- Email: [email protected]
Sa2068 Prevention of Fructose-Induced Obesity by Intestinal Alkaline Phosphatase
volumes (95% CI). More women had abnormal WLT compared with men (70.5% vs 29.5%, p,0.01). Tachygastria was the most common dysrhythmia evoked by the WLT, and patients with normal or abnormal WLT had similar incidences of gastric dysrhythmias overall (73.5% vs 71.1). However, patients with abnormal WLT are more likely to have tachygastria with a normal GES than with abnormal gastric emptying (64% vs. 49%, p=0.04). Additionally, 63% of the patients with abnormal WLTs reported increased nausea compared with 42% of patients who had normal WLTs (p=0.028). The incidence of delayed gastric emptying was similar in the two WLT groups (43% vs 40%). Conclusion: 1. Ingestion of a normal or abnormal water load test volume evoked gastric dysrhythmias in 70% of these patients. 2. Patients who ingested low WLT volumes are more likely to have tachygastria with normal gastric emptying. 3. Patients who ingested abnormally low volumes during the WLT experienced increased nausea, suggesting hypersensitivity to gastric distention. 4. Increased nausea and the onset of gastric dysrhythmias after the WLT indicates the stomach is the site of origin of nausea in these patients with or without gastroparesis. Sa2067 Obese Patients Have Higher Circulating Levels of Dipeptidyl Peptidase IV That May Impact on Satiety Signaling Under These Conditions Andreas Stengel, Tobias Hofmann, Anne Ahnis, Miriam Goebel-Stengel, Peter Kobelt, Burghard F. Klapp Background: Dipeptidyl peptidase IV (DPP IV) is a serine protease with broad distribution in tissue and circulation known to be involved in various homeostatic processes such as immune defense, psychoneuroendocrine functions as well as nutrition. Despite the fact that DPP IV levels have been investigated in patients with hyporectic disorders, the levels under conditions of obesity, a major health hazard in industrialized countries around the world, are not well studied. Aims: To investigate DPP IV concentration and total enzyme activity in obese subjects and a possible dependency on body weight across a broad range of body mass index (BMI). Methods: Obese (BMI .30 kg/m2, n=4 women and 5 men, age 46.2 ± 4.4 years), anorexic (BMI ,17.5 kg/m2, n=9 women, age 25.4 ± 2.7 years) and normal weight (BMI 19-25 kg/m2, n=4 women and 5 men, age 48.4 ± 3.8 years) hospitalized patients were recruited from the Division of Psychosomatic Medicine and gave informed consent to participate in the study. Blood was withdrawn from overnight fasted subjects between 07:00 and 08:00 am, plasma processed for Western blot, stained with an antihuman-DPP IV polyclonal antibody and semiquantitative analysis of pixel intensity was performed. DPP IV activity in serum was assessed using Gly-Pro p-nitroanilide as substrate. Results: DPP IV protein expression was detected in human plasma as indicated by a strong band at the expected size of 110 kDa. In normal weight patients (mean BMI 22.7 ± 0.8 kg/ m2) the intensity of the 110 kDa band was 9947.6 ± 1724.4. The expression was not different in anorexic patients (mean BMI 12.6 ± 0.6 kg/m2, intensity: 10418.5 ± 1348.8, P.0.05), while it was higher in the plasma of obese patients (mean BMI 68.9 ± 2.7 kg/m2, intensity: 21208.6 ± 1336.6, P,0.001). Plasma DPP IV protein expression showed a significant positive correlation with BMI (r=0.64, P ,0.001). Serum activity of DPP IV did not differ between normal weight (66.5 ± 4.7 U/l), anorexic (71.9 ± 5.0 U/l) and obese patients (69.1±5.5 U/l, P.0.05) and showed no correlation with BMI (r=0.05, P .0.05). The concentration/activity ratio was not different in anorexic patients (153.1 ± 27.5) compared to normal weight controls (153.5 ± 38.1, P .0.05) but was higher in obese (305.7 ± 34.9, P,0.05) and showed a positive correlation with BMI (r=0.63, P ,0.001). Conclusions: DPP IV protein levels, unlike serum activity, depend on the metabolic condition with increased levels in morbidly obese patients resulting in an increased concentration/activity ratio. Since DPP IV is involved in the deactivation of food intake inhibitory hormones such as glucagonlike peptide-1 these alterations of the concentration/activity ratio may reduce anorexigenic signaling under conditions of obesity and therefore contribute to the development or maintenance of a greatly elevated BMI.
Sa2065 Purinergic Pathway Is the Major Mechanism of Inhibitory Effect of Motilitone on the Rat Gastric Fundus Relaxation Yang Won Min, Kyu Choi, Eun-ju Ko, Ji Yeon Lee, Eun Ran Kim, Byung-Hoon Min, Jun Haeng Lee, Jae J Kim, Jong Chul Rhee, Sang D. Koh, Poong-Lyul Rhee Background/Aims: Motilitone, a new prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, has strong prokinetic effects. In a previous animal study, motilitone significantly improved gastric accommodation by increasing the postprandial gastric volume. In this study, we investigated how motilitone affects the muscle relaxation in the rat gastric fundus. Methods: Gastric fundus smooth muscle strips (12 longitudinal muscles and 9 circular muscles) were obtained from rats. Isometric force measurements were performed by using both longitudinal and circular muscle strips in response to the electrical field stimulation (EFS, 0.3 ms in trains of 5 Hz for 10 s, 150 V). Peak (the highest value) and nadir (the lowest value) were observed during the first 1 minute after the initiation of EFS in the control state and after administration of atropine (1 μM), motilitone (5 μg, 10 μg, 50 μg, and 100 μg), N-nitro-L-arginine (L-NNA, 100 μM), and MRS 2500 (1 μM), which were added in sequential order to the organ bath. Then tetrodotoxin (TTX, 1 μM) was administered to inhibit nerve-mediated relaxation. Results: EFS induced contractions in the control state, which were relaxed by atropine in the both longitudinal and circular muscles. When motilitone was added to the atropine group, the nadir dropped in a dose-dependent manner in the both longitudinal and circular muscles. The nadir did not increase significantly when L-NNA was added to the atropine and motilitone (100 μg) group in the both longitudinal and circular muscles. After addition of MRS 2500, the nadir increased to the level of atropine group (before administration of motilitone) in the circular muscles. In the longitudinal muscles, the nadir also increased but was not fully reversed to the level of atropine group after addition of MRS 2500. Addition of TTX increased the nadir further and completely abolished EFS-induced relaxation in the both longitudinal and circular muscles. However, there were no significant changes in the peak after administration of motilitone regardless of its dosage. Conclusion: Our data suggest that the inhibitory effect of motilitone on the rat gastric relaxation is largely mediated by purinergic rather than nitrergic pathway, and non-nitrergic non-purinergic component also appears to be involved in the EFS-induced relaxation in the presence of motilitone.
