Shino Ohno

Urocortin 1 reduces food intake and ghrelin secretion via CRF2 receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.

Su1253 Abnormal Dynamics of Endogenous Acylated Ghrelin Mediates Decreases in Gastric Phase III-Like Contraction, Leading to Delayed Gastric Emptying in Urocortin1-Induced Stress Rats

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

Mo2114 Enhancement of Ghrelin Secretion Is a Potential New Therapy Against Stress-Induced Functional Abnormality of the Upper Gastrointestinal Tract

G A A b st ra ct s higher stages of fibrosis (r=0.16, p,0.03). The levels of the apoptosis biomarker M30 also correlated with both the presence of histologic NASH (r=0.28, p,0.00016) and the presence of fibrosis (r=0.36, p,1.07e-6), but not with BMI. The levels of PTH were positively correlated with BMI (r=0.23, p,0.003) and negatively correlated with the levels of ALT (r=-0.18, p,0.025) and triglycerides (r=-0.213, p,0.01). The levels of the Vitamin D binding protein showed a positive correlation with the levels of triglycerides (r=0.336, p,0.0012) and a negative correlation with the degree of histologic hepatocyte ballooning (r=-0.231, p,0.02) in the liver biopsies. In addition, both Vitamin D levels (p=0.045) and Vitamin D binding protein levels (p=0.019) were significantly lower in the non-Caucasian group (n=135) within the cohort. Conclusions: The hormonal regulation of Vitamin D metabolism is substantially modified in very obese patients with NAFLD who are undergoing bariatric surgery. Further research is needed to elucidate the roles of individual components of the Vitamin D metabolic pathway and their molecular targets in morbidly obese patients with NAFLD.

Sa1461 Serotonin 2C Receptor Antagonism is a Potential New Candidate Treatment for Stress-Related Anorexia

Background/Aims: Stress is well known to suppress appetite, ultimately leading to chronic eating disorders that may require medical treatment. However, no treatment exists for stressinduced loss of appetite, because its detailed mechanism remains unknown. Serotonin regulates emotions and food intake, and serotonin 2c receptor (5-HT2cR) is known to control feeding behavior. In this study, we used an urocortin 1 (UCN)-induced stress model to evaluate the effects of central 5-HT2cR and changes in the expression of c-fos mRNA. 5-HT2bR, another serotonin receptor that controls food intake, was also evaluated, and the effects of both receptor antagonists on loss of appetite were compared to investigate the potential of 5-HT2cR antagonist drugs for anorexia treatment. Methods: Intraventricular (ICV) injection of UCN or phosphate-buffered saline was administered to Sprague-Dawley rats, and the brains were collected after perfusion fixation, and fixed whole brains were cut in the coronal plane. The mRNA expression of 5-HT2cR and c-fos was evaluated by in situ hybridization. In addition, double staining of 5-HT2cR was performed for a sample section in which an increased level of c-fos mRNA expression was observed. The selective 5-HT2cR antagonist SB242084, the selective 5-HT2bR antagonist SB215505 (intraperitoneal), or rikkunshito (oral), which shows properties of both 5-HT2cR and 5-HT2bR antagonism, was administered and their respective effects on food intake decreases were evaluated in order to clarify the role of 5-HT2cR activation on stress-induced anorexia. Results: The 5-HT2cR mRNA expression in rats under UCN-induced stress was increased primarily in the solitary tract nucleus (NTS) of the medulla oblongata and in the paraventricular nucleus (PVN) of the hypothalamus. Concurrent enhancement of c-fos mRNA expression was observed. Expression of 5-HT2cR mRNA in the arcuate nucleus was approximately the same in both stress-induced and non-stress-induced groups. Double staining revealed that 5-HT2cR and c-fos were expressed in the same cells in the solitary tract nucleus and PVN. Intraperitoneal administration of 5-HT2cR antagonist SB242084 and oral administration of rikkunshito significantly inhibited food intake decreases in rats exposed to UCN-induced stress; in contrast, administration of the 5-HT2bR antagonist SB215505 did not affect food intake. Conclusion: 5-HT2cR antagonism is a potential candidate for the treatment of stressrelated anorexia. When the corticotrophin-releasing factor (CRF) receptor is activated, stress responses may be enhanced by increased responsiveness of 5-HT2cR on CRF neurons in the PVN. Signals from the vagus nerve may increase responsiveness of 5-HT2cR in the NTS, leading to further negative regulation of food intake.

Inhibition of Gastric Ghrelin Secretion by Urocortin 1

G A A b st ra ct s cortex (pcc), mediodorsal thalamus (mdThal), M1, and vermis. In the rectal distention studies, results were found in the right and left aIns, left pIns, posterior thalamus (pulvinar), rOFC , M1, ACC, and PCC . Contrasting gastric with rectal distention demonstrated that gastric distention produced greater activation in the hippocampus (Hip), mdThal and cerebellar vermis. Conclusion: Brain activity in response to both rectal and gastric distention was shown in regions typically associated with emotional (amygdala, ACC, PCC, OFC), decision making (dlpfc, ofc, hippocampus), motor action (M1), and visceral sensation (insula, thalamus). However, gastric distention may induce greater activity within limbic (mdThal, Hip) and cerebellar emotional (vermis) circuitries. The greater brain response to gastric stimulation may have been due to distinct peripheral innervation pathways, as well as the different emotional valences placed on epigastric versus lower GI sensations. Future studies should investigate potential differences in functional GI disease populations, and compare these results to other GI related sensations such as nausea.

