- Email: [email protected]
308 Glucose-Dependent Insulinotropic Polypeptide (GIP) Enhances Adipocyte Development and Modulates Multiple Downstream Targets of AKT
AGA Abstracts
which increased to 10-12% of all DCs. However, the increase in CD103+DCs was shortlived. Instead, the healing phase of colitis was characterized by the influx of pDCs which increased over 10-fold during a 7 day recovery period and stayed elevated for over 3 weeks. The influx of pDCs was accompanied by the recruitment of Foxp3+Tregs in the colon which were found dispersed throughout the lamina propria and steadily increased during mucosal healing. Surprisingly, IL-10 deficient mice demonstrated the enhanced recruitment of pDCs (25-30% of all DCs) and Foxp3+Tregs even before the onset of colitis but without an increased in CD103+DCs. Onset of macroscopic disease in IL-10 deficient mice was characterized by the additional influx of CD11b-DCs. CONCLUSION: Mucosal defense activation involves the recruitment of specific DC subsets which have defined roles in the control of acute inflammation and support of mucosal healing. Surprisingly, DC subset recruitment was similar in colitis induced by IEC monolayer disruption and T cell mediated disease in IL10 deficient mice. This suggests common response programs to mucosal damage. We would like to propose a model in which a distinct CD103+DC subset in ILFs is responsible for the induction of Foxp3+Tregs but pDCs are required for the expansion and maintenance of Tregs in mucosal healing.
This potential pathway may well explain many of the findings of mucosal atrophy associated with TPN. Table
Results are mean ± SD, n=5 for each group, P<0.05*, P<0.01** compared to control group, using t-test 310 Aryl Hydrocarbon Receptor Pathway Modulates the Activity of Colitis Violeta Arsenescu, Jian Zhong, Razvan I. Arsenescu, Willem J. de Villiers, Hollie I. Swanson
308
Background: Increasing evidence supports the idea that gene-environment interactions may play a critical role in determining an individual's susceptibility to IBD. One of the strongest modifying factors identified by descriptive epidemiology is exposure to tobacco smoke. Aryl hydrocarbon receptor (AHR) regulates both xenobiotic metabolism and the inflammatory responses. Exposure to dietary environmental agents such as PCB 77 and cigarette smoke activates the AHR leading to increased expression of proinflammatory cytokines in a number of cell types relevant to Inflammatory Bowel Diseases (IBD). We hypothesized that downmodulation of this pathway will ameliorate colitis in a murine IBD model Methods: Acute Colitis was induced in C57/B6 AHR deficient mice and their wild type (WT) counterparts by feeding 3.5% dextran sodium sulfate (DSS) in drinking water for 7 fays followed by 3 days of water only. Disease progression was evaluated by measuring body weight, stool consistency and rectal bleeding. Colon lengths and histology were used to compare the groups. Serum and tissue levels of the acute phase protein serum amyloid A (SAA), pro-inflammatory cytokines IL-1β, IL-12 and adipokines osteopontin (OPN), angiotensin (ANG), rezistin, and the antiinflammatory cytokine IL-10, adiponectin. Apoptotic pathways were evaluated by measuring tissue expression of BCL2, BAD, COX2. Results: Following DSS exposure, AHR -/+ mice exhibited significantly decreased disease activity compared to control mice, as evidenced by reduced rectal bleeding, weight loss, and histological intestinal injury (P<0.001). The colonic epithelium in this group displayed reduced apoptosis and increased proliferation as judged by levels of COX-2, BCL2, BAD, and PCNA (proliferating cell nuclear antigen). Furthermore, serum level of SAA was significantly lowered in AHR -/+ group while proinflammatory adipokines and cytokines IL1, IL-12 decreased significantly in serum and tissue. IL-10 increased significantly in AHR -/+ group both in the serum and colon (P<0.001). Serum and fat adiponectin increased in heterozygote mice. Macrophage infiltration into colonic tissue was attenuated in AHR -/+ group. No significant differences were noted between control WT and AHR -/+ fed water only. Conclusion: activation of the AHR increases the expression of proinflammatory cytokines within the gut and mesenteric fat and thus contributes to the development and/or susceptibility of disease states such as IBD. Interestingly increased activation of AHR pathway in the context of inflammation promotes apoptosis. AHR agonist free diet, smoking cessation or AHR antagonists are viable strategies for the treatment of IBD.