Sa2068 Prevention of Fructose-Induced Obesity by Intestinal Alkaline Phosphatase Konstantinos P. Economopoulos, Kanakaraju Kaliannan, Nur Muhammad, Abeba Teshager, Sulaiman R. Hamarneh, Mussa Mohamed, Palak Patel, Qingsong Tao, Seyed M. Abtahi, Madhu S. Malo, Richard A. Hodin Introduction: The average consumption of fructose per capita in the U.S. has increased by approximately 200% over the past 30 years, mainly due to the dramatic increase in the consumption of fructose-sweetened beverages. It has been proposed that chronic fructose ingestion leads to intestinal bacterial dysbiosis and increased gut permeability. Intestinal Alkaline Phosphatase (IAP) is a brush border enzyme known to preserve intestinal epithelial barrier function and maintain the normal gut microbial homeostasis. Our aim in this study was to examine if IAP could prevent fructose-induced obesity and its deleterious effects. Methods: Three groups of eight-week old, C57BL/6 male mice (n=5) with unrestricted access to regular chow diet were administered either regular autoclaved water, or water enriched with 30% of fructose ± oral IAP (50 units/ml drinking water) for 16 weeks. Body weight and food intake were monitored weekly. The effects of IAP on serum glucose, intestinal permeability and normal flora were assessed. Data were expressed as mean ± standard error and were analyzed by one-way analysis of variance with Tukey's multiple comparison posttests. A significant difference was considered when p ,0.05. Results: The mice that received fructose gained significantly more weight compared to the controls (14.4±1.93 vs 7.2±0.35 g; p=0.016). Food intake was similar between mice receiving fructose+IAP and fructose alone (p=0.897). Fructose+IAP compared with fructose alone was associated with significantly less weight gain (8.3±1.81 vs 14.4±1.93 g; p=0.004), less visceral (1.1±0.10 vs 1.9±0.32 g; p=0.032) and subcutaneous fat weight (1.4±0.15 vs 2.4±0.26 g; p=0.011), lower pancreas weight (0.16±0.01 vs 0.22±0.02 g; p=0.022), lower fasting blood glucose levels (130±6.92 vs 181±15.71 mg/dl; p=0.017), lower area under the curve during glucose tolerance test (21,210±771.57 vs 24,394.5±668.72; p=0.011) and decreased intestinal permeability (0.3±0.08 vs 1.3±0.35; p=0.032). Mice receiving fructose+IAP compared to those receiving only fructose had increased number of aerobic bacteria (46.1E+05±9.75E+05 vs 4.48E+05±0.75E+05; p=0.001) and decreased number of gram negative bacteria (80±58.3 vs 2.77E+04±0.69E+04; p=0.001) in their stool. Conclusions: Oral supplementation with
Sa2066 The Provocative Water Load Test Evokes Nausea and Gastric Dysrhythmias in Patients With Unexplained Chronic Nausea and Vomiting Marcum Gillis, Kenneth L. Koch Background: The origins of nausea in patients with chronic nausea and vomiting syndromes can be difficult to elucidate. Ingestion of solids or liquids frequently precipitates symptomatic fullness and nausea. Aims: 1. To determine the incidence of abnormal water load tests (WLT) in patients with unexplained nausea and vomiting. 2. To determine the incidence of gastric dysrhythmias and severity of nausea symptoms evoked by the WLT. 3. To determine the relationship of water load volume ingested, gastric dysrhythmias, and gastric emptying results. Methods: Patients from Wake Forest Baptist Medical Center Gastroenterology Clinics who had an electrogastrogram (EGG) with WLT and solid phase gastric emptying studies were identified retrospectively. Baseline EGG recordings before and 10, 20, and 30 minutes after the WLT were evaluated. Patients drank water until they were "full" over a 5-minute period. The total volume of water ingested and visual analog scales for nausea, bloating, fullness, and hunger were completed at the time points above and overall EGG diagnoses were determined. An abnormal WLT volume was defined as less than 550 ml ingested in 5 minutes. Chi square analyses were used for statistical significance. Results: 113 patients were identified (72 women, 41 men, average age 50 years). 68/113 (60%) of patients had abnormal WLTs (average volume ingested 360 ± 29 ml) vs 702 ± 27 ml for normal WLT
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AGA Abstracts
AGA Abstracts
