- Email: [email protected]
Effects of A3309, an Ileal Bile Acid Transporter Inhibitor, on Colonic Transit and Symptoms in Patients with Functional Constipation
eradication must be strongly pursued in view of the high protective role of SVR from liverrelated death. Thus, new antivirals with improved efficacy and better tolerance profile are urgently needed
patients with pre-existing inflammatory bowel disease, family history of gastrointestinal malignancy, or personal history of colonic polyps. Similar to previous studies, CRCs diagnosed within 6 months after a lower endoscopy were categorized as detected cancers; CRCs diagnosed 6 to 36 months after a lower endoscopy were categorized as new or missed cancers. For flexible sigmoidoscopy, the study sample was limited to left-sided CRCs distal to the splenic flexure. For patients with multiple (N) lower endoscopies performed, each endoscopy was assigned the equal weight 1/N. The chi-square test and multivariate logistic model were used in statistical analysis. Results: Of the 52,236 patients included in the study, 3,523 flexible sigmoidoscopies and 57,412 colonoscopies were performed within 36 months prior to CRC diagnosis. The rate of new or missed left-sided CRCs after flexible sigmoidoscopy was 12%, with the highest rate in the descending colon (Figure 1). In contrast, the rate of new or missed CRCs after colonoscopy was 4% (p<0.001 vs. flexible sigmoidoscopy), increasing from 2-3% in the left-sided colon to 5-6% in the right-sided colon (Figure 1). In the left-sided colon, the risk of new or missed CRCs quadrupled after flexible sigmoidoscopy compared with colonoscopy (OR 4.02, 95% CI 3.53-4.38). In the right-sided colon, the risk of new or missed CRCs increased from the splenic flexure toward the cecum (odds ratio (OR) 1.86, 95% confidence interval (CI) 1.41 to 2.44) and was higher for nongastroenterologists (OR 1.36, 95% CI 1.23-1.50). Conclusions: Among older patients, the risk of new or missed left-sided CRCs quadrupled after flexible sigmoidoscopy compared with colonoscopy. The risk of new or missed CRCs after colonoscopy increased from the distal to proximal colon and was higher for non-gastroenterologists in the right-sided colon. Whether bowel preparation or endoscopist attributes led to these differences warrants further study.
AGA Abstracts
906 Teduglutide, a Novel Analogue of Glucagon-like Peptide 2 (GLP-2), Is Effective and Safe in Reducing Parenteral Support Volume in Short Bowel Syndrome–Intestinal Failure Subjects: Results From a 24-Week, PlaceboControlled Phase 3 Trial (STEPS) Palle B. Jeppesen, Marek Pertkiewicz, Douglas L. Seidner, Stephen O'Keefe, Hartmut Heinze, Bo Joelsson Background: Teduglutide, a dipeptidyl peptidase-IV degradation-resistant analogue of human GLP-2, repairs and restores functional and structural integrity of the intestinal mucosa. Improved fluid and nutrient balance is achieved by enhanced absorption resulting from increased villus height and crypt depth. Objective: In the largest placebo-controlled short bowel syndrome–intestinal failure (SBS-IF) study conducted to date, teduglutide was evaluated on its ability to reduce the volume of parenteral support (PS; intravenous fluids, nutrients, or both) in adult SBS-IF subjects. Methods: Eighty-six (86) SBS-IF subjects, dependent on PS for ≥1 year, were enrolled in a randomized, double-blind, placebocontrolled, parallel-group, multinational, multicenter study. After an initial optimization/ stabilization period of PS volume (ensuring stable urine output), subjects were randomized to subcutaneous teduglutide (0.05 mg/kg/d) or placebo, once daily, for 24 weeks. PS volumes were reduced at scheduled visits if urine volumes exceeded baseline by ≥10%. Responders were those who achieved a 20% to 100% reduction from baseline in weekly PS volume at Weeks 20 and 24. The primary efficacy endpoint of the study compared the percentage of responders on teduglutide versus placebo. Results: Seventy-eight (78) SBS-IF subjects completed the study (teduglutide 0.05 mg/kg/d, n=39; placebo, n=39). In the intention-totreat (ITT) population, 63% (27/43) of SBS-IF subjects randomized to teduglutide 0.05 mg/ kg/d were responders versus 30% (13/43) receiving placebo (p=0.002; ITT population). At Week 24, teduglutide reduced mean weekly PS volume by 4.4 L/wk (12.9 L/wk at baseline), whereas PS volume reduction with placebo treatment was 2.3 L/wk (13.2 L/wk at baseline; p≤0.001) (Figure 1). Significantly more teduglutide-treated subjects were able to reduce the number of infusion days by 1 or more days than placebo (54% versus 23%; p=0.0047). Despite the significant PS volume reduction, body weight remained constant. Teduglutide was well tolerated. Of the 8 dropouts, 3 of 4 in the placebo group and 2 of 4 in the teduglutide group were due to adverse events (AEs). AEs were found in 83% (35/42) treated with teduglutide versus 79% (34/43) in subjects treated with placebo. The most common AEs were abdominal pain, nausea, gastrointestinal stoma complications, and abdominal distension. More than 97% of the subjects who completed this study elected to enroll in an open-label 24-month continuation study. Conclusions: Treatment with teduglutide 0.05 mg/kg/d effectively and safely reduced parenteral support volume. Independence, characterized by fewer days of PS infusion, was significantly greater with teduglutide. Significant response was seen as early as at Week 8 and increased through Week 24 compared with placebo in short bowel syndrome–intestinal failure subjects.
