to 35 dph. The ghrelin mRNA expression was significantly upregulated in the brain and intestine during fasting and was downregulated after refeeding. The results suggest that ghrelin mRNA expression has obvious tissue specificity and may have a role in appetite regulation in grass carp.

The Preproghrelin 3056 TT Genotype is Associated with the Feeling of Hunger and Low Acylated Ghrelin Levels in Japanese Patients with <i>Helicobacter pylori</i>-negative Functional Dyspepsia

An impairment of gastric motility is strongly associated with the pathophysiology of functional dyspepsia (FD). Plasma ghrelin is one of the key molecules linked to gastric motility. Therefore, this study aimed to evaluate whether ghrelin (GHRL) gene polymorphisms are associated with clinical symptoms, the plasma ghrelin levels and gastric emptying in patients with FD as defined by the Rome III classification. We enrolled 74 Helicobacter pylori-negative patients presenting with typical symptoms of FD (epigastric pain syndrome (EPS), n=23; postprandial distress syndrome (PDS), n=51) and 102 healthy volunteers. Gastric motility was evaluated according to the Tmax value and T1/2 using the (13)C-acetate breath test. We used the Rome III criteria to evaluate upper abdominal symptoms and SRQ-D scores to determine the depression status. The Arg51Gln(346G->A), preproghrelin3056T->C, Leu72Met(408C->A) and Gln90Leu(3412T->A) polymorphisms were analyzed in DNA in blood samples obtained from the enrolled subjects. Genotyping was performed using polymerase chain reaction. There was a significant relationship (p=0.048) between the preproghrelin 3056TT genotype and the serum levels of acylated ghrelin in the H. pylori-negative FD patients. The preproghrelin 3056TT genotype was significantly (p=0.047) associated with the feeling of hunger in the H. pylori-negative FD patients. The preproghrelin 3056TT genotype is significantly associated with the acylated ghrelin levels and the feeling of hunger in H. pylori-negative FD patients. Further studies are needed to clarify the association between the preproghrelin 3056TT genotype and lower plasma acylated ghrelin levels and the impact of this relationship on the feeling of hunger in H. pylori-negative FD patients.

Ghrelin directly stimulates adult hippocampal neurogenesis: implications for learning and memory

Adult hippocampal neurogenesis is important in mediating hippocampal-dependent learning and memory. Exogenous ghrelin is known to stimulate progenitor cell proliferation in the dentate gyrus of adult hippocampus. The aim of this study was to investigate the role of endogenous ghrelin in regulating the in vivo proliferation and differentiation of the newly generating cells in the adult hippocampus using ghrelin knockout (GKO) mice. Targeted deletion of ghrelin gene resulted in reduced numbers of progenitor cells in the subgranular zone (SGZ) of the hippocampus, while ghrelin treatment restored progenitor cell numbers to those of wild-type controls. We also found that not only the number of bromodeoxyuridine (BrdU)-positive cells but also the fraction of immature neurons and newly generated neurons were decreased in the GKO mice, which were increased by ghrelin replacement. Additionally, in the GKO mice, we observed impairment of memory performance in Y-maze task and novel object recognition test. However, these functional deficiencies were attenuated by ghrelin administration. These results suggest that ghrelin directly induces proliferation and differentiation of adult neural progenitor cells in the SGZ. Our data suggest ghrelin may be a plausible therapeutic potential to enhance learning and memory processes.

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homoeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 amino acid preproghrelin isoform that codes for ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia have been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate-resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

Developmental transcription of genes putatively associated with growth in two sturgeon species of different growth rate