PGFS, and PGIS leading to the production of PGD, PGE2, PGF2 α, and PGI, respectively. Previous studies found that mechanical stretch in bowel obstruction markedly induced gene expression of COX-2, but not COX-1, in colonic smooth muscle cells (SMCs). The aims of the present study were to determine whether genes encoding individual prostaglandin synthases are mechanically responsive, and whether the composition of prostaglandins resulting from obstruction differs from that in inflammation. Methods: Partial colon obstruction was induced with a silicon band implanted surgically in the distal colon of male SpragueDawley rats. Static mechanical stretch (18% elongation) was mimicked in vitro in the culture of rat colonic circular SMCs with a Flexercell FX-4000 System. Rat colon inflammation was induced by manipulation of the colonic surface with wet cotton applicators. Results: 1. Quantitative RT-PCR found that the expression of PGDS and PGIS genes was not significantly changed on day 3 of obstruction. However, the membrane bound PGES-1 (mPGES-1) mRNA was significantly up-regulated in the obstructed segment, whereas mRNAs for other two types of PGES, cytosolic PGES and mPGES-2, were not changed. On the contrary, the PGFS mRNA was down-regulated in the obstructed colon. 2. PGE2, but not PGF2 α, was significantly increased in the colonic muscularis externae in obstruction. The PGE2 level increased to 4577±1249 pg/mg at day 3 of obstruction, compared to 1264±259 in sham (n=6, p,0.05). The PGF2 α level was 5718±910 pg /mg in obstruction compared to 6550±2178 pg/mg in the sham (n=5, p .0.05). 3. Direct stretch of rat colonic circular SMCs in vitro significantly up-regulated the mPGES-1 mRNA, but down-regulated that of PGFS. After stretch for 3 hrs, the mRNA levels of PGES and PGFS changed by 2.61±0.42 fold and 0.82±0.03 fold, respectively (n = 4 or 5, p ,0.05). 4. In colonic inflammation, both PGE2 and PGF2α were significantly increased in the colonic smooth muscle (5.2(±1.5)-fold and 4.7(±1.3)-fold of control, respectively, p ,0.05, n = 3). 5. Rat colonic circular muscle contractility was inhibited by exogenous PGE2 (10-9 ~10-6 M), but enhanced by PGF2 α (10-9 ~10-6 M) in a concentration-dependent manner (n = 4). Conclusions: Genes involved in the COX pathway of arachidonic acid metabolism are highly mechanosensitive in colonic SMCs, and play a critical role in motility function. Mechano-regulation of prostaglandin synthases accounts for increased PGE2 and unchanged PGF2 α in bowel obstruction, a pathological feature different from inflammation in which both PGE2 and PGF2 α are increased.
IAP may represent a novel therapeutic strategy to prevent fructose-induced obesity and diabetes. The beneficial effects of IAP may derive from the preservation of normal flora homeostasis and gut epithelial barrier function.
Sa2071 Overexpression of Gastric Leptin Precedes Fat Leptin Up-Regulation During Diet-Induced Obesity and Is Concomitant to Increased Number of Enterochromaffin Cells Johanne Le Beyec - Le Bihan, Anne-Laure Pelletier, Konstantinos Arapis, Maude Le Gall, Muriel Hourseau, Anne Couvelard, Thomas Aparicio, Francisca Joly, Jean-Pierre Marmuse, Andre Bado
AGA Abstracts
Sa2069 Mechanisms of Preprandial Ghrelin Release From Mouse Gastric X/A-like Cells - Molecular Basis for Regulation by Insulin Michelle Giffin, Janusz Jawien, Andreas Stengel, Miriam Goebel-Stengel, Nils W. Lambrecht
Background. Numerous gut hormones, secreted by enteroendocrine cells, participate in food intake control. Gastric leptin controls CCK or GLP-1 secretion, two peptides known to induce satiety. Gastric leptin could also regulate enteroendocrine cells differentiation. In this report, we studied the temporal changes in gastric leptin expression, serotonin (5HT) production by enterochromaffin (EC) cells as well as expression of Pax4, a critical transcription factor for the specification of EC cells during the induction of obesity. Methods. Gastric and/or duodenal mucosa biopsies were obtained from morbidly obese or lean subjects as well as from mice fed a normal (ND) or high-fat diet (HFD) for 12 weeks and from leptindeficient ob/ob mice, treated or not with oral leptin. Proteins and/or mRNA were prepared from these biopsies to quantify leptin, TPH-1 (an enzyme responsible of 5HT synthesis) and Pax4 levels in the oxyntic and duodenal mucosa. EC cells density was estimated following 5HT immunostaining of antral and duodenal biopies. Data were analysed using non-parametric tests. Results. Obese subjects exhibited a significant increase in leptin mRNA and protein levels in oxyntic mucosa and in TPH-1 and PAX-4 mRNA levels in antral mucosa (P,0.01 vs. lean subjects). One-week HFD fed mice exhibited an elevated insulinemia with no change in leptinemia but an increase in gastric Ob mRNA (4-fold, P ,0.01 vs. ND) and leptin content (2-fold, P,0.05 vs ND). These modifications were stable during 12 weeks. By contrast leptinemia significantly rose up at the third week of the HFD and correlated with fat mass increase (+15%, P ,0.05 vs ND). The number of EC cells, identified by 5HT staining and Pax4 mRNA levels were increased after one week of HFD. Furthermore, in leptin-deficient ob/ob mice, chronic (7 days) oral administration of leptin significantly increased Pax4 mRNA levels in antrum (1.5fold; P ,0.05 vs. control) and duodenum (3fold; P,0.05 vs. control). Conclusions. This is the first demonstration of gastric leptin overexpression in obese subjects concomitantly with an increased number of EC 5HTsecreting cells. Results obtained with HFD mice further suggest that these changes occur before the onset of obesity (i.e before any fat mass expansion) and that a leptin-serotonin pathway exists also in the gut. Furthermore, the increase in Pax4 levels in obese patients suggests a role for gastric leptin in enterochromaffin cell differentiation