Figure 1. Rate of new or missed colorectal cancers after colonoscopy or flexible sigmoidoscopy by location. 908 Effects of A3309, an Ileal Bile Acid Transporter Inhibitor, on Colonic Transit and Symptoms in Patients with Functional Constipation Banny S. Wong, Michael Camilleri, Sanna McKinzie, Duane D. Burton, Hans Graffner, Alan R. Zinsmeister Background & Aims: Delivery of bile acids to the colon stimulates propulsive motility and fluid secretion. We aimed to examine gastrointestinal (GI) transit effects of A3309, a small molecule inhibitor of the ileal bile acid (BA) transporter (IBAT), in patients with functional constipation (FC). Methods: The trial is registered in ClinicalTrials.gov under NCT01038687. In a double-blind, placebo-controlled study of 36 female FC patients randomized to placebo, 15mg A3309, or 20mg A3309 administered orally once daily for 14 consecutive days, we assessed GI and colonic transit of solids by a validated scintigraphic method, stool consistency by Bristol Stool Form (BSF) scale, symptoms of constipation (including patient perceived constipation rating relative to baseline, ease of passage, straining), patient-perceived treatment effectiveness, mean daily abdominal discomfort and bloating, fasting serum 7αC4 (surrogate of BA synthesis), and fasting serum total and LDL cholesterol (surrogates of inhibition of BA absorption). The study was powered to detect a 37% difference in colonic transit at 24 h between treatment groups. Following the ITT paradigm, we used ANCOVA analysis to assess overall treatment effects and Dunnett's test for pairwise comparisons. Results (see table): Overall colonic transit (geometric center at 24h, primary endpoint) was significantly accelerated with 20mg A3309 compared to placebo (overall effect, p=0.059; A3309 15mg p=0.18; and A3309 20mg p=0.04). Colonic transit at 48h was significantly accelerated with both A3309 dosages (overall effect p<0.001; A3309 15mg p=0.002; and A3309 20mg p<0.001). Significantly looser stool consistency was noted with both A3309 dosages compared to placebo (p<0.005); effect on stool frequency was not statistically significant (overall p=0.091). Significant effects of A3309 on constipation rating, ease of stool passage, and reduction of straining were also detected. The most common side effect was lower abdominal cramping/pain (p=0.005). A3309 treatment significantly and reversibly increased fasting 7αC4 (A3309 15mg p=0.05; A3309 20mg p<0.01). A3309 did not significantly affect gastric emptying, small bowel transit, or fasting total and LDL cholesterol levels in serum. Conclusions: A3309 accelerates colonic transit without significant changes in gastric emptying or small bowel transit and A3309 induced loosened stool consistency with beneficial effects on ease of passage and straining scores. In addition, there were significant effects on reduction of constipation severity and bloating and patients perceived effectiveness of A3309 treatment. These data support the potential use of A3309 in the treatment of patients with functional constipation.
