Role of the Energy Sensor AMP-Activated Protein Kinase (AMPK) and Its Downstream Effector Uncoupling Protein 2 (UCP2) in the Orexigenic Effect of Endogenous Ghrelin

after a 14 hour fast. Aims: Determine the signalling pathway used by leptin on gastric vagal afferents in mice fed ad libitum or fasted for 14 hours. Methods: In female C57/BL6 mice (n= 25/group) single fibre recordings of vagal mechanoreceptors were obtained (J Neurophysiol, 2002: 87; 2095) before and after application of antagonists to suspected second messenger molecules including janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), phosphodiesterase 3 (PDE3), phospholipase C (PLC), canonical transient receptor potential channel (TRPC) and large conductance calcium-activated potassium channels (BKCa). It was then determined if these antagonists could prevent the inhibitory or excitatory effect of leptin on vagal afferents. Results: In fed mice, leptin (10nM) increased the response of mucosal receptors to mucosal stroking with calibrated von Frey hairs (10-1000mg; P<0.001). This effect was blocked in the presence of the JAK2 inhibitor AG490 (5μM), the PI3K inhibitor wortmannin (5nM), the PDE3 inhibitor cilostamide (5μM), the PLC inhibitor U73122 (10μM) or the TRPC channel blocker 2-APB (10μM) applied individually. This suggests that leptin activates this series of signalling molecules to in turn activate an effector channel in TRPC to cause sensitization of the afferent ending. In fasted mice, leptin (10nM) reduced the response of tension receptors to circular tension (1-5g; P<0.001). This effect was blocked by AG490 (5μM) and wortmannin (5nM), but was unaffected by cilostamide (5μM), U73122 (10μM) or 2-APB (10μM) indicating a divergence from the second messenger system observed in gastric mucosal receptors. The BKCa channel inhibitor, iberiotoxin (100nM), inhibited the effect of leptin on tension receptors in fasted mice, identifying the BKCa channel as the effector channel for leptin's effect in tension receptors. Conclusion: Leptin has a dynamic effect on gastric vagal afferent mechanosensitivity which is changed under food deprivation. The dominant second messenger pathway used by leptin not only depends on food intake but also specific gastric mechanosensitive subclasses. These changes in the effect of leptin are consistent with an increase in drive to consume food under food deprivation conditions. Supported by University of Adelaide and NHMRC Australia

Role of the Energy Sensor AMP-Activated Protein Kinase (AMPK) and Its Downstream Effector Uncoupling Protein 2 (UCP2) in the Orexigenic Effect of Endogenous Ghrelin Pieter-Jan Verhulst, Sara Janssen, Jan F. Tack, Inge Depoortere Background. Ghrelin released from the stomach, stimulates food intake through stimulation of neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the hypothalamus. Several studies proposed a pivotal role for the energy sensor AMPK and UCP2 in ghrelin's effects on NPY/AgRP expression and food intake stimulation, although most of these studies focused on the effects of exogenous ghrelin. Aim. To investigate whether a rise in endogenous ghrelin levels is able to influence NPY/AgRP expression via activation of AMPK activity and UCP2 expression. Methods. Endogenous ghrelin levels were increased in wildtype (GHS-R+/+) and ghrelin receptor knockout (GHS-R-/-) mice by fasting (24h) or by induction of streptozotocin (STZ)-diabetes (15 days). Plasma octanoylated ghrelin levels were determined by radioimmunoassay. The mRNA expression of AgRP, NPY and UCP2 in the hypothalamus was measured by real-time PCR. Hypothalamic AMPK activity in tissue lysates was measured with an immunoprecipitation kinase assay. Results. Octanoylated ghrelin levels peaked (4.5fold, P<0.01) after 24h of fasting and declined thereafter due to a decrease (1.7-fold, P<0.01) in the mRNA expression of ghrelin-O-acyl transferase (GOAT). GHS-R+/+ mice showed a significant increase in AgRP mRNA (from 0.33±0.03 to 1.03±0.09; P<0.001) and NPY mRNA (from 0.54 ± 0.08 to 0.88 ± 0.05; P<0.01) expression after 24h-fasting. In GHS-R-/- mice no significant increase in AgRP and NPY mRNA expression was observed. Fasting did not affect AMPK activity in both genotypes but increased UCP2 mRNA expression (GHS-R+/+: from 0.35±0.05 to 0.50±0.04; P<0.05 and GHS-R-/-: from 0.41±0.04 to 0.55±0.08; P=0.058). The hyperghrelinemia associated with the induction of STZ-diabetes was accompanied by a significant increase in the expression of NPY and AgRP in GHS-R+/+ mice compared to nondiabetic controls (AgRP: from 0.22±0.12 to 2.74±0.45; P<0.001 and NPY: from 0.73±0.06 to 2.23±0.64; P<0.05) but not in GHS-R-/- mice. AMPK activity in the hypothalamus of GHSR+/+ mice after induction of diabetes (20.2±1.6 pmol/min/mg) was decreased (P<0.05) compared to non-diabetic littermates (16.3±1.3 pmol/min/mg) and there was no genotypic difference. Similarly, UCP2 mRNA levels were decreased after the induction of STZ-diabetes compared to control mice in both genotypes (GHS-R+/+: from 0.68±0.02 to 0.26±0.03; P<0.001 and GHS-R-/-: from 0.61±0.05 to 0.28±0.05; P<0.01). Conclusion. Increasing endogenous ghrelin levels by fasting and by induction of diabetes stimulates the expression of AgRP and NPY in the hypothalamus via interaction with the GHS-R. The change in AMPK activity and its downstream effector, UCP2, accompanying these changes in endogenous ghrelin levels occur independently from the GHS-R suggesting that AMPK and UCP2 do not play a major role in the orexigenic effect of endogenous ghrelin.

