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T1928 Anatomy of Gastric Vagal Afferent-Leptin Interactions in Normal and Obese Mice
AGA Abstracts
had no effect in IEC-6 cells, suggesting less functional GLUT2 in IEC-6 cells. CONCLUSION: Constitutive GLUT2 in the apical membrane and translocation of cytoplasmic GLUT2 to the apical membrane appears responsible for enhanced glc uptake at greater glc concentrations (≥20mM). GLUT2 translocation in intestinal cell lines appears to occur on an intact cytoskeletal system via activation of PKC.
T1927 Legume Lectin Rapidly Enters Intestinal Epithelial Cells and Disrupts Tight Junction Localization of ZO-1 Karol Dokladny, Balamurugan Ramadass, Henry C. Lin, Pope Moseley Legume lectin toxicity may represent the most common form of intestinal toxicant injury, causing diarrhea and altering gut epithelial functions. Since lectins are sugar-binding proteins, the most commonly understood mechanisms of legume lectin toxicity are those related to extracellular binding to carbohydrate moieties, disruption of digestive and absorptive mechanisms and interference with epithelial secretion. Previously, using immunohistochemistry of harvested gut samples, phytohemagglutinin (PHA), the lectin of red kidney beans, was reported to be visible in the enterocyte at 12h (B. J. Nutr. 2006; 95:105). However, how rapidly PHA is taken up by the intestinal epithelial cells and what effect PHA exerts on tight junction localization of ZO-1 is not known. Aim: Our aim was to test the hypothesis that PHA may enter the intestinal epithelial cells rapidly and this may have a profound effect on tight junction localization of ZO-1. Methods: Caco-2 cells were exposed to the PHA (200 μg/ml) conjugated with FITC with measurements made at 1, 3, 24 and 48h time points using immunofluorescent microscopy where blue was DAPI staining for nucleus and green was PHA-FITC. Results: PHA appeared inside cells as soon as 1h after lectin treatment (Figure). Interestingly, PHA concentrated mainly in the cytosol around the nucleus and appeared as small dots. At 3h time point, PHA intensity increased significantly within cells although the lectin was still cytosolic in location around a nucleus. At 24 and 48h timepoint, lectin was present all over the cell. Lectin exposure (200 μg/ml for 48 h) caused a marked disruption in junctional localization of ZO-1 protein with a visible discontinuity and gaps in ZO-1 localization. Exposure of Caco-2 cells to heat-denatured lectin produced no effect on ZO-1 appearance in tight junction localization. Lectin exposure resulted in a significant decrease in ZO-1 protein expression measured by Western blot analysis. Conclusions: The legume lectin PHA enters intestinal epithelial cell rapidly and the process of entering the cells progresses over time. The toxic effects of legume lectin may depend on its intracellular actions affecting tight junction proteins. Dr. Lin's research is supported by NIH, VA Research and the Department of Defense.
T1925 Anti-Apoptotic PI3K/AKT Signaling Activated by Sodium/Glucose Transporter 1 Prevents Epithelial Barrier Damage and Bacterial Translocation in Intestinal Ischemic Rats Ching-Ying Huang, Jong-Kai Hsiao, Yen-Zhen Lu, Linda C.H. Yu Intestinal ischemia/reperfusion (I/R)-induced sepsis remains a threat to the survival of critically ill patients and those undergoing major cardiovascular and abdominal surgery. I/R caused enterocytic cell death and bacterial translocation (BT) to extraintestinal organs. Our previous studies demonstrated that sodium/glucose transporter 1 (SGLT1) rescues enterocytes from permeability disruption after apoptosis stimulated by bacterial endotoxin and parasitic products In Vitro. The aim is to investigate whether SGLT1 glucose uptake protects against ischemia-induced epithelial barrier damage in rats and to explore the glucose-mediated cellular survival pathways. Ischemic challenge by occlusion of superior mesenteric artery caused rapid onset of enterocyte apoptosis prior to villous destruction. Intestinal infusion with a pan-caspase inhibitor (ZVAD) decreased I/R-triggered gut permeability rise determined by using a novel In Vivo assay based on magnetic resonance imaging technique. Augmentation of BT and mucosal inflammation, including heightened TNFα and MIP-1 levels and MPO activity, were also caspase-dependent. Luminal administration of glucose reduced the level of epithelial apoptosis caused by I/R, and ablated the subsequent increase of gut permeability, BT and inflammation. Phloridzin dose-dependently inhibited the glucose-mediated protection, suggesting a role for SGLT1. No effect was seen by mannitol, 3-OMG or glutamate when given in place of glucose, indicating that glycolytic pathway is partly involved in the protective mechanism. Glucose administration induced translocation of cytosolic Akt to brush border and subcellular organelles, and increased phosphorylation of Akt on villous epithelial cells. Akt activation leads to a number of anti-apoptotic events, including phosphorylation of mTOR and Bad, and dephosphorylation of p38 MAPK. Pharmacological inhibition of PI3K by wortmannin blocked epithelial Akt signaling and abolished the rescue against cell apoptosis and barrier damage following glucose uptake. Taken together, SGLT1 prevented I/R-induced gut barrier dysfunction and mucosal inflammation, via correcting epithelial apoptosis by activation of PI3K/Akt signaling pathways.