Background: Ghrelin is the only known peripherally produced and centrally acting peptide hormone that stimulates food intake in animals and humans. Despite the fact that the changes of circulating ghrelin have been investigated under various metabolic conditions, the physiological signals causing preprandial ghrelin release into the circulation on the cellular level are not known. These studies have been hampered by the difficult purification of ghrelin cells which are expressed in low abundance in the stomach. We have generated a novel transgenic mouse model expressing the red fluorescent protein mCherry specifically under the control of the ghrelin promoter. This allows for the first time the isolation and purification of ghrelin expressing X/A-like cells to homogeneity. Aims: To identify proteins highly and specifically expressed in mouse gastric X/A-like cells involved in preprandial ghrelin release. Methods: Gastric mucosal single cell suspensions from transgenic mice expressing mCherry as a X/A-like cell specific fluorophore were prepared and ghrelin expressing X/A-like cells isolated using FACS. RNA expression of homogenous X/A-like cell suspensions after FACS was compared to RNA expression of pre-purification primary gastric epithelial cell suspensions before FACS using Affymetrix GeneChip Mouse Genome 430 2.0 Array followed by in-silico subtractive expression analysis. Results: mCherry red fluorescent protein was exclusively expressed in ghrelin-expressing X/A-like enteroendocrine cells of the gastric oxyntic mucosa. FACS of primary gastric mucosal cell suspensions resulted in purification to near-purity X/A-like cell suspensions. Subtractive expression analysis followed by quantitative RT-qPCR and immunohistochemical antibody staining showed that X/Acells uniquely express the insulin receptor binding protein IRS4 (Table). The cells also show high but not unique expression of all three forms of the insulin receptor. Conclusions: Our findings provide a molecular basis of the recently reported hypothesis that X/A-like cell ghrelin release is stimulated by the pre-prandial fall of insulin concentrations in the circulation (Endocrinology 2012, 153, 3646-56). Funding: Supported by a VA Merit 5I01BX000309-03
Sa2072 Simeprevir (TMC435) With Peginterferon/Ribavirin for Chronic HCV Genotype–1 Infection in Treatment-Nai;auve Patients: Results From QUEST–1, a Phase III Trial Ira M. Jacobson, Gregory J. Dore, Graham Foster, Michael W. Fried, Monica N. Radu, Vladimir V. Rafalskiy, Larysa Moroz, Antonio Craxi;ag, Monika Peeters, Oliver Lenz, Sivi Ouwerkerk-Mahadevan, Ronald Kalmeijer, Maria Beumont-Mauviel
Subtractive microarray expression analysis of X/A like cell suspensions POST FACS compared to PRE FACS gastric mucosal cells (* p ,0.001, ** p,0.0001).
Background and aims: Simeprevir (TMC435) is a potent, once-daily, oral, investigational, HCV NS3/4A protease inhibitor. QUEST–1 (TMC435–C208; NCT01289782) is a Phase III, randomized, double-blind, placebo-controlled trial assessing simeprevir plus peginterferon α–2a/ribavirin (PR) versus placebo plus PR in treatment-nai;auve patients with genotype– 1 infection. Safety and SVR12 results from a primary (Week 60) analysis are presented.Methods: HCV genotype–1–infected patients with METAVIR score F0–F4 (n=394), stratified by HCV subtype and host IL28B genotype, were randomized 2:1 to receive simeprevir (150 mg QD) or placebo, plus PR for 12 weeks, followed by PR alone. Total treatment duration was 24 or 48 weeks (simeprevir group) based on response-guided therapy (RGT) criteria (HCV RNA ,25 IU/mL Week 4 and undetectable Week 12) or 48 weeks (placebo group).Results: Patient disease characteristics: 18% METAVIR F3; 12% F4; 29% CC IL28B genotype; and 56% infected with HCV genotype–1a. Simeprevir/PR was superior to placebo/PR, with SVR12 rates of 80 vs 50%, respectively (p ,0.001). The majority (85%) of patients in the simeprevir group met RGT criteria and completed treatment at Week 24. Overall, 80% of simeprevir- and 12% of placebo-treated patients achieved RVR. Treatment with simeprevir/ PR led to a lower on-treatment failure rate, compared to placebo/PR (9 vs 34%), and a lower relapse rate (9 vs 21%). In the simeprevir group, AEs led to discontinuation of simeprevir in 3% of patients. The most common AEs were fatigue, pruritus and headache. The prevalence of anemia and rash was similar between the simeprevir and placebo groups.Conclusions: Simeprevir 150 mg QD with PR was generally well tolerated, leading to a high SVR12 rate of 80%. The majority of patients (85%) receiving simeprevir was able to shorten therapy to 24 weeks.