Fig 1. PS Volume Reduction (Liters/Week) 907 Increased Risk of New or Missed Colorectal Cancers after Flexible Sigmoidoscopy Compared with Colonoscopy among Older Patients in the United States: A Population-Based Analysis of the SEER-Medicare Linked Database, 1998-2005 Yize R. Wang, John R. Cangemi, Edward V. Loftus, Michael F. Picco Background: Previous studies on the rate of new or missed colorectal cancers (CRCs) after colonoscopy were limited to regional studies. It is unknown how flexible sigmoidoscopy compares with colonoscopy in the rate of new or missed CRCs. Objective: To compare the rate of new or missed CRCs after flexible sigmoidoscopy compared with colonoscopy among older patients in the United States. Data and Methods: All patients in the Surveillance, Epidemiology and End Results Medicare (SEER-Medicare) linked database who were 67 years or older at the time of a lower endoscopy during 1998-2005 and subsequently diagnosed with CRC within 36 months (through 12/31/2005) were identified. We excluded high-risk
AGA Abstracts
S-146
910
Canonical Wnt signaling plays a critical role in intestinal homeostasis and stem cell maintenance. As cells migrate up the crypt villus axis to differentiate, the Wnt pathway becomes inactive and cells exit the cell cycle. The function of Wnt signaling during differentiation of enteroendocrine cells is not well understood. Neurogenin 3 (Ngn3) and its direct target NeuroD1 are two basic helix-loop-helix transcription factors that control enteroendocrine early cell fate specification (Ngn3) and later stages differentiation (NeuroD1). Previously, we reported that conditional activation of Wnt signaling in Ngn3 expressing early enteroendocrine precursors resulted in widespread development of adenomas with neuroendocrine features in mice. However, conditional activation of β-catenin in maturing NeuroD1+ enteroendocrine cells failed to activate the Wnt pathway or produce tumors. The aim of this study was to determine whether NeuroD1 functions to inhibit Wnt-β−catenin signaling. We initially examined the effects of NeuroD1 on Wnt signaling dependent transcription using transfection assays with the TCF dependent reporter gene, TOPFLASH in HEK293 cells and in the human colon cancer line LS174T. We found that NeuroD1 was a potent inhibitor of TOPFLASH in both cell types, requiring amino acids 1-138 of NeuroD1, which includes the bHLH protein: protein interaction domain. We subsequently identified NeuroD1 in βcatenin containing protein complexes by coimmunoprecipitation, suggesting that NeuroD1 is associated with transcriptional effector components of Wnt signaling. To determine the functional effects of NeuroD1 in inhibiting Wnt signaling In Vivo, we established a subline of DLD1 colon cancer cells that stably expressed NeuroD1. Expression of the endogenous Wnt target gene, cMyc, was significantly reduced in NeuroD1-DLD1 cells. Chromatin immunoprecipitation (ChIP) assays showed the presence of NeuroD1 at the cMyc enhancer In Vivo. The absence of a consensus NeuroD1 binding site in the cMyc enhancer suggests that the recruitment of NeuroD1 may occur by interactions with other proteins occupying the enhancer rather than by direct DNA binding. Consistent with inhibited Wnt signaling, NeuroD1-DLD1 cells showed reduced [3H] Thymidine incorporation and delayed cell cycle progression by FACS, with no significant change in apoptosis. A limited number of transcription factors, including MTGR1, RUNX3 and Sox9 have been shown to inhibit Wnt activity by interacting with Wnt transcriptional effector proteins. We conclude that in addition to the established role of NeuroD1 as a transcriptional activator, inhibition of Wnt signaling may represent a novel mechanism for NeuroD1 to further regulate enteroendocrine differentiation.
*Covariates for responses were BMI and baseline GC24 value for GC24 and GC48, and corresponding baseline value for stool frequency, consistency, and ease of passage.†Lower scores are associated with lower symptoms/better efficacy.
911 909
Bile Acid-Induced Stimulation of ERK1/2 Activity, GLP-1 and PYY Release in Enteroendocrine Cells is Mediated by the Activation of Epac/PLC-ε Signaling Pathway via GS-Coupled TGR5 Vanitha Bala, Sunila Mahavadi, Senthilkumar Rajagopal, Ruizhe Zhou, John F. Kuemmerle, Arun J. Sanyal, Karnam S. Murthy
MTORC1 Inhibition Increases Neurotensin Secretion and Gene Expression Through Activation of MEK/ERK/C-Jun Pathway in the Human Endocrine Cell Line BON Jing Li, Jianyu Liu, Jun Song, Heidi Weiss, Courtney M. Townsend, Tianyan Gao, B. M. Evers