In the present study, we surveyed developmental changes in the transcription of growth hormone (gh), insulin-like growth factor-I (igf-I), ghrelin (ghrl) and vascular endothelial growth factor (vegf) genes in the largest freshwater fish, European sturgeon (Beluga, Huso huso) and compared the same parameters to that of its phylogenically close moderate-sized species, Persian sturgeon (Acipenser persicus). The transcripts of gh, igf-I, ghrl and vegf were detected at all developmental time-points of Persian sturgeon and Beluga from embryos to juvenile fish. Changes in normalized gh, igf-I, ghrl and vegf transcription by using the geometric average of genes encoding ribosomal protein L6 (RPL6) and elongation factor (EF1A) over the time of development of Persian sturgeon and Beluga were statistically significant (P<0.05). Our results showed that the mRNA expression levels of both igf-I and ghrl were low during early larval development and then increased significantly to the late larval time-points when larvae started exogenous feeding. In both Beluga and Persian sturgeon, after a low mRNA expression during the embryonic stage, the transcript levels of vegf displayed an increasing trend during yolk-sac fry, consistent with organogenesis. The vegf level remained constantly high in the time of exogenous feeding. The highest detection of gh transcripts coincided with the end of the embryonic stage (hatching time) in Persian sturgeon and 3days-post-hatching (dph) in Beluga. In Persian sturgeon, the gh transcript started to decrease to the rest of the developmental time-points, whereas in Beluga gh transcript had a marked second increase from the time of exogenous feeding (20-dph). This Beluga specific increase in gh transcription may be associated with the marked growth rate and extraordinary size of this fish species.

Letter to “Mapping of Ghrelin Gene Expression and Cell Distribution in the Stomach of Morbidly Obese Patients—a Possible Guide for Efficient Sleeve Gastrectomy Construction”—Authors’ Reply

In our study [1], we examined tissue samples from three different regions of the resected stomach segment of obese individuals who underwent laparoscopic sleeve gastrectomy. The regions were the fundus, body, and pre-antral area. The “pre-antral” region was at the distal end of the specimen, i.e., the area closest to the true antrum that was included in the resection. As specified in the “Materials and Methods” section, the stomach was divided using linear endoscopic staplers, commencing 5 cm proximally to the pylorus. This correlates to the antrum in most patients, albeit the proximal part thereof. We used “pre-antral” to differentiate from endoscopic/histologic specimens obtained in other reports from the prepyloric region endoscopically in healthy patients [2] or from resected antra of gastric cancer patients [3]. The presence of transitional gastric mucosa was not investigated and could potentially lead to a difference from the “true” antrum, but the optimal distance from the pylorus is still in debate (as elaborated in the discussion)—a distance of 5 cm was chosen which is consistent with the 2–6 cm from the pylorus recommended in the International Sleeve Gastrectomy Expert Panel Consensus Statement [4]. Histologic examination of our specimens did not find noteworthy inflammation. Of the 20 patients enrolled, only 2 suffered from comorbidities (diabetes in one and hyperlipidemia in the other). Therefore, we did not comment on the potential role of the metabolic syndrome in the behavior of gastric endocrine cells. We hope that our study, which helps define the borders of significant ghrelin-producing tissue, will assist surgeons in optimizing sleeve gastrectomy construction.

Obestatin stimulates differentiation and regulates lipolysis and leptin secretion in rat preadipocytes.

Obestatin is a 23-amino acid peptide encoded by the ghrelin gene, which regulates food intake, body weight and insulin sensitivity. Obestatin influences glucose and lipid metabolism in mature adipocytes in rodents. However, the role of this peptide in rat preadipocytes remains to be fully understood. The current study characterized the effects of obestatin on lipid accumulation, preadipocyte differentiation, lipolysis and leptin secretion in rat primary preadipocytes. Obestatin enhanced lipid accumulation in rat preadipocytes and increased the expression of surrogate markers of preadipocyte differentiation. At the early stage of differentiation, obestatin suppressed lipolysis. By contrast, lipolysis was stimulated at the late stage of adipogenesis. Furthermore, obestatin stimulated the release of leptin, a key satiety hormone. Overall, the results indicated that obestatin promotes preadipocyte differentiation. Obestatin increased leptin release in preadipocytes, while the modulation of lipolysis appears to depend upon the stage of differentiation.

Ghrelin: a new treatment for non-alcoholic fatty liver disease?