197 Suboptimal Gastroprotective Coverage of NSAID Use and the Risk of Upper Gastrointestinal Complications Eva M. van Soest, Vera E. Valkhoff, Giampiero Mazzaglia, Rene Schade, Mariam Molokhia, Jay L. Goldstein, Sonia Hernandez-Diaz, Gianluca Trifiro, Jeanne Dieleman, Ernst J. Kuipers, Miriam C. Sturkenboom Background: Non-steroidal anti-inflammatory drugs (NSAIDs) have known associated upper gastrointestinal (UGI) risks . Gastro-protective agents (GPAs) are co-prescribed to lower the risk of UGI complications, but patient adherence is suboptimal. Aim: To study the association between GPA adherence and UGI complications in non-selective (ns)NSAID users ≥ 50 yrs. Methods: A multi-database nested case-control study was conducted using primary care data from the General Practice Research Database (United Kingdom (UK), 1998-2008), the Integrated Primary Care Information database (The Netherlands (NL), 1996-2007) and the Health Search Database (Italy (IT), 2000-2007). The study was nested within a cohort of new nsNSAID users ≥ 50 yrs. Episodes of nsNSAID use were defined as a period of nsNSAID use with gaps between subsequent prescriptions that were no longer than the duration of the previous prescription. Patients could have more than one episode of nsNSAID use during follow-up, but only if at least a 180-day NSAID-free period was present prior to the start of each episode. Cases with UGI complications (UGI bleeding and (un)complicated UGI ulcers) during or within 60 days after nsNSAID use were matched to controls on age, gender, database, and calendar time. Adherence to GPAs was calculated over the most recent episode of nsNSAID use prior to the index date as the proportion of NSAID treatment days covered (PDC) by a GPA prescription. Adjusted odds ratios (OR) with 95% confidence intervals (95%CI) were calculated using multivariate conditional logistic regression analysis. Results: 1,107,266 nsNSAID episodes were counted in the nsNSAID user cohort (UK: 647,615; NL: 78,853, IT: 380,798). In 117,307 (10.6%) of the nsNSAID episodes a GPA was prescribed (UK: 11.6%; NL: 7.8%, IT: 9.5%). Of the GPA users, 4.9% had a PDC ratio <20% (low adherence), 27.0% had a PDC ratio between 20-80% (partial adherence) and 68.1% had a PDC ratio >80% (high adherence). Mean PDC was 0.81±0.28. Twenty-one percent used nsNSAIDs >30 days. In those episodes, mean adherence dropped to 0.66±0.32. Within the nsNSAID+GPA users, we identified 339 cases with an UGI complication (187 UGI bleedings and 152 (un)complicated UGI ulcers). For patients with low adherence, the risk was 1.89 (95%CI 1.09-3.28) for UGI bleeding alone and 2.39 (95%CI 1.66-3.44) for all UGI complications, compared to patients with high adherence. For every 10% decrease in adherence, the risk of UGI bleeding increased by 6% (OR: 1.06, 95%CI: 1.01-1.12) and the risk of all UGI complications increased by 9% (OR 1.09, 95%CI: 1.05-1.13). Conclusions: The risk of UGI complications in nsNSAID users significantly depends on the level of GPA adherence. This suggests that improvement of GPA adherence could be beneficial in reducing nsNSAIDrelated UGI complications.