In addition to having a role in dietary nutrient absorption, bile acids are signaling molecules with paracrine and endocrine functions. Recent studies have demonstrated that bile acids are ligands, for the novel membrane-bound G protein-coupled receptor TGR5 in addition to nuclear receptors. Activation of TGR5 is implicated in secretion and motility of gastrointestinal tract as well as in energy expenditure. Several signaling pathways including cAMP/ PKA, Ca2+/PKC, MAP kinase and tyrosine kinase have been shown to contribute to the overall responses induced by bile acids. Understanding the molecular mechanisms underlying TGR5-induced functions has therapeutic potential to curb weight gain and improve glucose homeostasis. AIM. To identify the signaling pathways activated by TGR5 in enteroendocrine cells and their role in hormone release. METHODS. Murine enteroendocrine STC-1 cells were obtained from ATCC and maintained in culture. Expression of TGR5 was determined by RT-PCR, western blot and immunocytochemistry. G protein activation in response to TGR5 selective ligand oleanolic acid (OA) was measured as increase in Gα binding to [35S] GTPγS, cAMP formation was measured by ELISA, phosphoinositide (PI) hydrolysis was measured by ion-exchange chromatography. Glucagon like peptide-1 (GLP-1), Peptide YY (PYY) and ERK1/2 activity were measured by ELISA and immunokinase assay. RESULTS. TGR5 expression was detected in STC-1 cells by RT-PCR, western blot and immunocytochemistry. Expression of TGR5 was also detected in human colon mucosa by RT-PCR and western blot. OA activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαs, OA increased cAMP formation. OA also elicited a significant increase in ERK1/2 activity that was blocked by the selective MEK inhibitor PD98059, but not by the selective PKA inhibitor, myristoylated PKI. These results suggest that activation of ERK1/2 was independent of PKA, and probably involve cAMP-activated exchange factor Epac and activation of PLCε. STC-1 cells expressed both Epac and PLC-ε. The Epac selective ligand 8CPT-2Me-cAMP stimulated ERK1/2 activity. OA and 8CPT-2Me-cAMP also stimulated PI hydrolysis and elicited the release of GLP-1 and PYY. PI-PLC inhibitor, U73122 blocked PI hydrolysis and the release of GLP-1 and PYY in response to both OA and 8CPT-2Me-cAMP. CONCLUSION. Gs-coupled TGR5 receptors are expressed in enteroendocrine cells and activation of these receptors by bile acids is coupled to stimulation of PI hydrolysis and ERK1/2 activity, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε pathway. Supported by training grant DK-07150
Mammalian target of rapamycin (mTOR) exists in two complexes: mTORC1 and mTORC2; mTORC1 is a sensor of nutrients and growth factors and regulates cellular homeostasis. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, facilitates fatty acid translocation from the intestinal lumen and regulates gut motility. Previously, we showed that the inhibition of mTORC1 increased NT gene expression and secretion in BON cells, an endocrine cell line that synthesizes and secretes NT peptide in a manner analogous to that of enteroendocrine cells. In the present study, we sought to determine the signaling molecules involved in mTORC1-regulated NT gene expression and secretion. METHODS. i) BON and Hep3B, a hepatoma cell line which expresses high levels of NT mRNA similar to BON, were used; stable p70S6K shRNA knockdown cell lines were established. Cells were treated for 24 h with rapamycin, an mTORC1 inhibitor, or torin1, a dual inhibitor of mTORC1 and mTORC2; NT secretion was measured by NT EIA; ii) total RNA was isolated and real-time PCR performed. NT and c-Jun promoter activities were determined by dual luciferase assays; iii) Western blot was performed to determine protein phosphorylation and expression. RESULTS. We demonstrate that the inhibition of mTORC1 by rapamycin, torin1 or shRNA-mediated knockdown of p70S6K, a substrate of mTORC1, increased phosphorylation of ERK1/2 and c-Jun (S63) in BON and Hep3B cells. c-Jun promoter activity was increased by rapamycin treatment, but decreased by p70S6K overexpression in BON cells. Consistently, overexpression of ERK1 or MEK1-DD, a constitutively active form of MEK1, increased phosphorylation of c-Jun (S63), and ERK1 overexpression increased NT promoter activity and NT secretion. c-Jun overexpression dramatically increased NT promoter activity. Treatment with PD98059, an MEK inhibitor, blocked rapamycin- or torin1-enhanced NT secretion, and NT promoter activity increased by c-Jun overexpression. Importantly, activation of mTORC1 signaling by serum or high glucose decreased phosphorylation of ERK1/2 and c-Jun as well as NT secretion compared with serum-free and low glucose conditions, respectively. In contrast, inhibition of mTORC1 signaling by AICAR, an activator of AMP-activated protein kinase (AMPK), increased phosphorylation of ERK1/ 2 and c-Jun as well as NT secretion. CONCLUSIONS. mTORC1 inhibition increases NT gene expression and secretion by up-regulating the MEK/ERK/c-Jun signaling pathway. Our results highlight an interaction of mTORC1 and the MEK/ERK/c-Jun signaling pathways in the regulation of NT gene induction and NT peptide secretion; these data establish a novel negative role for mTORC1 signaling in nutrient-mediated intestinal hormone release.