Glucose-Dependent Insulinotropic Polypeptide (GIP) Enhances Adipocyte Development and Modulates Multiple Downstream Targets of AKT Diane H. Song, T. Carlton Moore, M. Michael Wolfe Background and Objectives: In addition to its role as an incretin hormone, we have previously reported that GIP possesses insulin-mimetic properties by virtue of its ability to inhibit isoproterenol-induced lipolysis in adipocytes. To further delineate the biological significance of GIP, the present studies were directed to characterize the effects of GIP and GIPR in adipocyte development and signaling. Methods: The effects of GIP and its receptor (GIPR) on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-RedO staining, cAMP RIA, immunofluorescence microscopy, and glucose uptake measurements. Results: GIPR protein expression was enhanced early (after 1-2 h) during the differentiation process, and co-incubation with GIP augmented the expression of aP2, a fat cell marker, at the end of the differentiation process (Day 9). Conversely, the suppression of GIPR expression by a specific shRNA attenuated Oil-Red-O staining and aP2 expression, suggesting that GIPR induction may be requisite for adipocyte development. In separate studies, GIP, like insulin, induced Akt activation in a concentration-dependent manner in both 3T3-L1 and human adipocytes. Furthermore, GIP promoted membrane accumulation of GLUT-4 and enhanced [3H]-2-deoxyglucose uptake, both of which are Akt-dependent processes. A downstream target of Akt involves the mammalian target of rapamycin (mTOR) pathway, which incorporates signals from growth factors and nutrients to regulate many biological processes, including ribosome biogenesis and protein synthesis. Dysregulation of the mTOR pathway has been linked to various disorders, such as type 2 diabetes mellitus and malignancy. When adipocytes were incubated in the presence of GIP, the phosphorylation of two downstream components of the mTOR complex 1, ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP-1), were enhanced. These stimulatory effects were significantly attenuated by rapamycin, an mTOR pathway inhibitor. Finally, the effects of GIP on Akt and p70S6K phosphorylation were abolished in cells with repressed GIPR expression, indicating GIP/GIPR-specific signaling events. Summary and Conclusions: GIP signaling appears to play an instrumental role in fat cell development and appears to enhance glucose uptake in adipocytes, at least in part, through modulation of the critical mediator Akt. Furthermore, GIP appears to stimulate Akt downstream targets of the mTOR pathway. Thus, GIP signaling may serve as an important hormone linking nutrient storage, lipid homeostasis, enhanced protein translation, and cell survival, and may contribute to the pathogenesis of obesity.
