Ghrelin Gene Expression Research Articles

A body-wide transcriptome screen reveals high ghrelin gene (GHRL) expression in monocytes and supports a hypothesis of monocyte GHRL-derived hormones as critical mediators of the brain-gut axis and monocyte-adipocyte cross-talk in health and obesity

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

RNAi-mediated Ghrelin affects gastric H+–K+-ATPase activity and expression of GOAT–Ghrelin system in vitro

Ghrelin has been implicated in the regulation of gastric functional development, and its physiological functions are mediated by Ghrelin-O-acyltransferase (GOAT) which is capable of generating the active form of this polypeptide hormone. However, whether and how ghrelin gene silencing may modify gastric acid secretion and GOAT–Ghrelin system is yet to be explored. The study was performed in gastric mucosal cells from weanling piglets in vitro. We evaluated the effect of ghrelin on gastric acid secretion, gene expression of GOAT and ghrelin as well as ghrelin levels by RNA interference assay. shGhrelin triggered the down-regulation of ghrelin mRNA expression (P<0.05) via an RNAi mechanism, as observed by real-time RT-PCR. In addition, shGhrelin showed reduced total ghrelin production and secretion (P<0.05) using ELISA in vitro. We also detected that GOAT mRNA expression was reduced in shGhrelin group (P<0.05), compared with control groups. In accordance with the GOAT expression, acylated ghrelin production and secretion were reduced in gastric mucosal cells and culture medium (P<0.05). Silencing of ghrelin gene achieved by RNAi-mediation inhibited the activity of H+–K+-ATPase and pepsin (P<0.05) in gastric mucosal cells. These results indicated that RNAi of Ghrelin gene inhibited the gastric acid secretion with decreased GOAT mRNA and acylated Ghrelin in gastric mucosal cells.

Expression of Ghrelin and GHSR-1a in Long Term Diabetic Rat's Kidney

The aim of this work was to study the relative ghrelin and growth hormone secretagogue receptor (GHS-R)1a gene expression in the kidney of long-term diabetic rats. Forty male Wistar albino rats were divided into four groups: C- control group, DI- one month diabetic rats group, DII- two months diabetic rats group, and DIII- three months diabetic rats group. Diabetes was induced by streptozotocin STZ (40mg/kg i.p). The rats were decapitated under ketamine anesthesia and their kidney tissues were removed. Tissue GHS-R mRNA levels, ghrelin expression, and histopathological damage scores were compared. Dilatation in the distal tubules, epithelial desquamation into the lumen of the tubules and transparent tubules showing glycogen vacuolation were observed in all the diabetic groups. Ghrelin immunoreactivity was significantly higher in group DI compared to group C, whereas in groups DII and DIII, ghrelin immunoreactivity was similar with group C. GHSR-1a mRNA level in group DIII was significantly lower than in group C. As a result, ghrelin immunoreactivity increased at the beginning of diabetes; however, with increase in the duration of diabetes ghrelin immunoreactivity approached to the control values. The expression of GHSR-1a mRNA decreased with increase in diabetes duration. It seemed that down-regulation of GHSR-1a contributed to the renal damage induced by long-term diabetes.

Effect of Zinc on Appetite Regulatory Peptides in the Hypothalamus of Salmonella-Challenged Broiler Chickens.

The effects of dietary Zinc (Zn) supplementation on the gene expression of appetite regulatory peptides were investigated in Salmonella-infected broiler chickens. Broiler chickens (Arbor Acres, 1day old) were allocated randomly into 24 pens of 10 birds. The chickens from 12 pens were fed with basal diet and the other with basal diet supplemented with Zn (ZnSO4·H2O, 120mg/kg). At 5days of age, the chickens were divided into 4 treatments with 6 pens: basal diet; basal diet and Salmonella challenge; Zn-supplemented diet; Zn-supplemented diet and Salmonella challenge. At 42days of age, the hypothalamus from 6 chickens per treatment (1 chicken per pen) was individually collected for gene expression determination. Results showed that dietary supplementation of Zn reduced the gene expression of hypothalamic ghrelin and tumor necrosis factor alpha (TNF-α) (P < 0.05). Salmonella infection upregulated the messenger RNA (mRNA) levels of hypothalamic neuropeptide Y (NPY) and TNF-α. Zn supplementation and Salmonella inoculation were significantly correlated with the mRNA levels of toll-like receptor 2-1 (P < 0.05). However, neither dietary Zn supplementation nor Salmonella inoculation had significant effect on hypothalamic agouti-related protein, cocaine- and amphetamine-regulated transcript, and pro-opiomelanocortin. This study shows that dietary Zn supplementation promoted orexigenic appetite regulatory peptides and reduced the expression of the inflammatory cytokine TNF-α in the hypothalamus of Salmonella-challenged broilers.