912 Bitter Taste Receptor Ligands Activate Gut Enteroendocrine Cells and Enterochromaffin Cells in the Rat In Vivo Helen Raybould, James W. Sharp, Catia Sternini Bitter taste in the oral cavity is detected by a vast array of taste type 2 (TAS2Rs) receptors. The TAS2R gene family of G-protein coupled receptors contains about 30 different members, each with relative specificity for different bitter-tasting ligands. TAS2Rs are expressed in the gut of rodents and humans, and the associated G proteins (e.g. α-gustducin and transducin) have been localized to enteroendocrine cells (EECs). In rodents, administration of ligands
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AGA Abstracts
AGA Abstracts
Novel Function of NeuroD1 in Inhibiting Canonical Wnt Signaling to Regulate Enteroendocrine Differentiation Joyce Li, Subir K. Ray, Andrew B. Leiter
patients with pre-existing inflammatory bowel disease, family history of gastrointestinal malignancy, or personal history of colonic polyps. Similar to previous studies, CRCs diagnosed within 6 months after a lower endoscopy were categorized as detected cancers; CRCs diagnosed 6 to 36 months after a lower endoscopy were categorized as new or missed cancers. For flexible sigmoidoscopy, the study sample was limited to left-sided CRCs distal to the splenic flexure. For patients with multiple (N) lower endoscopies performed, each endoscopy was assigned the equal weight 1/N. The chi-square test and multivariate logistic model were used in statistical analysis. Results: Of the 52,236 patients included in the study, 3,523 flexible sigmoidoscopies and 57,412 colonoscopies were performed within 36 months prior to CRC diagnosis. The rate of new or missed left-sided CRCs after flexible sigmoidoscopy was 12%, with the highest rate in the descending colon (Figure 1). In contrast, the rate of new or missed CRCs after colonoscopy was 4% (p<0.001 vs. flexible sigmoidoscopy), increasing from 2-3% in the left-sided colon to 5-6% in the right-sided colon (Figure 1). In the left-sided colon, the risk of new or missed CRCs quadrupled after flexible sigmoidoscopy compared with colonoscopy (OR 4.02, 95% CI 3.53-4.38). In the right-sided colon, the risk of new or missed CRCs increased from the splenic flexure toward the cecum (odds ratio (OR) 1.86, 95% confidence interval (CI) 1.41 to 2.44) and was higher for nongastroenterologists (OR 1.36, 95% CI 1.23-1.50). Conclusions: Among older patients, the risk of new or missed left-sided CRCs quadrupled after flexible sigmoidoscopy compared with colonoscopy. The risk of new or missed CRCs after colonoscopy increased from the distal to proximal colon and was higher for non-gastroenterologists in the right-sided colon. Whether bowel preparation or endoscopist attributes led to these differences warrants further study.
AGA Abstracts
906 Teduglutide, a Novel Analogue of Glucagon-like Peptide 2 (GLP-2), Is Effective and Safe in Reducing Parenteral Support Volume in Short Bowel Syndrome–Intestinal Failure Subjects: Results From a 24-Week, PlaceboControlled Phase 3 Trial (STEPS) Palle B. Jeppesen, Marek Pertkiewicz, Douglas L. Seidner, Stephen O'Keefe, Hartmut Heinze, Bo Joelsson Background: Teduglutide, a dipeptidyl peptidase-IV degradation-resistant analogue of human GLP-2, repairs and restores functional and structural integrity of the intestinal mucosa. Improved fluid and nutrient balance is achieved by enhanced absorption resulting from increased villus height and crypt depth. Objective: In the largest placebo-controlled short bowel syndrome–intestinal failure (SBS-IF) study conducted to date, teduglutide was evaluated on its ability to reduce the volume of parenteral support (PS; intravenous fluids, nutrients, or both) in adult SBS-IF subjects. Methods: Eighty-six (86) SBS-IF subjects, dependent on PS for ≥1 year, were enrolled in a randomized, double-blind, placebocontrolled, parallel-group, multinational, multicenter study. After an initial optimization/ stabilization period of PS volume (ensuring stable urine output), subjects were randomized to subcutaneous teduglutide (0.05 mg/kg/d) or placebo, once daily, for 24 weeks. PS volumes were reduced at scheduled visits if urine volumes exceeded baseline by ≥10%. Responders were those who achieved a 20% to 100% reduction from baseline in weekly PS volume at Weeks 20 and 24. The primary efficacy endpoint of the study compared the percentage of responders on teduglutide versus placebo. Results: Seventy-eight (78) SBS-IF subjects completed the study (teduglutide 0.05 mg/kg/d, n=39; placebo, n=39). In the intention-totreat (ITT) population, 63% (27/43) of SBS-IF subjects randomized to teduglutide 0.05 mg/ kg/d were responders versus 30% (13/43) receiving placebo (p=0.002; ITT population). At Week 24, teduglutide reduced mean weekly PS volume by 4.4 L/wk (12.9 L/wk at baseline), whereas PS volume reduction with placebo treatment was 2.3 L/wk (13.2 L/wk at baseline; p≤0.001) (Figure 1). Significantly more teduglutide-treated subjects were able to reduce the number of infusion days by 1 or more days than placebo (54% versus 23%; p=0.0047). Despite the significant PS volume reduction, body weight remained constant. Teduglutide was well tolerated. Of the 8 dropouts, 3 of 4 in the placebo group and 2 of 4 in the teduglutide group were due to adverse events (AEs). AEs were found in 83% (35/42) treated with teduglutide versus 79% (34/43) in subjects treated with placebo. The most common AEs were abdominal pain, nausea, gastrointestinal stoma complications, and abdominal distension. More than 97% of the subjects who completed this study elected to enroll in an open-label 24-month continuation study. Conclusions: Treatment with teduglutide 0.05 mg/kg/d effectively and safely reduced parenteral support volume. Independence, characterized by fewer days of PS infusion, was significantly greater with teduglutide. Significant response was seen as early as at Week 8 and increased through Week 24 compared with placebo in short bowel syndrome–intestinal failure subjects.