311 Altered Duodenal Sweet Taste Receptor Expression in Diabetic Hyperglycemia Kate Sutherland, Stuart M. Brierley, Chris Rayner, Michael Horowitz, L. Ashley Blackshaw, Richard L. Young
309
Background: The molecular mechanisms for sensing carbohydrate in the human gut are poorly understood and may be particularly relevant in diabetes where blood glucose control is impaired. Sweet taste mechanisms in the tongue, in contrast, are well defined and involve activation of heterodimeric G protein coupled receptors (T1R2 and T1R3), signaling via the taste G-protein α-gustducin (α-GD) and gating of the transient receptor potential ion channel, TRPM5. We recently showed that sweet taste receptors are also expressed in a region and cell specific manner in the gastrointestinal epithelium of mice [1], consistent with a role in nutrient-evoked vagal reflexes. However, no reports exist on the quantitative regional expression of sweet taste receptors in the small intestine in healthy humans or type-2 diabetic patients. Methods: Biopsies were obtained with consent from the distal esophagus, gastric fundus, body and antrum, duodenum and proximal jejunum of type-2 diabetic (n = 7) and non-diabetic patients (n = 6) undergoing endoscopy and enteroscopy, and were histologically normal. RNA was extracted from these biopsies and the absolute copy number of transcripts for sweet taste receptors was measured by quantitative RT-PCR (QPCR). Results: T1R2, αGD and TRPM5 transcripts were expressed in a region-specific manner in the human gut, with peak expression in the duodenum. Absolute levels of transcript in the duodenum correlated inversely with fasting blood glucose (5-10 mmol/l) in diabetic patients (p < 0.05 for each). In contrast, transcript levels did not differ between diabetic and non-diabetic patients, nor were they related to age, gender, body mass index, duration of diabetes or glycated hemoglobin. Conclusions: We provide quantitative evidence that sweet taste receptors are expressed in a region specific manner in the human gastrointestinal epithelium, and that hyperglycemia in diabetes is associated with reduced expression of duodenal taste receptors. We speculate that this may be an adaptive response to hyperglycemia, to reduce the stimulation of luminal glucose absorption, but ongoing studies are required to investigate the regulation and functional consequences of changes in sweet taste receptor expression in diabetes. 1. Sutherland K.S. et al Am J Physiol 2007 292(5):G1420-5. Supported by the University of Adelaide.
E-Cadherin/Beta-Catenin Dissociation Results in Altered Wnt/β-Catenin Signaling: A Mechanism for Villus Atrophy in a Mouse Model of Total Parental Nutrition Yongjia Feng, Xiaoyi Sun, Hua Yang, Keisuke Nose, Hiroyuki Koga, Daniel H. Teitelbaum Background: Total Parenteral nutrition (TPN) induces a decline in epithelial cell (EC) proliferation, leading to atrophy of the small intestinal mucosa. Previously we noted that EC surface expression of E-cadherin was decreased in a mouse TPN model. E-cadherin is a critical for maintaining barrier function and recruiting mucosal lymphocytes. As a large portion of α- and β-catenin are tightly associated with E-cadherin, we wondered whether alterations in these two catenins could contribute to the loss of E-cadherin. We further hypothesized that altered β-catenin expression would affect the Wnt signaling pathway, which could help explain the loss of EC proliferation. Methods: E-Cadhernin, β-catenin, αcatenin and downstream transcription factors of tcf-4 activation, (c-myc and cyclin D1) were measured with real-time PCR and western blot in C57BL/6J male mice which received enteral nutrition (Control) or TPN for 7 days. Immunofluence and confocal microscopy were used to localize factors. Mucosal morphology and EC proliferation were measured with H&E and BrdU staining. Results: TPN significantly down-regulated RNA and protein expression of E-Cadherin, β-catenin and α-catenin (Table). TPN also up-regulated phosphorylated (p)-β-catenin expression (which provides for a recognition site on GSK3 and βcatenin ubiquination). Immunofluorescent staining confirmed this by showing that EC cytoplasmic β-catenin was diminished in TPN mice, along with a near total loss of cell membrane β-catenin. We next examined the implications of β-catenin loss by looking at down-stream products of tcf-4 activation. We noted a significant down-regulation in mRNA expression of c-myc and cyclin D1 (Table), suggesting decreased tcf4 transcription. We also noted increased Axin1 expression, which could also contribute to the loss of β-catenin. Finally, we looked for morphologic implications of decreased tcf-4 activation, and noted a decline in villus height (397.4±49.5 vs 308.4±65.2µm), crypt depth (121.4±27.2 vs 76.1±7.4µm),and EC proliferation rates (24.4±4.2% vs 13.5±2.4%) (Control vs TPN). Conclusions: TPN led to a marked up-regulation of β-catenin phosphorylation. This suggests a mechanism for loss of cytosolic β-catenin and the subsequent decline in tcf-4 activation.