The effects of streptozotocin-induced diabetes on ghrelin expression in rat testis: biochemical and immunohistochemical study.

Ghrelin is a hormone which has effects on the secretion of growth hormone, gastrointestinal system, cardiovascular system, cell proliferation and reproductive system. The present study we focused on the relation between ghrelin and GHS-R1a gene expression and the regulation of their expression in the testes of diabetic rats. 40 male Wistar albino rats were divided into four groups: control, and sampled 4, 8 and 12 weeks after induction of diabetes by streptozotocin (STZ) intraperitoneal injection (40 mg/kg). The rats were decapitated under ketamine anesthesia and their testes were removed. Blood was obtained from heart and serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels were measured by ELISA. Tissue ghrelin and GHS-R mRNA levels were determined by qRT-PCR, while ghrelin protein expression was studied by immunohistochemistry. Histopathological damage scores were also assessed. Eight weeks after diabetes induction serum FSH level was increased, whereas LH and testosterone concentrations decreased. The ghrelin and GHS-R1a gene expression and ghrelin immunohistochemistry score first tended to increase after first four weeks of diabetes, and then tended to decrease. Ghrelin-immunopositive cells were detected in Leydig cells in all groups of rats, however, not in the germinal epithelium. Congestion of vessels and hemorrhage, formation of the vacuoles in spermatogonia and spermatocytes, desquamation of spermatids in the lumen and disorganization of seminiferous tubule germinal epithelium were observed in testis of all the diabetic rats. In addition, mean testicular biopsy score and mean seminiferous tubule diameter were getting lower in diabetic animals. Our results suggest that diabetes affects ghrelin expression in rat testis.

Acylated ghrelin is not required for the surge in pituitary growth hormone observed in pregnant mice

Objective: Ghrelin is produced by the stomach, hypothalamus and pituitary. It circulates as acylated ghrelin (AG, which stimulates growth hormone (GH) secretion) and unacylated ghrelin (UAG). Acylation is mediated by the enzyme ghrelin O-acyltransferase (GOAT). In mice, pregnancy is associated with a marked increase in circulating pituitary GH. We investigated the role of AG and UAG in the surge of plasma GH concentrations in pregnant mice at the end of pregnancy. Design: Using a mouse model generated on a C57BL/6 background (wild type, WT) in which the GOAT gene has been deleted (KO), we measured plasma AG, UAG and GH concentrations and tissue (stomach, pituitary and hypothalamus) preproghrelin and GOAT mRNA in non-pregnant (NP) and pregnant (P), WT and KO mice. Results: GOAT deletion was associated with undetectable concentrations of AG. UAG concentrations were similar in all groups. In both WT and KO animals, mean GH concentrations increased 30 to 50 times during pregnancy. There was a tendency toward lower median GH concentrations in KO (301ng/mL) compared to WT (428ng/mL) mice (p=0.059). Preproghrelin expression was not affected by GOAT deletion or by pregnancy in the stomach. In contrast, pituitary and hypothalamic ghrelin gene expression were lower in KO-NP and KO-P mice compared to their WT counterparts. Conclusion: The complete absence of ghrelin acylation, which is associated with undetectable AG concentrations, does not prevent the marked increase in pituitary GH concentrations observed in pregnant mice, suggesting that AG is not the major mediator of GH secretion during pregnancy.

Ghrelin gene expression in rats with ethanol-induced gastric ulcers: arole of melatonin.

The aim of the present work was to reveal the mechanisms of melatonin treatment on ethanol-induced gastric mucosal lesions in rats, including its role in the induction of ghrelin biosynthesis. Sixty male Wistar rats were divided into 3 groups (20 in each group): a) control group, b) ulcer group (100% ethanol was given intragastrically (i.g.) in a dose of 1 ml/100 g of body weight), and 3) melatonin-treated group, which received a single dose (25 mg/kg) of melatonin (Biovea) i.g. 30 min before ulcer induction with ethanol. Reduced glutathione (GSH) and malondialdehyde (MDA) were measured in tissues and ghrelin levels determined in the serum. RNA isolation and RT-PCR expression of ghrelin were performed. Both macroscopic and microscopic examinations of gastric mucosa were done in all groups. Significant decrease in ghrelin levels and mRNA expression and reduced levels of GSH were observed in ulcer group of rats in comparison with controls. All parameters studied were significantly increased after treatment with melatonin in comparison with ulcer bearing group of rats. On the other hand, the tissue levels of MDA were significantly increased in ulcer group of rats in comparison with controls and significantly decreased after melatonin treatment in comparison with the ulcer group of rats. Histological examinations revealed severe mucosal lesions induced by ethanol which were significantly improved by melatonin administration. The present data indicate that melatonin may have a potential impact in the treatment of peptic ulcer not only via its known antioxidant effect but also via induction of the ghrelin biosynthesis, as it was documented by significant increase in ghrelin mRNA expression.