Sa2070 Specific Pathways of Nutrient Activation in Human and Mouse Enteroendocrine Eells Madusha Peiris, Abigail Masding, Vasileios Galanakis, David C. Bulmer, Charles H. Knowles, L. Ashley Blackshaw Background: Bypass surgery for obesity and type II diabetes works by exposing the distal gut to undigested nutrients, which in turn results in increased release of Peptide YY and GLP-1. We are investigating the mechanism of this effect, and have shown that in the distal ileum and colon of humans and mice, receptors for fatty and amino acids are expressed in enteroendocrine (EE) cells. This points to a functional role for lower gastrointestinal nutrient receptors in weight loss and glucose homeostasis. Here we determined the pathways by which nutrients activate major EE cell subtypes: L cells and enterochromaffin (EC) cells. Methods: Healthy human ascending colon samples were obtained from surgical resection specimens. Mouse proximal colon was obtained from C57BL/6 mice. Nutrient exposure studies were performed on freshly dissected mucosa in an Ussing chamber. Cell activation was measured by induction of phospho-ERK and phospho-CAMKII immunoreactivity (IR). Results: Specific EE cell activation in mouse colon was seen in response to the following, in rank order of potency, in terms of the number of EE cells showing pERK activation: lauric acid (agonist of GPR84) (0.88 ± 0.077 cells/crypt) . protein hydrolysate (agonist of GPR93) (0.33 ± 0.048 cells/crypt) . phenylalanine (Phe)/tryptophan (Trp) (selective activators of Calcium Sensing Receptor - CaSR) (0.36 ± 0.012 cells/crypt). Similar patterns were fould in human. We further investigated the intracellular pathway involved in activation and its relationship with mediator release. Interference with G-protein coupling using pertussis toxin (PT) abrogated pCAMKII-IR in response to Phe/Trp in human colon (-PT: 1.45 ± 0.16 cells/crypt vs. +PT: 0.12 ± 0.11 cells/crypt). Specific signalling pathways appeared to be associated with specific mediators: Phe/Trp evoked pCAMKII-IR was seen only in 5-HTIR EE cells, and not in GLP-1-IR cells. Correspondingly, 91% of CaSR-IR cells were 5-HTIR. CaSR also co-localised with GLP-1 and PYY in 14% and 26% of L-cells respectively, and yet Phe/Trp did not activate pERK in human colon. This suggests L-cells respond to activation of CaSR via a distinct, unknown pathway. Conclusions: In-depth study of CaSR coupling showed that it activates human EE cells via G-proteins, which are in turn coupled with either pCAMKII in enterochromaffin cells, or a different mechanism in L-cells, but not to pERK. However, pERK expression was activated by other nutrients in human and mouse, with the largest population responding to lauric acid. Therefore therapeutic effects of bypass surgery resulting from nutrient diversion are mediated via a range of intracellular signalling pathways in EE cells of the distal gut, and these may constitute pharmacological targets. Supported by the Wellcome Trust
AGA Abstracts
Sa2073 SVR4 Results of a Once Daily Regimen of Simeprevir (TMC435) Plus Sofosbuvir (GS–7977) With or Without Ribavirin (RBV) in HCV GT 1 Null Responders Eric Lawitz, Reem Ghalib, Maribel Rodriguez-Torres, Zobair M. Younossi, Ana Corregidor, Ira M. Jacobson, Katleen Callewaert, William T. Symonds, Gaston Picchio, Karen Lindsay Background and aims: Simeprevir (TMC435), an HCV NS3/4A protease inhibitor, is being studied with sofosbuvir (GS–7977), an HCV nucleotide NS5B polymerase inhibitor, in COSMOS, an exploratory, Phase IIa, randomized, open-label study investigating the efficacy and safety of 12 or 24 weeks of simeprevir + sofosbuvir with or without ribavirin (RBV) in HCV genotype (GT) 1 null responders to prior peginterferon (PegIFN)/RBV therapy with
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Sa2065 Purinergic Pathway Is the Major Mechanism of Inhibitory Effect of Motilitone on the Rat Gastric Fundus Relaxation Yang Won Min, Kyu Choi, Eun-ju Ko, Ji Yeon Lee, Eun Ran Kim, Byung-Hoon Min, Jun Haeng Lee, Jae J Kim, Jong Chul Rhee, Sang D. Koh, Poong-Lyul Rhee Background/Aims: Motilitone, a new prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, has strong prokinetic effects. In a previous animal study, motilitone significantly improved gastric accommodation by increasing the postprandial gastric volume. In this study, we investigated how motilitone affects the muscle relaxation in the rat gastric fundus. Methods: Gastric fundus smooth muscle strips (12 longitudinal muscles and 9 circular muscles) were obtained from rats. Isometric force measurements were performed by using both longitudinal and circular muscle strips in response to the electrical field stimulation (EFS, 0.3 ms in trains of 5 Hz for 10 s, 150 V). Peak (the highest value) and nadir (the lowest value) were observed during the first 1 minute after the initiation of EFS in the control state and after administration of atropine (1 μM), motilitone (5 μg, 10 μg, 50 μg, and 100 μg), N-nitro-L-arginine (L-NNA, 100 μM), and MRS 2500 (1 μM), which were added in sequential order to the organ bath. Then tetrodotoxin (TTX, 1 μM) was administered to inhibit nerve-mediated relaxation. Results: EFS induced contractions in the control state, which were relaxed by atropine in the both longitudinal and circular muscles. When motilitone was added to the atropine group, the nadir dropped in a dose-dependent manner in the both longitudinal and circular muscles. The nadir did not increase significantly when L-NNA was added to the atropine and motilitone (100 μg) group in the both longitudinal and circular muscles. After addition of MRS 2500, the nadir increased to the level of atropine group (before administration of motilitone) in the circular muscles. In the longitudinal muscles, the nadir also increased but was not fully reversed to the level of atropine group after addition of MRS 2500. Addition of TTX increased the nadir further and completely abolished EFS-induced relaxation in the both longitudinal and circular muscles. However, there were no significant changes in the peak after administration of motilitone regardless of its dosage. Conclusion: Our data suggest that the inhibitory effect of motilitone on the rat gastric relaxation is largely mediated by purinergic rather than nitrergic pathway, and non-nitrergic non-purinergic component also appears to be involved in the EFS-induced relaxation in the presence of motilitone.