T1928 Anatomy of Gastric Vagal Afferent-Leptin Interactions in Normal and Obese Mice Nicole J. Isaacs, Claudine L. Flach, Richard L. Young, Gary Wittert, Ashley Blackshaw, Amanda J. Page Introduction: The adipocyte derived hormone leptin regulates energy balance. In addition, leptin is also a gut peptide that interacts with vagal afferents. We have recently shown, in mice, that leptin markedly enhances the mechanosensitivity of vagal sensory endings that innervate the gastric mucosa, an effect that is profoundly influenced by changes in even short-term food intake [1]. It is unknown whether long-term alterations in food intake leading to obesity, alters expression of leptin or its receptor in gastric vagal pathways. Aims: To determine in normal and obese mice: 1. leptin protein distribution in gastric epithelium 2. leptin receptor protein distribution and mRNA expression in vagal afferent cell bodies. 3. The presence and distribution of gastric vagal afferent endings. Methods: Mice were fed either a high fat diet (HFD, 60% energy from fat) or a standard laboratory diet (SLD, 7% energy from fat) for 12 weeks. Thereafter vagal afferent endings were identified in the gastric mucosa by anterograde tracing from the nodose ganglion by WGA-HRP. Leptin and leptin receptors were identified in gastric mucosa and nodose ganglion by immunolabeling and quantitative RT-PCR respectively. Results: HFD mice gained 37% weight at 12 weeks compared to 29% weight gain in SLD mice (p < 0.05). Immunolabeling for leptin was similar in both SLD and HFD mice and was localized to the crypts of columnar epithelium at the lesser curvature and at both the apex and crypts of columnar epithelium in regions approaching the greater curvature. Vagal afferent endings in muscular layers of the stomach were reliably detected in both SLD and HFD mice. Vagal endings were also abundant in the gastric mucosa of SLD mice, where they were closely associated with leptin immunopositive cells, but were sparse in HFD mice. Leptin receptor was expressed in 90% of vagal afferent cell bodies, in SLD mice. However, mRNA expression of leptin receptor in the nodose ganglia of HFD and SLD mice was not significantly different. Conclusions: This study demonstrates leptin distribution in the mouse gastric epithelium. Although a HFD does not alter expression of gastric leptin, or leptin receptor in vagal afferents, vagal afferent density and connections with leptin immunopositive cells in the gastric mucosa is profoundly reduced in obese mice. This altered anatomical relationship may lead to changes in vagally mediated satiety signals in obesity. References: [1] Page et al. Neurogastro. Motil. 2008; 2(s2): 33 Supported by University of Adelaide and NHMRC Australia
T1926 Removal of Enteral Nutrition With Total Parenteral Nutrition in Mice Leads to Changes in Bacterial Flora and an Associated Increased Toll-Like Receptors in the Small Intestinal Lamina Propria Eiichi A. Miyasaka, Katie Lynn Mason, Gary B. Huffnagle, Daniel H. Teitelbaum Introduction: Healthy intestinal epithelium is in equilibrium with its resident microbial flora, with which it interacts via host toll-like receptors (TLR). We have previously shown that the mouse intestinal epithelium is in a pro-inflammatory state when deprived of enteral nutrition by giving total parenteral nutrition (TPN). We hypothesized that TPN disrupts normal bacterial flora, leading to changes in TLR signaling, which may contribute to this pro-inflammatory state. Methods: Male C57BL/6 mice were intravenously cannulated and given