Regulation of the Human Ghrelin Promoter Activity by Transcription Factors, NF-κB and Nkx2.2.

To examine the gene expression of ghrelin, a growth hormone releasing and appetite stimulating hormone from stomach, we constructed human ghrelin promoter-reporter vectors and analyzed the promoter activity. The ghrelin promoter activity was high when cultured cells that express ghrelin mRNA endogenously like TT or ECC10 cells were used, indicating that these cells contain factors necessary for full expression of the human ghrelin gene. The human ghrelin promoter contains both positive and negative regulatory regions. A transient decrease of the promoter activity was found when the reporter vector with the −1600 fragment of the human ghrelin promoter was transfected into cultured cells. We then examined the effect of several transcription factors on the ghrelin promoter activity and found that NF-κB suppressed and that Nkx2.2, a homeodomain-containing transcription factor that is important for ghrelin cell development in pancreas, activates the promoter activity. These transcription factors may be possible targets for the control of ghrelin gene expression.

The Effects of Long-Term Diabetes on Ghrelin Expression in Rat Stomachs.

Ghrelin is ahormone which has effects on the secretion of growth hormone, the gastrointestinal system, the cardiovascular system, cell proliferation and the reproductive system. This study focused on relative ghrelin and GHS-R1a gene expression in the stomach of long-term diabetic rats. Atotal of 36 male Wistar albino rats were divided into four groups: acontrol group, one-month diabetic rats, two-month diabetic rats and three-month diabetic rats. Diabetes was induced by streptozotocin STZ (40 mg/kg i.p). The rats were decapitated under ketamine anesthesia and their stomach tissues were removed. Tissue ghrelin expression, ghrelin and GHS-RmRNA levels were then compared using immunohistochemistry and qRT-PCR. After one month of diabetes, the number of ghrelin-immunopositive cells decreased significantly compared to those of the control rats. However, the ghrelin-immunopositive cells increased numerically in the two- and three-month diabetic rats compared to those of the control rats. It was also observed that there were high levels of ghrelin mRNA in the one- and two-month diabetic rats, and asubsequent decrease of ghrelin mRNA levels in the three-month diabetic rats compared to the control rats. However, ghrelin receptor mRNA expression levels decreased in the one-month diabetic rats, and ghrelin levels subsequently increased in the two- and three-month diabetic rats compared to the control rats. The two- and three-month diabetic rats became cachectic due to the large amount of weight lost. The authors therefore concluded that ghrelin-immunopositive cells increased in these rats due to their cachectic state.

Neonatal oxytocin administration and supplemental milk ameliorate the weaning transition and alter hormonal expression in the gastrointestinal tract in pigs

The aim of this study was to investigate the influences of milk supplementation during lactation, over 1 wk after weaning, and oxytocin administration for the first 14 d of life on the pigs' response to weaning. Pigs from 20 litters were allocated to each of these 3 treatments in a randomized factorial design. Oxytocin was administered subcutaneously daily from 0 to 14 d of age at a rate of 10 I.U. per kg. The milk supplement consisted of a mixture of 25% skim milk powder offered either during lactation between 10 and 20 d of age or for the first week after weaning as a transitional diet along with dry pellets. Pigs were weaned at 21 d of age. Growth rate was measured from birth to slaughter at 140 d of age and feed intake of supplemental milk or feed from 10 to 56 d of age. Organ weights (heart, liver, stomach, and kidneys) and the gene expression of ghrelin, leptin, and glucagon-like peptides (glucagon-like peptide-1 and glucagon-like peptide-2) were measured in the stomach, ileum, and duodenum at 10, 21, and 28 d of age. Milk supplementation after weaning resulted in immediate feed intake and partially alleviated the depression in growth rate over the first 7 d postweaning (P < 0.001), but milk supplementation during lactation had no effects (P > 0.1). However, effects were only transient and disappeared once the milk liquid diet was removed. Neonatal oxytocin administration reduced weight loss over the first 2 d after weaning (P = 0.03), without affecting feed intake (P > 0.1), hence possibly reducing weaning stress. Seven days after weaning, oxytocin-treated pigs had greater stomach ghrelin and leptin expression (both P = 0.02), and pigs supplemented with milk after weaning had greater stomach leptin and glucagon-like peptide-2 expression (P = 0.02 and P = 0.05, respectively). Hence, neonatal oxytocin administration or postweaning milk supplementation are both effective means of enhancing gastric leptin expression and reducing weight loss at weaning, likely improving gut health during this critical period.

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.

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.

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.

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.