AGA Abstracts
A-44
which increased to 10-12% of all DCs. However, the increase in CD103+DCs was shortlived. Instead, the healing phase of colitis was characterized by the influx of pDCs which increased over 10-fold during a 7 day recovery period and stayed elevated for over 3 weeks. The influx of pDCs was accompanied by the recruitment of Foxp3+Tregs in the colon which were found dispersed throughout the lamina propria and steadily increased during mucosal healing. Surprisingly, IL-10 deficient mice demonstrated the enhanced recruitment of pDCs (25-30% of all DCs) and Foxp3+Tregs even before the onset of colitis but without an increased in CD103+DCs. Onset of macroscopic disease in IL-10 deficient mice was characterized by the additional influx of CD11b-DCs. CONCLUSION: Mucosal defense activation involves the recruitment of specific DC subsets which have defined roles in the control of acute inflammation and support of mucosal healing. Surprisingly, DC subset recruitment was similar in colitis induced by IEC monolayer disruption and T cell mediated disease in IL10 deficient mice. This suggests common response programs to mucosal damage. We would like to propose a model in which a distinct CD103+DC subset in ILFs is responsible for the induction of Foxp3+Tregs but pDCs are required for the expansion and maintenance of Tregs in mucosal healing.
This potential pathway may well explain many of the findings of mucosal atrophy associated with TPN. Table
Results are mean ± SD, n=5 for each group, P<0.05*, P<0.01** compared to control group, using t-test 310 Aryl Hydrocarbon Receptor Pathway Modulates the Activity of Colitis Violeta Arsenescu, Jian Zhong, Razvan I. Arsenescu, Willem J. de Villiers, Hollie I. Swanson
308
Background: Increasing evidence supports the idea that gene-environment interactions may play a critical role in determining an individual's susceptibility to IBD. One of the strongest modifying factors identified by descriptive epidemiology is exposure to tobacco smoke. Aryl hydrocarbon receptor (AHR) regulates both xenobiotic metabolism and the inflammatory responses. Exposure to dietary environmental agents such as PCB 77 and cigarette smoke activates the AHR leading to increased expression of proinflammatory cytokines in a number of cell types relevant to Inflammatory Bowel Diseases (IBD). We hypothesized that downmodulation of this pathway will ameliorate colitis in a murine IBD model Methods: Acute Colitis was induced in C57/B6 AHR deficient mice and their wild type (WT) counterparts by feeding 3.5% dextran sodium sulfate (DSS) in drinking water for 7 fays followed by 3 days of water only. Disease progression was evaluated by measuring body weight, stool consistency and rectal bleeding. Colon lengths and histology were used to compare the groups. Serum and tissue levels of the acute phase protein serum amyloid A (SAA), pro-inflammatory cytokines IL-1β, IL-12 and adipokines osteopontin (OPN), angiotensin (ANG), rezistin, and the antiinflammatory cytokine IL-10, adiponectin. Apoptotic pathways were evaluated by measuring tissue expression of BCL2, BAD, COX2. Results: Following DSS exposure, AHR -/+ mice exhibited significantly decreased disease activity compared to control mice, as evidenced by reduced rectal bleeding, weight loss, and histological intestinal injury (P<0.001). The colonic epithelium in this group displayed reduced apoptosis and increased proliferation as judged by levels of COX-2, BCL2, BAD, and PCNA (proliferating cell nuclear antigen). Furthermore, serum level of SAA was significantly lowered in AHR -/+ group while proinflammatory adipokines and cytokines IL1, IL-12 decreased significantly in serum and tissue. IL-10 increased significantly in AHR -/+ group both in the serum and colon (P<0.001). Serum and fat adiponectin increased in heterozygote mice. Macrophage infiltration into colonic tissue was attenuated in AHR -/+ group. No significant differences were noted between control WT and AHR -/+ fed water only. Conclusion: activation of the AHR increases the expression of proinflammatory cytokines within the gut and mesenteric fat and thus contributes to the development and/or susceptibility of disease states such as IBD. Interestingly increased activation of AHR pathway in the context of inflammation promotes apoptosis. AHR agonist free diet, smoking cessation or AHR antagonists are viable strategies for the treatment of IBD.