W1364 Role of Hypothalamic Ghs-R1a on Appetite Loss During Chemotherapy

Background/Aim: Because ghrelin is a strong orexigenic peptide, targeted depletion of the ghrelin receptor is expected to have a significant effect on feeding behavior. Although recent loss-of-function studies using GHS-R1a knockout animals have raised questions regarding the physiological significance of ghrelin on feeding, the roles of central GHS-R1a in anorexia models have not been investigated. To clarify that the gut-brain axis is associated with the regulation of appetite and the hypothalamic ghrelin signal, we investigated hypothalamic GHS-R1a gene expression and studied chemotherapy-induced dyspepsia in rats. Methods: We first examined the effects of an intracerebroventricular (ICV) injection of exogenous ghrelin on food intake with or without cisplatin treatment, as well as those of 48-h fasting and cisplatin or a 5-HT receptor agonist treatment on hypothalamic GHS-R1a mRNA expression. To identify the mechanism of the cisplatin-induced decrease in hypothalamic GHSR1a mRNA expression, we evaluated the effects of SB242084, a 5-HT2C receptor antagonist, and Rikkunshito (RKT), a ghrelin receptor activator (Endocrinology, 151, 2009) on hypothalamic GHS-R1a gene expression, as well as the effect of coadministration of a GHS-R1a antagonist on decreased food intake. Results: Compared to vehicle controls, ICV ghrelininjected rats (2 nmol/head) that were treated with cisplatin failed to reduce the decrease in food intake. Hypothalamic GHS-R1a gene expression was significantly enhanced during the 48-h fasting but was reduced after treatment with cisplatin or mCPP, a 5-HT2C receptor agonist (saline; 1.0 ± 0.08 vs. mCPP; 0.77 ± 0.06 relative mRNA, p = 0.0157, Steels test), but not with 1B/2B receptor agonist treatment. The induced decrease was reversed by a 5HT2C receptor antagonist (cisplatin plus saline; 0.83 ± 0.06, and vs. relative mRNA level in cisplatin plus SB242084HCl, 1.2 ± 0.06, p = 0.026) or RKT (0.71 ± 0.06; cisplatin plus 500 mg/kg, 1.12 ± 0.09, p = 0.009; cisplatin plus 1000 mg/kg, 1.06 ± 0.09, p = 0.03), but not by granisetron or ondansetron, which are 5-HT3 receptor antagonists. The suppressive effect of cisplatin and mCPP on the decrease in food intake was abolished by coadministration of the GHS-R1a antagonist. Conclusion: Cisplatin-induced anorexia may worsen because of decreased hypothalamic GHS-R1a gene expression. Activation of the cerebral 5-HT2C receptor partially contributed to the decrease in GHS-R1a gene expression after cisplatin treatment.

W2052 Urocortin Reduces Food Intake via Inhibition of Ghrelin Secretion in Rats

Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomach region. However, to date there have been few studies on the gastric contractility of the human stomach. Objectives: The aim of the present study was therefore to examine the response to acetylcholine and electrical field stimulation (EFS) of the human fundus, corpus and antrum. Methods: Gastric muscles were obtained from 71 patients (44 males, 27 females with an average age of 60.7 yrs; 34 85 yrs) undergoing gastric cancer surgery. Muscle strips (n = 71) were isolated and attached to a fixed mount and to an isometric strain gauge. The muscle strips were studied In Vitro for their contractile responses to acetylcholine (n=25) and electrical field stimulation (EFS, n=46). N-nitro-L-arginine (L-NA), atropine and tetrodotoxin (TTX) were added to assess the nitro oxide-mediated changes and cholinergic neural pathway involved in EFS-induced contractions. Results: Spontaneous contractile activity was observed in muscle strips of stomach regions. In the antrum, acetylcholine induced increase in contraction frequency and peak contraction in dose dependent pattern, but no significant change was noticed in muscle tone. In corpus and fundus, acetylcholine induced dose-dependent increase in peak contraction and muscle tone, but there was no significant change in frequency. The response to EFS also differed by stomach region. EFS produced no significant change in contractile activity in 22 of 46 muscle strips. When EFS evoked frequency-dependent contraction or relaxation in muscle strips, contraction was primarily observed in the muscle strips from the antrum, and relaxation was primarily observed in the muscle strips of the corpus and fundus. The addition of LNA (100 uM) to the muscle bath converted the EFS-induced relaxation to contractions and increased the EFS-induced contractions. EFS-induced contractions were abolished by atropine (1uM) or TTX (1uM). Conclusions: Our study demonstrated that the response to acetylcholine and electrical field stimulation on human gastric fundus and corpus is different with that of human gastric antrum.