Sa2068 Prevention of Fructose-Induced Obesity by Intestinal Alkaline Phosphatase Konstantinos P. Economopoulos, Kanakaraju Kaliannan, Nur Muhammad, Abeba Teshager, Sulaiman R. Hamarneh, Mussa Mohamed, Palak Patel, Qingsong Tao, Seyed M. Abtahi, Madhu S. Malo, Richard A. Hodin Introduction: The average consumption of fructose per capita in the U.S. has increased by approximately 200% over the past 30 years, mainly due to the dramatic increase in the consumption of fructose-sweetened beverages. It has been proposed that chronic fructose ingestion leads to intestinal bacterial dysbiosis and increased gut permeability. Intestinal Alkaline Phosphatase (IAP) is a brush border enzyme known to preserve intestinal epithelial barrier function and maintain the normal gut microbial homeostasis. Our aim in this study was to examine if IAP could prevent fructose-induced obesity and its deleterious effects. Methods: Three groups of eight-week old, C57BL/6 male mice (n=5) with unrestricted access to regular chow diet were administered either regular autoclaved water, or water enriched with 30% of fructose ± oral IAP (50 units/ml drinking water) for 16 weeks. Body weight and food intake were monitored weekly. The effects of IAP on serum glucose, intestinal permeability and normal flora were assessed. Data were expressed as mean ± standard error and were analyzed by one-way analysis of variance with Tukey's multiple comparison posttests. A significant difference was considered when p ,0.05. Results: The mice that received fructose gained significantly more weight compared to the controls (14.4±1.93 vs 7.2±0.35 g; p=0.016). Food intake was similar between mice receiving fructose+IAP and fructose alone (p=0.897). Fructose+IAP compared with fructose alone was associated with significantly less weight gain (8.3±1.81 vs 14.4±1.93 g; p=0.004), less visceral (1.1±0.10 vs 1.9±0.32 g; p=0.032) and subcutaneous fat weight (1.4±0.15 vs 2.4±0.26 g; p=0.011), lower pancreas weight (0.16±0.01 vs 0.22±0.02 g; p=0.022), lower fasting blood glucose levels (130±6.92 vs 181±15.71 mg/dl; p=0.017), lower area under the curve during glucose tolerance test (21,210±771.57 vs 24,394.5±668.72; p=0.011) and decreased intestinal permeability (0.3±0.08 vs 1.3±0.35; p=0.032). Mice receiving fructose+IAP compared to those receiving only fructose had increased number of aerobic bacteria (46.1E+05±9.75E+05 vs 4.48E+05±0.75E+05; p=0.001) and decreased number of gram negative bacteria (80±58.3 vs 2.77E+04±0.69E+04; p=0.001) in their stool. Conclusions: Oral supplementation with
Sa2066 The Provocative Water Load Test Evokes Nausea and Gastric Dysrhythmias in Patients With Unexplained Chronic Nausea and Vomiting Marcum Gillis, Kenneth L. Koch Background: The origins of nausea in patients with chronic nausea and vomiting syndromes can be difficult to elucidate. Ingestion of solids or liquids frequently precipitates symptomatic fullness and nausea. Aims: 1. To determine the incidence of abnormal water load tests (WLT) in patients with unexplained nausea and vomiting. 2. To determine the incidence of gastric dysrhythmias and severity of nausea symptoms evoked by the WLT. 3. To determine the relationship of water load volume ingested, gastric dysrhythmias, and gastric emptying results. Methods: Patients from Wake Forest Baptist Medical Center Gastroenterology Clinics who had an electrogastrogram (EGG) with WLT and solid phase gastric emptying studies were identified retrospectively. Baseline EGG recordings before and 10, 20, and 30 minutes after the WLT were evaluated. Patients drank water until they were "full" over a 5-minute period. The total volume of water ingested and visual analog scales for nausea, bloating, fullness, and hunger were completed at the time points above and overall EGG diagnoses were determined. An abnormal WLT volume was defined as less than 550 ml ingested in 5 minutes. Chi square analyses were used for statistical significance. Results: 113 patients were identified (72 women, 41 men, average age 50 years). 68/113 (60%) of patients had abnormal WLTs (average volume ingested 360 ± 29 ml) vs 702 ± 27 ml for normal WLT
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AGA Abstracts
AGA Abstracts
PGFS, and PGIS leading to the production of PGD, PGE2, PGF2 α, and PGI, respectively. Previous studies found that mechanical stretch in bowel obstruction markedly induced gene expression of COX-2, but not COX-1, in colonic smooth muscle cells (SMCs). The aims of the present study were to determine whether genes encoding individual prostaglandin synthases are mechanically responsive, and whether the composition of prostaglandins resulting from obstruction differs from that in inflammation. Methods: Partial colon obstruction was induced with a silicon band implanted surgically in the distal colon of male SpragueDawley rats. Static mechanical stretch (18% elongation) was mimicked in vitro in the culture of rat colonic circular SMCs with a Flexercell FX-4000 System. Rat colon inflammation was induced by manipulation of the colonic surface with wet cotton applicators. Results: 1. Quantitative RT-PCR found that the expression of PGDS and PGIS genes was not significantly changed on day 3 of obstruction. However, the membrane bound PGES-1 (mPGES-1) mRNA was significantly up-regulated in the obstructed segment, whereas mRNAs for other two types of PGES, cytosolic PGES and mPGES-2, were not changed. On the contrary, the PGFS mRNA was down-regulated in the obstructed colon. 2. PGE2, but not PGF2 α, was significantly increased in the colonic muscularis externae in obstruction. The PGE2 level increased to 4577±1249 pg/mg at day 3 of obstruction, compared to 1264±259 in sham (n=6, p,0.05). The PGF2 α level was 5718±910 pg /mg in obstruction compared to 6550±2178 pg/mg in the sham (n=5, p .0.05). 3. Direct stretch of rat colonic circular SMCs in vitro significantly up-regulated the mPGES-1 mRNA, but down-regulated that of PGFS. After stretch for 3 hrs, the mRNA levels of PGES and PGFS changed by 2.61±0.42 fold and 0.82±0.03 fold, respectively (n = 4 or 5, p ,0.05). 4. In colonic inflammation, both PGE2 and PGF2α were significantly increased in the colonic smooth muscle (5.2(±1.5)-fold and 4.7(±1.3)-fold of control, respectively, p ,0.05, n = 3). 5. Rat colonic circular muscle contractility was inhibited by exogenous PGE2 (10-9 ~10-6 M), but enhanced by PGF2 α (10-9 ~10-6 M) in a concentration-dependent manner (n = 4). Conclusions: Genes involved in the COX pathway of arachidonic acid metabolism are highly mechanosensitive in colonic SMCs, and play a critical role in motility function. Mechano-regulation of prostaglandin synthases accounts for increased PGE2 and unchanged PGF2 α in bowel obstruction, a pathological feature different from inflammation in which both PGE2 and PGF2 α are increased.
IAP may represent a novel therapeutic strategy to prevent fructose-induced obesity and diabetes. The beneficial effects of IAP may derive from the preservation of normal flora homeostasis and gut epithelial barrier function.