High Lever Dietary Copper Promote Ghrelin Gene Expression in the Fundic Gland of Growing Pigs

This experiment was conducted to examine the effect of dietary copper supplementation on ghrelin mRNA expression level in the fundic gland of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs, five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5mg/kg copper (as the control group), 125mg/kg copper sulfate, or 125mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group, slaughtered, and collected the fundic gland for determination of ghrelin mRNA expression level. The results showed that average daily gain, average daily feed intake, absolute weight, serum growth hormone (GH) concentration, and ghrelin mRNA level were higher in pigs fed the diets with 125mg/kg copper methionine and 125mg/kg copper sulfate (P < 0.05), than in pigs fed a diet with 5mg/kg copper. These data suggest that high dietary copper (125mg/kg) appears to increase feed intake and promote weight gain by enhancing the secretion of GH and ghrelin mRNA level in growing pigs.

Cloning and prokaryotic expression of ghrelin gene in crucian carp (Carassius auratus)

To make up the flaw that there is no available information about ghrelin gene in crucian carp. The ghrelin gene was amplified by reverse transcription-PCR (RT-PCR) using total RNA extracted from intestine of crucian carp. PCR product was cloned into the pMD®19-T vector to construct pMD®19-T-ghrlein for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a and was transformed into hostEscherichia coli strain Rosetta for expression. In this study, 490 bp fragment of ghrelinwas obtained by RT-PCR. In comparison with other fishes, the amino acid sequences ofghrelin in crucian carp showed a high similarity to that of goldfish (99%). The high expression of ghrelin gene was detected in the intestine and liver by real-time PCR. IPTG at concentrations of 0, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mmol/L could efficiently induce the expression of pGh-32. The result showed that the optimal concentration of IPTG was 0.3 mmol/L by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The ghrelin gene expressed as early as 1 h after IPTG induction, and reached peak levels after 3 h. Successful expression of ghrelin fusion protein in prokaryotic cell could lay a basis for further study of industrial production. Key words: Crucian carp, ghrelin, cloning, prokaryotic expression.

202 Hydrogen Sulfide (H2S) Inhibits Bile Acid Receptor TGR5-Mediated GLP-1 Release From Enteroendrocrine Cells: Possible Involvement of H2S in Altered Glucose Metabolism in Diabetes

G A A b st ra ct s gene expression was lower in the Ift88ΔS mice. Despite the rapid induction of gastrin secretion, Ift88ΔS mice showed delayed induction of acid secretion with food intake, and lower mRNA levels of both H+K+ATPase α and β-subunits. In these mice, gastrin, ghrelin, and somatostatin gene expression did not change in response to food intake as in controls. At age 5 months, Ift88ΔS stomachs were histologically normal. However when analyzed at 8 months, mice developed gastric pathology associated with endocrine dysfunction including foveolar hyperplasia, hypergastrinemia, and hypochlorhydria. Epithelial deletion of Kif3a, another gene required for cilia formation, recapitulated the phenotypes observed in Ift88ΔS mice. Conclusion: Overall, our results demonstrated that gastric epithelial cilia regulate endocrine cell function and gastric homeostasis by sensing luminal food.

Mapping of Ghrelin Gene Expression and Cell Distribution in the Stomach of Morbidly Obese Patients—a Possible Guide for Efficient Sleeve Gastrectomy Construction

Ghrelin is secreted mainly in the stomach and plays a role in food intake regulation. Morbidly obese (MO) individuals report a decline in appetite after sleeve gastrectomy (SG), presumably due, in part, to ghrelin cell removal. Ghrelin cell distribution and expression were determined in three areas of resected stomach specimens from MO patients subjected to SG. Resected stomach specimens from 20 MO patients undergoing SG were analyzed. Real-time polymerase chain reaction of ghrelin mRNA and immunohistostaining for ghrelin cells in three stomach regions (fundus, body, and pre-antral areas) were performed. Body mass index (BMI) and total plasma ghrelin levels were obtained before and 3 months postoperatively. Ghrelin mRNA was detected throughout the stomach, its expression decreasing from the fundus towards the antrum. The relative quantification for ghrelin mRNA expression was 0.043, 0.026, and 0.015 at the fundus, body, and pre-antral region, respectively (P = 0.05, fundus vs. pre-antral region). Average ghrelin cell counts declined from 60 ± 40 to 45 ± 20 and 39 ± 13 cells/high power fields in the fundus, body, and pre-antral region, respectively. Three months after surgery, total plasma ghrelin levels decreased from 1,676 ± 470 to 1,179 ± 188 pg/ml (P < 0.00001) and BMI dropped from 46 ± 6 to 38 ± 5 kg/m2 (P < 0.00001). Distribution and expression of ghrelin-secreting cells throughout the stomach were defined, emphasizing the importance of meticulous resection of the fundus during SG for maximal ghrelin cell removal.

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin–GHSR1a system is activated by glucocorticoids in the hypothalamic cells.