Glucose-Dependent Insulinotropic Polypeptide (GIP) Enhances Adipocyte Development and Modulates Multiple Downstream Targets of AKT Diane H. Song, T. Carlton Moore, M. Michael Wolfe Background and Objectives: In addition to its role as an incretin hormone, we have previously reported that GIP possesses insulin-mimetic properties by virtue of its ability to inhibit isoproterenol-induced lipolysis in adipocytes. To further delineate the biological significance of GIP, the present studies were directed to characterize the effects of GIP and GIPR in adipocyte development and signaling. Methods: The effects of GIP and its receptor (GIPR) on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-RedO staining, cAMP RIA, immunofluorescence microscopy, and glucose uptake measurements. Results: GIPR protein expression was enhanced early (after 1-2 h) during the differentiation process, and co-incubation with GIP augmented the expression of aP2, a fat cell marker, at the end of the differentiation process (Day 9). Conversely, the suppression of GIPR expression by a specific shRNA attenuated Oil-Red-O staining and aP2 expression, suggesting that GIPR induction may be requisite for adipocyte development. In separate studies, GIP, like insulin, induced Akt activation in a concentration-dependent manner in both 3T3-L1 and human adipocytes. Furthermore, GIP promoted membrane accumulation of GLUT-4 and enhanced [3H]-2-deoxyglucose uptake, both of which are Akt-dependent processes. A downstream target of Akt involves the mammalian target of rapamycin (mTOR) pathway, which incorporates signals from growth factors and nutrients to regulate many biological processes, including ribosome biogenesis and protein synthesis. Dysregulation of the mTOR pathway has been linked to various disorders, such as type 2 diabetes mellitus and malignancy. When adipocytes were incubated in the presence of GIP, the phosphorylation of two downstream components of the mTOR complex 1, ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP-1), were enhanced. These stimulatory effects were significantly attenuated by rapamycin, an mTOR pathway inhibitor. Finally, the effects of GIP on Akt and p70S6K phosphorylation were abolished in cells with repressed GIPR expression, indicating GIP/GIPR-specific signaling events. Summary and Conclusions: GIP signaling appears to play an instrumental role in fat cell development and appears to enhance glucose uptake in adipocytes, at least in part, through modulation of the critical mediator Akt. Furthermore, GIP appears to stimulate Akt downstream targets of the mTOR pathway. Thus, GIP signaling may serve as an important hormone linking nutrient storage, lipid homeostasis, enhanced protein translation, and cell survival, and may contribute to the pathogenesis of obesity.