either TPN or saline with enteral chow (Control) for 7 days (n=6/group). Terminal restriction fragment length polymorphism (T-RFLP) techniques were used to assess the bacterial fingerprint in small and large bowel mucosa. Extracted DNA underwent PCR amplification using 6-carboxyfluorescein labeled 8F forward primer and unlabeled 1492R primer for the 16S ribosome. PRC products were digested by MspI, then fragments were analyzed to generate dendrograms and Bray-Curtis distances. The lamina propria lymphocytes (LPL) were isolated and examined for mRNA abundance of TLR and cytokines shown in the table. CD4+ FOXP3+ T-regulatory cell (Treg) percentage was assessed by flow cytometry. Results were analyzed using analysis of similarities or the unpaired T-test. Results: TRFLP analysis showed significant differences in mucosal bacterial composition between control and TPN samples in both the small (p = 0.003) and large bowel (p = 0.005). mRNA abundance of selected LPL TLRs and cytokines are shown in the Table. A marked increase in pro-inflammatory cytokines was seen. To further examine the potential etiology of the cytokine changes, the Treg population of the LPL was examined. TPN mice had significant decreases in both FOXP3 mRNA expression and Treg numbers (12.7±1.4% versus 4.7±1.3%, p = 0.003). Conclusions: The bacterial fingerprint in the intestinal mucosa changed significantly with TPN administration. There was an associated increase in TLR-signaling in the LPL, suggesting that changes in the intestinal flora may lead to up-regulated TLR signaling and a pro-inflammatory state, potentially by depleting the Treg population. These results suggest that methods for controlling these bacterial population shifts may lead to safer administration of TPN to patients. TLR and cytokine mRNA expression
T1929 Role of the Cell Cycle Regulators in Both Intestinal Mucosal Hypertrophy and Atrophy Mouse Models Chaojun Zhang, Hua Yang, Hiroyuki Koga, Daniel H. Teitelbaum Background: Total parenteral nutrition (TPN) results in intestinal mucosal atrophy due to an absence of enteral nutrition, however short bowel syndrome (SBS) induces intestinal mucosal hypertrophy owing to an adaptive response to the loss of epithelium. The mechanisms of those morphological changes are not well understood. To our knowledge, it is currently unknown if the cell cycle regulators are involved in those mechanisms. This study examined the role of cell cycle regulators in both intestinal mucosal hypertrophy and atrophy models. Methods: Mouse model of villous atrophy was induced by the administration of TPN, oral feeding as control group. Villous hypertrophy was created by removal of 55% of the mid-small intestine (SBS), transaction without bowel resection served as sham-operated group. All mice were sacrificed at 7 days. Real time PCR was used to detect the cell cycle regulators mRNA expressions. Western blotting was used to determine the abundance expressions of cell cycle regulators. Intestinal mucosal proliferation was assessed by using
Expression normalized to β-actin. FOXP3 further normalized to stable portion of the T-cell receptor. Values are 10^-2.
AGA Abstracts
S-608
had no effect in IEC-6 cells, suggesting less functional GLUT2 in IEC-6 cells. CONCLUSION: Constitutive GLUT2 in the apical membrane and translocation of cytoplasmic GLUT2 to the apical membrane appears responsible for enhanced glc uptake at greater glc concentrations (≥20mM). GLUT2 translocation in intestinal cell lines appears to occur on an intact cytoskeletal system via activation of PKC.