Li et al. [1] report in this issue of the journal that administration of ghrelin might improve inflammation, oxidative stress, and apoptosis during and after non-alcoholic fatty liver disease development (NAFLD). Non-alcoholic fatty liver disease development is becoming an increasingly important disease condition [2], as NAFLD and non-alcoholic steatohepatitis (NASH) are the most common chronic liver disease in the western world, which is associated with end-stage liver disease and hepatocellular carcinoma. Moreover, an increasing number of liver transplantations is performed due to NASH and transplantation in this cohort seems to be associated with high mortality and postoperative complications, most likely due to associated obesity and diabetes [3]. In a recent review by Takamura et al. [4], they nicely addressed the remarkable changes in the liver in NAFLD by addressing NAFLD both as a consequence and as a cause of insulin resistance through lessons learned from the liver of patients with type 2 diabetes. The development of NAFLD is linked to food intake, as diet seems to be an important contributor to the pathogenesis of NAFLD [5]. In a recent review by de Wit et al. [5], they specifically mentioned that saturated fat and fructose seem to stimulate hepatic lipid accumulation and progression into NASH, whereas unsaturated fat, choline, antioxidants, and highprotein diets rich in isoflavones seem to have a more preventive effect. Therefore, it is not surprising that gut hormones that are known to control the uptake of nutrients by organs are now increasingly investigated in the light of NAFLD. Interestingly, the report by Yan Li in this journal adds up to the rapidly increasing number of publications on the effects of ghrelin (and its analogs) on a multitude of physiological and pathophysiological conditions. Both ghrelin and des-acyl ghrelin (DAG) appear to have many functions and effects that are much wider than the direct control over food intake [6]. In a recent publication by Estep et al. [7], they report on the relation of the three ghrelin gene products (ghrelin, DAG, and obestatin) and their involvement in those metabolic and inflammatory pathways that are linked with the development of NAFLD. They studied 75 morbidly obese patients with biopsy-proven NAFLD (41 with histologic NASH) and found that patients with NASH had twofold higher concentration of DAG than patients with nonNASH. Ghrelin and obestatin concentrations positively correlated with fibrosis stage. Apparently, products of the ghrelin gene may be important for the pathogenesis of NASH and fibrosis [7]. In another report on this topic by Okamatsu et al. [8], again the relation between appetite regulating hormones and NAFLD was studied, but in this case in 49 prepubertal overweight children and 49 age-matched controls of normal weight. The incidence of presumed NAFLD was 28.6 % in overweight children, and in these subjects, serum leptin levels were associated with aspartate aminotransferase/alanine aminotransferase ratios and serum insulin levels. Interestingly, also plasma ghrelin levels significantly correlated with liver function index [8]. In line with the observations by Okamatsu, GutierrezGrobe et al. reported that serum ghrelin and obestatin concentrations are correlated with a low risk of developing NAFLD. However, ghrelin/obestatin ratio was not correlated with NAFLD [9] in their cross-sectional study in 98 subjects (51 NAFLD patients and 47 controls), in which P. J. D. Delhanty A. J. van der Lely (&) Department of Medicine, Erasmus University MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands e-mail: a.vanderlelij@erasmusmc.nl

The Obestatin/GPR39 System Is Up-regulated by Muscle Injury and Functions as an Autocrine Regenerative System

The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.

The association between testicular ghrelin receptor mRNA and serum testosterone levels in immunocastrated boars

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), has various functions. The expression of ghrelin and growth hormone secretagogue receptor type 1a (GHS-R1a) has been demonstrated in rat and human testis, and ghrelin also affects testosterone (T) secretion in vitro, suggesting a role for this molecule in the direct control of testis function. However, whether this signaling system is present in pig testis, and the association with serum T remains largely unexplored. In this study we investigated the relationship between serum T levels and ghrelin and GHS-R1a gene expression in the testes of intact and immunocastrated boars. Testicular tissue and serum samples were collected from seven intact boars and seven recombinant GnRH-I immunocastrated boars. GHS-R1a gene expression in the testis was higher in immunocastrated than intact boars (2.22±0.71 vs. 0.53±0.23; P<0.01). Mean serum T levels were markedly lower in immunocastrated than intact boars (0.40±0.04 vs. 2.10±0.94ng/mL, P<0.01). Ghrelin gene expression in testis did not differ between both groups of boars. GHS-R1a gene expression was positively correlated with ghrelin gene expression (r=0.68, P<0.001), but negatively correlated with serum T concentrations (r=−0.83, P<0.001). Overall, the existence of ghrelin and GHS-R1a gene expression in pig testis, and the increase in GHS-R1a gene expression in the testes and the inverse correlation with serum T in immunocastrated boars are highly suggestive of a role for ghrelin in the regulation of mammalian testicular function.