Sa2071 Overexpression of Gastric Leptin Precedes Fat Leptin Up-Regulation During Diet-Induced Obesity and Is Concomitant to Increased Number of Enterochromaffin Cells Johanne Le Beyec - Le Bihan, Anne-Laure Pelletier, Konstantinos Arapis, Maude Le Gall, Muriel Hourseau, Anne Couvelard, Thomas Aparicio, Francisca Joly, Jean-Pierre Marmuse, Andre Bado
AGA Abstracts
Sa2069 Mechanisms of Preprandial Ghrelin Release From Mouse Gastric X/A-like Cells - Molecular Basis for Regulation by Insulin Michelle Giffin, Janusz Jawien, Andreas Stengel, Miriam Goebel-Stengel, Nils W. Lambrecht
Background. Numerous gut hormones, secreted by enteroendocrine cells, participate in food intake control. Gastric leptin controls CCK or GLP-1 secretion, two peptides known to induce satiety. Gastric leptin could also regulate enteroendocrine cells differentiation. In this report, we studied the temporal changes in gastric leptin expression, serotonin (5HT) production by enterochromaffin (EC) cells as well as expression of Pax4, a critical transcription factor for the specification of EC cells during the induction of obesity. Methods. Gastric and/or duodenal mucosa biopsies were obtained from morbidly obese or lean subjects as well as from mice fed a normal (ND) or high-fat diet (HFD) for 12 weeks and from leptindeficient ob/ob mice, treated or not with oral leptin. Proteins and/or mRNA were prepared from these biopsies to quantify leptin, TPH-1 (an enzyme responsible of 5HT synthesis) and Pax4 levels in the oxyntic and duodenal mucosa. EC cells density was estimated following 5HT immunostaining of antral and duodenal biopies. Data were analysed using non-parametric tests. Results. Obese subjects exhibited a significant increase in leptin mRNA and protein levels in oxyntic mucosa and in TPH-1 and PAX-4 mRNA levels in antral mucosa (P,0.01 vs. lean subjects). One-week HFD fed mice exhibited an elevated insulinemia with no change in leptinemia but an increase in gastric Ob mRNA (4-fold, P ,0.01 vs. ND) and leptin content (2-fold, P,0.05 vs ND). These modifications were stable during 12 weeks. By contrast leptinemia significantly rose up at the third week of the HFD and correlated with fat mass increase (+15%, P ,0.05 vs ND). The number of EC cells, identified by 5HT staining and Pax4 mRNA levels were increased after one week of HFD. Furthermore, in leptin-deficient ob/ob mice, chronic (7 days) oral administration of leptin significantly increased Pax4 mRNA levels in antrum (1.5fold; P ,0.05 vs. control) and duodenum (3fold; P,0.05 vs. control). Conclusions. This is the first demonstration of gastric leptin overexpression in obese subjects concomitantly with an increased number of EC 5HTsecreting cells. Results obtained with HFD mice further suggest that these changes occur before the onset of obesity (i.e before any fat mass expansion) and that a leptin-serotonin pathway exists also in the gut. Furthermore, the increase in Pax4 levels in obese patients suggests a role for gastric leptin in enterochromaffin cell differentiation
Background: Ghrelin is the only known peripherally produced and centrally acting peptide hormone that stimulates food intake in animals and humans. Despite the fact that the changes of circulating ghrelin have been investigated under various metabolic conditions, the physiological signals causing preprandial ghrelin release into the circulation on the cellular level are not known. These studies have been hampered by the difficult purification of ghrelin cells which are expressed in low abundance in the stomach. We have generated a novel transgenic mouse model expressing the red fluorescent protein mCherry specifically under the control of the ghrelin promoter. This allows for the first time the isolation and purification of ghrelin expressing X/A-like cells to homogeneity. Aims: To identify proteins highly and specifically expressed in mouse gastric X/A-like cells involved in preprandial ghrelin release. Methods: Gastric mucosal single cell suspensions from transgenic mice expressing mCherry as a X/A-like cell specific fluorophore were prepared and ghrelin expressing X/A-like cells isolated using FACS. RNA expression of homogenous X/A-like cell suspensions after FACS was compared to RNA expression of pre-purification primary gastric epithelial cell suspensions before FACS using Affymetrix GeneChip Mouse Genome 430 2.0 Array followed by in-silico subtractive expression analysis. Results: mCherry red fluorescent protein was exclusively expressed in ghrelin-expressing X/A-like enteroendocrine cells of the gastric oxyntic mucosa. FACS of primary gastric mucosal cell suspensions resulted in purification to near-purity X/A-like cell suspensions. Subtractive expression analysis followed by quantitative RT-qPCR and immunohistochemical antibody staining showed that X/Acells uniquely express the insulin receptor binding protein IRS4 (Table). The cells also show high but not unique expression of all three forms of the insulin receptor. Conclusions: Our findings provide a molecular basis of the recently reported hypothesis that X/A-like cell ghrelin release is stimulated by the pre-prandial fall of insulin concentrations in the circulation (Endocrinology 2012, 153, 3646-56). Funding: Supported by a VA Merit 5I01BX000309-03
Sa2072 Simeprevir (TMC435) With Peginterferon/Ribavirin for Chronic HCV Genotype–1 Infection in Treatment-Nai;auve Patients: Results From QUEST–1, a Phase III Trial Ira M. Jacobson, Gregory J. Dore, Graham Foster, Michael W. Fried, Monica N. Radu, Vladimir V. Rafalskiy, Larysa Moroz, Antonio Craxi;ag, Monika Peeters, Oliver Lenz, Sivi Ouwerkerk-Mahadevan, Ronald Kalmeijer, Maria Beumont-Mauviel
Subtractive microarray expression analysis of X/A like cell suspensions POST FACS compared to PRE FACS gastric mucosal cells (* p ,0.001, ** p,0.0001).