T1927 Legume Lectin Rapidly Enters Intestinal Epithelial Cells and Disrupts Tight Junction Localization of ZO-1 Karol Dokladny, Balamurugan Ramadass, Henry C. Lin, Pope Moseley Legume lectin toxicity may represent the most common form of intestinal toxicant injury, causing diarrhea and altering gut epithelial functions. Since lectins are sugar-binding proteins, the most commonly understood mechanisms of legume lectin toxicity are those related to extracellular binding to carbohydrate moieties, disruption of digestive and absorptive mechanisms and interference with epithelial secretion. Previously, using immunohistochemistry of harvested gut samples, phytohemagglutinin (PHA), the lectin of red kidney beans, was reported to be visible in the enterocyte at 12h (B. J. Nutr. 2006; 95:105). However, how rapidly PHA is taken up by the intestinal epithelial cells and what effect PHA exerts on tight junction localization of ZO-1 is not known. Aim: Our aim was to test the hypothesis that PHA may enter the intestinal epithelial cells rapidly and this may have a profound effect on tight junction localization of ZO-1. Methods: Caco-2 cells were exposed to the PHA (200 μg/ml) conjugated with FITC with measurements made at 1, 3, 24 and 48h time points using immunofluorescent microscopy where blue was DAPI staining for nucleus and green was PHA-FITC. Results: PHA appeared inside cells as soon as 1h after lectin treatment (Figure). Interestingly, PHA concentrated mainly in the cytosol around the nucleus and appeared as small dots. At 3h time point, PHA intensity increased significantly within cells although the lectin was still cytosolic in location around a nucleus. At 24 and 48h timepoint, lectin was present all over the cell. Lectin exposure (200 μg/ml for 48 h) caused a marked disruption in junctional localization of ZO-1 protein with a visible discontinuity and gaps in ZO-1 localization. Exposure of Caco-2 cells to heat-denatured lectin produced no effect on ZO-1 appearance in tight junction localization. Lectin exposure resulted in a significant decrease in ZO-1 protein expression measured by Western blot analysis. Conclusions: The legume lectin PHA enters intestinal epithelial cell rapidly and the process of entering the cells progresses over time. The toxic effects of legume lectin may depend on its intracellular actions affecting tight junction proteins. Dr. Lin's research is supported by NIH, VA Research and the Department of Defense.
T1925 Anti-Apoptotic PI3K/AKT Signaling Activated by Sodium/Glucose Transporter 1 Prevents Epithelial Barrier Damage and Bacterial Translocation in Intestinal Ischemic Rats Ching-Ying Huang, Jong-Kai Hsiao, Yen-Zhen Lu, Linda C.H. Yu Intestinal ischemia/reperfusion (I/R)-induced sepsis remains a threat to the survival of critically ill patients and those undergoing major cardiovascular and abdominal surgery. I/R caused enterocytic cell death and bacterial translocation (BT) to extraintestinal organs. Our previous studies demonstrated that sodium/glucose transporter 1 (SGLT1) rescues enterocytes from permeability disruption after apoptosis stimulated by bacterial endotoxin and parasitic products In Vitro. The aim is to investigate whether SGLT1 glucose uptake protects against ischemia-induced epithelial barrier damage in rats and to explore the glucose-mediated cellular survival pathways. Ischemic challenge by occlusion of superior mesenteric artery caused rapid onset of enterocyte apoptosis prior to villous destruction. Intestinal infusion with a pan-caspase inhibitor (ZVAD) decreased I/R-triggered gut permeability rise determined by using a novel In Vivo assay based on magnetic resonance imaging technique. Augmentation of BT and mucosal inflammation, including heightened TNFα and MIP-1 levels and MPO activity, were also caspase-dependent. Luminal administration of glucose reduced the level of epithelial apoptosis caused by I/R, and ablated the subsequent increase of gut permeability, BT and inflammation. Phloridzin dose-dependently inhibited the glucose-mediated protection, suggesting a role for SGLT1. No effect was seen by mannitol, 3-OMG or glutamate when given in place of glucose, indicating that glycolytic pathway is partly involved in the protective mechanism. Glucose administration induced translocation of cytosolic Akt to brush border and subcellular organelles, and increased phosphorylation of Akt on villous epithelial cells. Akt activation leads to a number of anti-apoptotic events, including phosphorylation of mTOR and Bad, and dephosphorylation of p38 MAPK. Pharmacological inhibition of PI3K by wortmannin blocked epithelial Akt signaling and abolished the rescue against cell apoptosis and barrier damage following glucose uptake. Taken together, SGLT1 prevented I/R-induced gut barrier dysfunction and mucosal inflammation, via correcting epithelial apoptosis by activation of PI3K/Akt signaling pathways.