Figure 1. Rate of new or missed colorectal cancers after colonoscopy or flexible sigmoidoscopy by location. 908 Effects of A3309, an Ileal Bile Acid Transporter Inhibitor, on Colonic Transit and Symptoms in Patients with Functional Constipation Banny S. Wong, Michael Camilleri, Sanna McKinzie, Duane D. Burton, Hans Graffner, Alan R. Zinsmeister Background & Aims: Delivery of bile acids to the colon stimulates propulsive motility and fluid secretion. We aimed to examine gastrointestinal (GI) transit effects of A3309, a small molecule inhibitor of the ileal bile acid (BA) transporter (IBAT), in patients with functional constipation (FC). Methods: The trial is registered in ClinicalTrials.gov under NCT01038687. In a double-blind, placebo-controlled study of 36 female FC patients randomized to placebo, 15mg A3309, or 20mg A3309 administered orally once daily for 14 consecutive days, we assessed GI and colonic transit of solids by a validated scintigraphic method, stool consistency by Bristol Stool Form (BSF) scale, symptoms of constipation (including patient perceived constipation rating relative to baseline, ease of passage, straining), patient-perceived treatment effectiveness, mean daily abdominal discomfort and bloating, fasting serum 7αC4 (surrogate of BA synthesis), and fasting serum total and LDL cholesterol (surrogates of inhibition of BA absorption). The study was powered to detect a 37% difference in colonic transit at 24 h between treatment groups. Following the ITT paradigm, we used ANCOVA analysis to assess overall treatment effects and Dunnett's test for pairwise comparisons. Results (see table): Overall colonic transit (geometric center at 24h, primary endpoint) was significantly accelerated with 20mg A3309 compared to placebo (overall effect, p=0.059; A3309 15mg p=0.18; and A3309 20mg p=0.04). Colonic transit at 48h was significantly accelerated with both A3309 dosages (overall effect p<0.001; A3309 15mg p=0.002; and A3309 20mg p<0.001). Significantly looser stool consistency was noted with both A3309 dosages compared to placebo (p<0.005); effect on stool frequency was not statistically significant (overall p=0.091). Significant effects of A3309 on constipation rating, ease of stool passage, and reduction of straining were also detected. The most common side effect was lower abdominal cramping/pain (p=0.005). A3309 treatment significantly and reversibly increased fasting 7αC4 (A3309 15mg p=0.05; A3309 20mg p<0.01). A3309 did not significantly affect gastric emptying, small bowel transit, or fasting total and LDL cholesterol levels in serum. Conclusions: A3309 accelerates colonic transit without significant changes in gastric emptying or small bowel transit and A3309 induced loosened stool consistency with beneficial effects on ease of passage and straining scores. In addition, there were significant effects on reduction of constipation severity and bloating and patients perceived effectiveness of A3309 treatment. These data support the potential use of A3309 in the treatment of patients with functional constipation.