195 Bitter Taste Receptors (T2Rs) Expression and Regulation in the Gastrointestinal Tract in Mice Gaia Vegezzi, Laura Anselmi, Elisabetta Barocelli, Enrique Rozengurt, Helen Raybould, Catia Sternini Background and aim: Bitter taste receptors (T2Rs) are a large family of GPCRs that perceive bitter taste in the mouth. T2Rs and their signaling molecules that transmit bitter taste are also expressed in a variety of other organs, including the gastrointestinal (GI) tract mucosa. Taste signaling molecules are found in enteroendocrine cells producing peptides that are involved in food intake and GI function. The aim of this study was to evaluate the distribution of two T2Rs subtypes, T2R138, the preferred receptor for phenylthiocarbamide (PTC) and phenylthiourea (PTU), and T2R108, activated by denatonium benzoate (DB), in the mouse GI tract, and the effect of fasting/re-feeding and different diets on their expression. Methods: Fasting/re-feeding experiments: male C57BL mice were fasted for 18 hours and re-fed 2 or 4 hours later. Cholesterol-lowering diet: mice were fed for 7 days with chow supplemented with Lovastatin (100 mg/100 mg chow w/w) and Ezetimibe (21 mg/100 mg chow w/w). High fat diet: Mice were fed high fat diet (60% fat by calories) or low fat diet for 14 days. Specimens of the GI tract were collected for qRT-PCR analysis.Results: T2R138 and T2R108 mRNAs were expressed along the entire GI tract; T2R138 transcript was significantly more abundant in the duodenum and distal colon, and T2R108 transcript was more abundant in the stomach. Both transcripts were abundant in STC-1 cells, the enteroendocrine cell line used as positive control, and absent in 3T3 fibroblast cells, which served as negative control. T2R138 mRNA levels were significantly reduced by fasting in the antrum only (0.07±0.03 vs. 1.59±0.39, p<0.05) and restored to higher than normal levels after 4 hrs re-feeding. T2R108 mRNA levels were also reduced only in the antrum of fasted animals and significantly increased 4 hrs after re-feeding. Cholesterol-lowering diet resulted in a significant increase in T2R138 mRNA levels in the duodenum (1.26±0.26 vs 2.55±0.37 chow vs. Cholesterol lowering, p<0.05), but not in other regions of the small intestine or colon; there were no changes in T2R108 mRNA in any region. Finally, a high fat diet resulted in increased T2R138, but not T2R108 mRNA in the large intestine.Conclusions: These data indicate that T2Rs subtypes with different ligand selectivity are differentially expressed and regulated in the GI tract. These observations further support a functional role of T2Rs in chemosensing in the GI tract, where they might serve as physiological regulators of diet-induced responses. Supported by NIH grants DK79155 and DK41301

198 What Role Does Surgery Play in the Modern Day Management of Non-Variceal Upper Gastrointestinal Bleeding? Vipul Jairath, Richard F. Logan, Sarah Hearnshaw, Brennan Kahan, Simon Travis, Michael F. Murphy, Kelvin Palmer Introduction: Combinations of endoscopic, pharmacological and radiological intervention appears to have reduced the need for surgery in non-variceal upper gastrointestinal bleeding (NVUGIB), for what was once a condition primarily managed by surgeons. The place of surgery in today's management of NVUGIB remains poorly characterized. Methods: We examined the clinical characteristics, sequence of intervention and outcomes of NVUGIB in a nationwide study, by analyzing patients coming to surgery in the 2007 national audit of Acute Upper Gastrointestinal Bleeding(1). Results: The study recruited 6,750 patients. Only 1.9% (127/6750) underwent surgery. The mean age was 69.4 (sd 15) years, 60% (75/127) male, with median of 1 co-morbid illnesses. Indications for surgery were uncontrolled bleeding/high risk stigmata of hemorrhage in 82% (104/127), peritonitis/perforation in 12% (15/127), malignancy in 4% (5/127), and other indications in 9% (12/127). Surgical procedures were oversew or under-run of an ulcer in 65% (83/127), partial gastrectomy in 9% (12/127), excision of an ulcer with vagotomy/pyloroplasty (2%) and other procedure

196 The “Leptin Switch” Mechanism in Gastric Vagal Afferents Stephen Kentish, Tracey A. O'Donnell, Gary Wittert, L. Ashley Blackshaw, Amanda J. Page Background: We have shown that leptin potentiates mechanosensory function of gastric mucosal vagal afferents, but after a 14 hour fast inhibits mechanosensory function of gastric tension receptors (Gastroenterology 2008: 134(4); A-97). In addition to the activation of the JAK/STAT pathway leptin receptors have been associated with numerous other intracellular pathways. Activation of different pathways may explain the switch in effect of leptin observed

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AGA Abstracts

AGA Abstracts

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