311 Altered Duodenal Sweet Taste Receptor Expression in Diabetic Hyperglycemia Kate Sutherland, Stuart M. Brierley, Chris Rayner, Michael Horowitz, L. Ashley Blackshaw, Richard L. Young
309
Background: The molecular mechanisms for sensing carbohydrate in the human gut are poorly understood and may be particularly relevant in diabetes where blood glucose control is impaired. Sweet taste mechanisms in the tongue, in contrast, are well defined and involve activation of heterodimeric G protein coupled receptors (T1R2 and T1R3), signaling via the taste G-protein α-gustducin (α-GD) and gating of the transient receptor potential ion channel, TRPM5. We recently showed that sweet taste receptors are also expressed in a region and cell specific manner in the gastrointestinal epithelium of mice [1], consistent with a role in nutrient-evoked vagal reflexes. However, no reports exist on the quantitative regional expression of sweet taste receptors in the small intestine in healthy humans or type-2 diabetic patients. Methods: Biopsies were obtained with consent from the distal esophagus, gastric fundus, body and antrum, duodenum and proximal jejunum of type-2 diabetic (n = 7) and non-diabetic patients (n = 6) undergoing endoscopy and enteroscopy, and were histologically normal. RNA was extracted from these biopsies and the absolute copy number of transcripts for sweet taste receptors was measured by quantitative RT-PCR (QPCR). Results: T1R2, αGD and TRPM5 transcripts were expressed in a region-specific manner in the human gut, with peak expression in the duodenum. Absolute levels of transcript in the duodenum correlated inversely with fasting blood glucose (5-10 mmol/l) in diabetic patients (p < 0.05 for each). In contrast, transcript levels did not differ between diabetic and non-diabetic patients, nor were they related to age, gender, body mass index, duration of diabetes or glycated hemoglobin. Conclusions: We provide quantitative evidence that sweet taste receptors are expressed in a region specific manner in the human gastrointestinal epithelium, and that hyperglycemia in diabetes is associated with reduced expression of duodenal taste receptors. We speculate that this may be an adaptive response to hyperglycemia, to reduce the stimulation of luminal glucose absorption, but ongoing studies are required to investigate the regulation and functional consequences of changes in sweet taste receptor expression in diabetes. 1. Sutherland K.S. et al Am J Physiol 2007 292(5):G1420-5. Supported by the University of Adelaide.
E-Cadherin/Beta-Catenin Dissociation Results in Altered Wnt/β-Catenin Signaling: A Mechanism for Villus Atrophy in a Mouse Model of Total Parental Nutrition Yongjia Feng, Xiaoyi Sun, Hua Yang, Keisuke Nose, Hiroyuki Koga, Daniel H. Teitelbaum Background: Total Parenteral nutrition (TPN) induces a decline in epithelial cell (EC) proliferation, leading to atrophy of the small intestinal mucosa. Previously we noted that EC surface expression of E-cadherin was decreased in a mouse TPN model. E-cadherin is a critical for maintaining barrier function and recruiting mucosal lymphocytes. As a large portion of α- and β-catenin are tightly associated with E-cadherin, we wondered whether alterations in these two catenins could contribute to the loss of E-cadherin. We further hypothesized that altered β-catenin expression would affect the Wnt signaling pathway, which could help explain the loss of EC proliferation. Methods: E-Cadhernin, β-catenin, αcatenin and downstream transcription factors of tcf-4 activation, (c-myc and cyclin D1) were measured with real-time PCR and western blot in C57BL/6J male mice which received enteral nutrition (Control) or TPN for 7 days. Immunofluence and confocal microscopy were used to localize factors. Mucosal morphology and EC proliferation were measured with H&E and BrdU staining. Results: TPN significantly down-regulated RNA and protein expression of E-Cadherin, β-catenin and α-catenin (Table). TPN also up-regulated phosphorylated (p)-β-catenin expression (which provides for a recognition site on GSK3 and βcatenin ubiquination). Immunofluorescent staining confirmed this by showing that EC cytoplasmic β-catenin was diminished in TPN mice, along with a near total loss of cell membrane β-catenin. We next examined the implications of β-catenin loss by looking at down-stream products of tcf-4 activation. We noted a significant down-regulation in mRNA expression of c-myc and cyclin D1 (Table), suggesting decreased tcf4 transcription. We also noted increased Axin1 expression, which could also contribute to the loss of β-catenin. Finally, we looked for morphologic implications of decreased tcf-4 activation, and noted a decline in villus height (397.4±49.5 vs 308.4±65.2µm), crypt depth (121.4±27.2 vs 76.1±7.4µm),and EC proliferation rates (24.4±4.2% vs 13.5±2.4%) (Control vs TPN). Conclusions: TPN led to a marked up-regulation of β-catenin phosphorylation. This suggests a mechanism for loss of cytosolic β-catenin and the subsequent decline in tcf-4 activation.
AGA Abstracts
A-44