Fig 1. PS Volume Reduction (Liters/Week) 907 Increased Risk of New or Missed Colorectal Cancers after Flexible Sigmoidoscopy Compared with Colonoscopy among Older Patients in the United States: A Population-Based Analysis of the SEER-Medicare Linked Database, 1998-2005 Yize R. Wang, John R. Cangemi, Edward V. Loftus, Michael F. Picco Background: Previous studies on the rate of new or missed colorectal cancers (CRCs) after colonoscopy were limited to regional studies. It is unknown how flexible sigmoidoscopy compares with colonoscopy in the rate of new or missed CRCs. Objective: To compare the rate of new or missed CRCs after flexible sigmoidoscopy compared with colonoscopy among older patients in the United States. Data and Methods: All patients in the Surveillance, Epidemiology and End Results Medicare (SEER-Medicare) linked database who were 67 years or older at the time of a lower endoscopy during 1998-2005 and subsequently diagnosed with CRC within 36 months (through 12/31/2005) were identified. We excluded high-risk
AGA Abstracts
S-146
910
Canonical Wnt signaling plays a critical role in intestinal homeostasis and stem cell maintenance. As cells migrate up the crypt villus axis to differentiate, the Wnt pathway becomes inactive and cells exit the cell cycle. The function of Wnt signaling during differentiation of enteroendocrine cells is not well understood. Neurogenin 3 (Ngn3) and its direct target NeuroD1 are two basic helix-loop-helix transcription factors that control enteroendocrine early cell fate specification (Ngn3) and later stages differentiation (NeuroD1). Previously, we reported that conditional activation of Wnt signaling in Ngn3 expressing early enteroendocrine precursors resulted in widespread development of adenomas with neuroendocrine features in mice. However, conditional activation of β-catenin in maturing NeuroD1+ enteroendocrine cells failed to activate the Wnt pathway or produce tumors. The aim of this study was to determine whether NeuroD1 functions to inhibit Wnt-β−catenin signaling. We initially examined the effects of NeuroD1 on Wnt signaling dependent transcription using transfection assays with the TCF dependent reporter gene, TOPFLASH in HEK293 cells and in the human colon cancer line LS174T. We found that NeuroD1 was a potent inhibitor of TOPFLASH in both cell types, requiring amino acids 1-138 of NeuroD1, which includes the bHLH protein: protein interaction domain. We subsequently identified NeuroD1 in βcatenin containing protein complexes by coimmunoprecipitation, suggesting that NeuroD1 is associated with transcriptional effector components of Wnt signaling. To determine the functional effects of NeuroD1 in inhibiting Wnt signaling In Vivo, we established a subline of DLD1 colon cancer cells that stably expressed NeuroD1. Expression of the endogenous Wnt target gene, cMyc, was significantly reduced in NeuroD1-DLD1 cells. Chromatin immunoprecipitation (ChIP) assays showed the presence of NeuroD1 at the cMyc enhancer In Vivo. The absence of a consensus NeuroD1 binding site in the cMyc enhancer suggests that the recruitment of NeuroD1 may occur by interactions with other proteins occupying the enhancer rather than by direct DNA binding. Consistent with inhibited Wnt signaling, NeuroD1-DLD1 cells showed reduced [3H] Thymidine incorporation and delayed cell cycle progression by FACS, with no significant change in apoptosis. A limited number of transcription factors, including MTGR1, RUNX3 and Sox9 have been shown to inhibit Wnt activity by interacting with Wnt transcriptional effector proteins. We conclude that in addition to the established role of NeuroD1 as a transcriptional activator, inhibition of Wnt signaling may represent a novel mechanism for NeuroD1 to further regulate enteroendocrine differentiation.
*Covariates for responses were BMI and baseline GC24 value for GC24 and GC48, and corresponding baseline value for stool frequency, consistency, and ease of passage.†Lower scores are associated with lower symptoms/better efficacy.
911 909
Bile Acid-Induced Stimulation of ERK1/2 Activity, GLP-1 and PYY Release in Enteroendocrine Cells is Mediated by the Activation of Epac/PLC-ε Signaling Pathway via GS-Coupled TGR5 Vanitha Bala, Sunila Mahavadi, Senthilkumar Rajagopal, Ruizhe Zhou, John F. Kuemmerle, Arun J. Sanyal, Karnam S. Murthy
MTORC1 Inhibition Increases Neurotensin Secretion and Gene Expression Through Activation of MEK/ERK/C-Jun Pathway in the Human Endocrine Cell Line BON Jing Li, Jianyu Liu, Jun Song, Heidi Weiss, Courtney M. Townsend, Tianyan Gao, B. M. Evers
In addition to having a role in dietary nutrient absorption, bile acids are signaling molecules with paracrine and endocrine functions. Recent studies have demonstrated that bile acids are ligands, for the novel membrane-bound G protein-coupled receptor TGR5 in addition to nuclear receptors. Activation of TGR5 is implicated in secretion and motility of gastrointestinal tract as well as in energy expenditure. Several signaling pathways including cAMP/ PKA, Ca2+/PKC, MAP kinase and tyrosine kinase have been shown to contribute to the overall responses induced by bile acids. Understanding the molecular mechanisms underlying TGR5-induced functions has therapeutic potential to curb weight gain and improve glucose homeostasis. AIM. To identify the signaling pathways activated by TGR5 in enteroendocrine cells