Background and aims: Simeprevir (TMC435) is a potent, once-daily, oral, investigational, HCV NS3/4A protease inhibitor. QUEST–1 (TMC435–C208; NCT01289782) is a Phase III, randomized, double-blind, placebo-controlled trial assessing simeprevir plus peginterferon α–2a/ribavirin (PR) versus placebo plus PR in treatment-nai;auve patients with genotype– 1 infection. Safety and SVR12 results from a primary (Week 60) analysis are presented.Methods: HCV genotype–1–infected patients with METAVIR score F0–F4 (n=394), stratified by HCV subtype and host IL28B genotype, were randomized 2:1 to receive simeprevir (150 mg QD) or placebo, plus PR for 12 weeks, followed by PR alone. Total treatment duration was 24 or 48 weeks (simeprevir group) based on response-guided therapy (RGT) criteria (HCV RNA ,25 IU/mL Week 4 and undetectable Week 12) or 48 weeks (placebo group).Results: Patient disease characteristics: 18% METAVIR F3; 12% F4; 29% CC IL28B genotype; and 56% infected with HCV genotype–1a. Simeprevir/PR was superior to placebo/PR, with SVR12 rates of 80 vs 50%, respectively (p ,0.001). The majority (85%) of patients in the simeprevir group met RGT criteria and completed treatment at Week 24. Overall, 80% of simeprevir- and 12% of placebo-treated patients achieved RVR. Treatment with simeprevir/ PR led to a lower on-treatment failure rate, compared to placebo/PR (9 vs 34%), and a lower relapse rate (9 vs 21%). In the simeprevir group, AEs led to discontinuation of simeprevir in 3% of patients. The most common AEs were fatigue, pruritus and headache. The prevalence of anemia and rash was similar between the simeprevir and placebo groups.Conclusions: Simeprevir 150 mg QD with PR was generally well tolerated, leading to a high SVR12 rate of 80%. The majority of patients (85%) receiving simeprevir was able to shorten therapy to 24 weeks.
Sa2070 Specific Pathways of Nutrient Activation in Human and Mouse Enteroendocrine Eells Madusha Peiris, Abigail Masding, Vasileios Galanakis, David C. Bulmer, Charles H. Knowles, L. Ashley Blackshaw Background: Bypass surgery for obesity and type II diabetes works by exposing the distal gut to undigested nutrients, which in turn results in increased release of Peptide YY and GLP-1. We are investigating the mechanism of this effect, and have shown that in the distal ileum and colon of humans and mice, receptors for fatty and amino acids are expressed in enteroendocrine (EE) cells. This points to a functional role for lower gastrointestinal nutrient receptors in weight loss and glucose homeostasis. Here we determined the pathways by which nutrients activate major EE cell subtypes: L cells and enterochromaffin (EC) cells. Methods: Healthy human ascending colon samples were obtained from surgical resection specimens. Mouse proximal colon was obtained from C57BL/6 mice. Nutrient exposure studies were performed on freshly dissected mucosa in an Ussing chamber. Cell activation was measured by induction of phospho-ERK and phospho-CAMKII immunoreactivity (IR). Results: Specific EE cell activation in mouse colon was seen in response to the following, in rank order of potency, in terms of the number of EE cells showing pERK activation: lauric acid (agonist of GPR84) (0.88 ± 0.077 cells/crypt) . protein hydrolysate (agonist of GPR93) (0.33 ± 0.048 cells/crypt) . phenylalanine (Phe)/tryptophan (Trp) (selective activators of Calcium Sensing Receptor - CaSR) (0.36 ± 0.012 cells/crypt). Similar patterns were fould in human. We further investigated the intracellular pathway involved in activation and its relationship with mediator release. Interference with G-protein coupling using pertussis toxin (PT) abrogated pCAMKII-IR in response to Phe/Trp in human colon (-PT: 1.45 ± 0.16 cells/crypt vs. +PT: 0.12 ± 0.11 cells/crypt). Specific signalling pathways appeared to be associated with specific mediators: Phe/Trp evoked pCAMKII-IR was seen only in 5-HTIR EE cells, and not in GLP-1-IR cells. Correspondingly, 91% of CaSR-IR cells were 5-HTIR. CaSR also co-localised with GLP-1 and PYY in 14% and 26% of L-cells respectively, and yet Phe/Trp did not activate pERK in human colon. This suggests L-cells respond to activation of CaSR via a distinct, unknown pathway. Conclusions: In-depth study of CaSR coupling showed that it activates human EE cells via G-proteins, which are in turn coupled with either pCAMKII in enterochromaffin cells, or a different mechanism in L-cells, but not to pERK. However, pERK expression was activated by other nutrients in human and mouse, with the largest population responding to lauric acid. Therefore therapeutic effects of bypass surgery resulting from nutrient diversion are mediated via a range of intracellular signalling pathways in EE cells of the distal gut, and these may constitute pharmacological targets. Supported by the Wellcome Trust
AGA Abstracts
Sa2073 SVR4 Results of a Once Daily Regimen of Simeprevir (TMC435) Plus Sofosbuvir (GS–7977) With or Without Ribavirin (RBV) in HCV GT 1 Null Responders Eric Lawitz, Reem Ghalib, Maribel Rodriguez-Torres, Zobair M. Younossi, Ana Corregidor, Ira M. Jacobson, Katleen Callewaert, William T. Symonds, Gaston Picchio, Karen Lindsay Background and aims: Simeprevir (TMC435), an HCV NS3/4A protease inhibitor, is being studied with sofosbuvir (GS–7977), an HCV nucleotide NS5B polymerase inhibitor, in COSMOS, an exploratory, Phase IIa, randomized, open-label study investigating the efficacy and safety of 12 or 24 weeks of simeprevir + sofosbuvir with or without ribavirin (RBV) in HCV genotype (GT) 1 null responders to prior peginterferon (PegIFN)/RBV therapy with
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