Lack of Association of the &lt;i&gt;Ghrelin&lt;/i&gt; Gene Arg51Gln Single Nucleotide Polymorphism with Obesity and Metabolic Syndrome among Multi-ethnic Malaysian Subjects

Obesity and metabolic syndrome has become a public health concern because of its association with a number of med ical co mplications that lead to increased morb idity and mo rtality. Gh relin is a hormone that is primarily secreted in the stomach, which plays an important role to increase hunger through its action on hypothalamic feed ing. The Ghrelin gene Arg51Gln single nucleotide poly morphism (SNP) (rs34911341) has been associated with obesity and metabolic syndrome in previous studies. Therefore, this study was to examine the prevalence of this SNP and its association with obesity, obesity-related traits and metabolic syndrome among 184 mult i-ethnic Malaysian subjects (67 males, 117 females; 76 obese, 108 non obese; 52 Malay, 91 ethnic Ch inese, 41 ethnic Indians) fro m the Kampar Health Clinic cohort. Demographic data, anthropometric and clinical measurements of subjects were collected. Genotyping was performed b y using the geno mic DNA extracted fro m leukocytes, followed by Poly merase Chain Reaction and SacI Restriction Frag ment Length Poly morphism, revealing 113 GG, 70 GA and 1 AA subjects ; minor allele frequency 0.196. Arg 51Gln alleles did not show any association with obesity (p = 0.643), gender (p = 0.064) and ethnicity (p = 0.390). Besides, it d id not show any association with the presence of metabolic syndrome according to 3 criteria in the modified NCEP ATP III for Asians ( p = 0.931). Anthropometric and clinical measurements indicative of obesity and metabolic syndrome were also all not significantly different between the alleles. In conclusion, the Ghrelin Arg51Gln gene variant was not associated with obesity, obesity-related traits and metabolic syndrome among Malaysian subjects in this study.

Food restriction, ghrelin, its antagonist and obestatin control expression of ghrelin and its receptor in chicken hypothalamus and ovary

The purpose of the present study was to identify the role of age, nutritional state and some metabolic hormones in control of avian hypothalamic and ovarian ghrelin/ghrelin receptor system. We examined the effect of food restriction, administration of ghrelin 1–18, ghrelin antagonistic analogue (D-Lys-3)-GHRP-6, obestatin and combinations of them on the expression of ghrelin and ghrelin receptor (GHS-R1a) in hypothalamus and ovary of old (23months of age) and young (7months of age) chickens. Expression of mRNAs for ghrelin and GHS-R1a in both hypothalamus and largest ovarian follicle was measured by RT-PCR. It was observed that food restriction could promote the expression of ghrelin and GHS-R1a in hypothalamus and ovary of the old chickens, but in the young chickens it reduced expression of ghrelin and did not affect expression of GHS-R1a in the ovary. Administration of ghrelin 1–18 did not affect hypothalamic or ovarian ghrelin mRNA, but significantly increased the expression of GHS-R1a in hypothalamus, but not in ovary. (D-Lys-3)-GHRP-6, significantly stimulated accumulation of ghrelin, but not GHS-R1a mRNA in hypothalamus or ghrelin or GHS-R1a in the ovary. Ghrelin 1–18 and (D-Lys-3)-GHRP-6, when given together, were able either to prevent or to induce effect of these hormones. Obestatin administration increased expression of ghrelin gene in the hypothalamus, but not expression of hypothalamic GHS-R1a, ovarian ghrelin and GHS-R1a. Furthermore, obestatin was able to modify effect of both ghrelin and fasting on hypothalamic and ovarian mRNA for ghrelin GHS-R1a. Our results (1) confirm the existence of ghrelin and its functional receptors GHS-R1a in the chicken hypothalamus and ovary (2) confirm the age-dependent control of ovarian ghrelin by feeding, (3) demonstrate, that nutritional status can influence the expression of both ghrelin and GHS-R1a in hypothalamus and in the ovary (3) demonstrates for the first time, that ghrelin can promote generation of its functional receptor in the hypothalamus, but not in the ovary, (4) show that ghrelin1–18 and (D-Lys-3)-GHRP-6 could not only be antagonists in the action on chicken hypothalamus and ovaries, but also independent regulators and even agonists, and (5) provide first evidence for action of obestatin on hypothalamic ghrelin and on the response of hypothalamic and ovarian ghrelin/GHS-R1a system to food restriction. These data indicate the involvement of both hypothalamic and ovarian ghrelin/GHS-R1 systems in mediating the effects of nutritional status, ghrelin and obestatin on reproductive processes.