and their role in hormone release. METHODS. Murine enteroendocrine STC-1 cells were obtained from ATCC and maintained in culture. Expression of TGR5 was determined by RT-PCR, western blot and immunocytochemistry. G protein activation in response to TGR5 selective ligand oleanolic acid (OA) was measured as increase in Gα binding to [35S] GTPγS, cAMP formation was measured by ELISA, phosphoinositide (PI) hydrolysis was measured by ion-exchange chromatography. Glucagon like peptide-1 (GLP-1), Peptide YY (PYY) and ERK1/2 activity were measured by ELISA and immunokinase assay. RESULTS. TGR5 expression was detected in STC-1 cells by RT-PCR, western blot and immunocytochemistry. Expression of TGR5 was also detected in human colon mucosa by RT-PCR and western blot. OA activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαs, OA increased cAMP formation. OA also elicited a significant increase in ERK1/2 activity that was blocked by the selective MEK inhibitor PD98059, but not by the selective PKA inhibitor, myristoylated PKI. These results suggest that activation of ERK1/2 was independent of PKA, and probably involve cAMP-activated exchange factor Epac and activation of PLCε. STC-1 cells expressed both Epac and PLC-ε. The Epac selective ligand 8CPT-2Me-cAMP stimulated ERK1/2 activity. OA and 8CPT-2Me-cAMP also stimulated PI hydrolysis and elicited the release of GLP-1 and PYY. PI-PLC inhibitor, U73122 blocked PI hydrolysis and the release of GLP-1 and PYY in response to both OA and 8CPT-2Me-cAMP. CONCLUSION. Gs-coupled TGR5 receptors are expressed in enteroendocrine cells and activation of these receptors by bile acids is coupled to stimulation of PI hydrolysis and ERK1/2 activity, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε pathway. Supported by training grant DK-07150
Mammalian target of rapamycin (mTOR) exists in two complexes: mTORC1 and mTORC2; mTORC1 is a sensor of nutrients and growth factors and regulates cellular homeostasis. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, facilitates fatty acid translocation from the intestinal lumen and regulates gut motility. Previously, we showed that the inhibition of mTORC1 increased NT gene expression and secretion in BON cells, an endocrine cell line that synthesizes and secretes NT peptide in a manner analogous to that of enteroendocrine cells. In the present study, we sought to determine the signaling molecules involved in mTORC1-regulated NT gene expression and secretion. METHODS. i) BON and Hep3B, a hepatoma cell line which expresses high levels of NT mRNA similar to BON, were used; stable p70S6K shRNA knockdown cell lines were established. Cells were treated for 24 h with rapamycin, an mTORC1 inhibitor, or torin1, a dual inhibitor of mTORC1 and mTORC2; NT secretion was measured by NT EIA; ii) total RNA was isolated and real-time PCR performed. NT and c-Jun promoter activities were determined by dual luciferase assays; iii) Western blot was performed to determine protein phosphorylation and expression. RESULTS. We demonstrate that the inhibition of mTORC1 by rapamycin, torin1 or shRNA-mediated knockdown of p70S6K, a substrate of mTORC1, increased phosphorylation of ERK1/2 and c-Jun (S63) in BON and Hep3B cells. c-Jun promoter activity was increased by rapamycin treatment, but decreased by p70S6K overexpression in BON cells. Consistently, overexpression of ERK1 or MEK1-DD, a constitutively active form of MEK1, increased phosphorylation of c-Jun (S63), and ERK1 overexpression increased NT promoter activity and NT secretion. c-Jun overexpression dramatically increased NT promoter activity. Treatment with PD98059, an MEK inhibitor, blocked rapamycin- or torin1-enhanced NT secretion, and NT promoter activity increased by c-Jun overexpression. Importantly, activation of mTORC1 signaling by serum or high glucose decreased phosphorylation of ERK1/2 and c-Jun as well as NT secretion compared with serum-free and low glucose conditions, respectively. In contrast, inhibition of mTORC1 signaling by AICAR, an activator of AMP-activated protein kinase (AMPK), increased phosphorylation of ERK1/ 2 and c-Jun as well as NT secretion. CONCLUSIONS. mTORC1 inhibition increases NT gene expression and secretion by up-regulating the MEK/ERK/c-Jun signaling pathway. Our results highlight an interaction of mTORC1 and the MEK/ERK/c-Jun signaling pathways in the regulation of NT gene induction and NT peptide secretion; these data establish a novel negative role for mTORC1 signaling in nutrient-mediated intestinal hormone release.
912 Bitter Taste Receptor Ligands Activate Gut Enteroendocrine Cells and Enterochromaffin Cells in the Rat In Vivo Helen Raybould, James W. Sharp, Catia Sternini Bitter taste in the oral cavity is detected by a vast array of taste type 2 (TAS2Rs) receptors. The TAS2R gene family of G-protein coupled receptors contains about 30 different members, each with relative specificity for different bitter-tasting ligands. TAS2Rs are expressed in the gut of rodents and humans, and the associated G proteins (e.g. α-gustducin and transducin) have been localized to enteroendocrine cells (EECs). In rodents, administration of ligands
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AGA Abstracts
AGA Abstracts
Novel Function of NeuroD1 in Inhibiting Canonical Wnt Signaling to Regulate Enteroendocrine Differentiation Joyce Li, Subir K. Ray, Andrew B. Leiter