T1928 Anatomy of Gastric Vagal Afferent-Leptin Interactions in Normal and Obese Mice Nicole J. Isaacs, Claudine L. Flach, Richard L. Young, Gary Wittert, Ashley Blackshaw, Amanda J. Page Introduction: The adipocyte derived hormone leptin regulates energy balance. In addition, leptin is also a gut peptide that interacts with vagal afferents. We have recently shown, in mice, that leptin markedly enhances the mechanosensitivity of vagal sensory endings that innervate the gastric mucosa, an effect that is profoundly influenced by changes in even short-term food intake [1]. It is unknown whether long-term alterations in food intake leading to obesity, alters expression of leptin or its receptor in gastric vagal pathways. Aims: To determine in normal and obese mice: 1. leptin protein distribution in gastric epithelium 2. leptin receptor protein distribution and mRNA expression in vagal afferent cell bodies. 3. The presence and distribution of gastric vagal afferent endings. Methods: Mice were fed either a high fat diet (HFD, 60% energy from fat) or a standard laboratory diet (SLD, 7% energy from fat) for 12 weeks. Thereafter vagal afferent endings were identified in the gastric mucosa by anterograde tracing from the nodose ganglion by WGA-HRP. Leptin and leptin receptors were identified in gastric mucosa and nodose ganglion by immunolabeling and quantitative RT-PCR respectively. Results: HFD mice gained 37% weight at 12 weeks compared to 29% weight gain in SLD mice (p < 0.05). Immunolabeling for leptin was similar in both SLD and HFD mice and was localized to the crypts of columnar epithelium at the lesser curvature and at both the apex and crypts of columnar epithelium in regions approaching the greater curvature. Vagal afferent endings in muscular layers of the stomach were reliably detected in both SLD and HFD mice. Vagal endings were also abundant in the gastric mucosa of SLD mice, where they were closely associated with leptin immunopositive cells, but were sparse in HFD mice. Leptin receptor was expressed in 90% of vagal afferent cell bodies, in SLD mice. However, mRNA expression of leptin receptor in the nodose ganglia of HFD and SLD mice was not significantly different. Conclusions: This study demonstrates leptin distribution in the mouse gastric epithelium. Although a HFD does not alter expression of gastric leptin, or leptin receptor in vagal afferents, vagal afferent density and connections with leptin immunopositive cells in the gastric mucosa is profoundly reduced in obese mice. This altered anatomical relationship may lead to changes in vagally mediated satiety signals in obesity. References: [1] Page et al. Neurogastro. Motil. 2008; 2(s2): 33 Supported by University of Adelaide and NHMRC Australia
T1926 Removal of Enteral Nutrition With Total Parenteral Nutrition in Mice Leads to Changes in Bacterial Flora and an Associated Increased Toll-Like Receptors in the Small Intestinal Lamina Propria Eiichi A. Miyasaka, Katie Lynn Mason, Gary B. Huffnagle, Daniel H. Teitelbaum Introduction: Healthy intestinal epithelium is in equilibrium with its resident microbial flora, with which it interacts via host toll-like receptors (TLR). We have previously shown that the mouse intestinal epithelium is in a pro-inflammatory state when deprived of enteral nutrition by giving total parenteral nutrition (TPN). We hypothesized that TPN disrupts normal bacterial flora, leading to changes in TLR signaling, which may contribute to this pro-inflammatory state. Methods: Male C57BL/6 mice were intravenously cannulated and given either TPN or saline with enteral chow (Control) for 7 days (n=6/group). Terminal restriction fragment length polymorphism (T-RFLP) techniques were used to assess the bacterial fingerprint in small and large bowel mucosa. Extracted DNA underwent PCR amplification using 6-carboxyfluorescein labeled 8F forward primer and unlabeled 1492R primer for the 16S ribosome. PRC products were digested by MspI, then fragments were analyzed to generate dendrograms and Bray-Curtis distances. The lamina propria lymphocytes (LPL) were isolated and examined for mRNA abundance of TLR and cytokines shown in the table. CD4+ FOXP3+ T-regulatory cell (Treg) percentage was assessed by flow cytometry. Results were analyzed using analysis of similarities or the unpaired T-test. Results: TRFLP analysis showed significant differences in mucosal bacterial composition between control and TPN samples in both the small (p = 0.003) and large bowel (p = 0.005). mRNA abundance of selected LPL TLRs and cytokines are shown in the Table. A marked increase in pro-inflammatory cytokines was seen. To further examine the potential etiology of the cytokine changes, the Treg population of the LPL was examined. TPN mice had significant decreases in both FOXP3 mRNA expression and Treg numbers (12.7±1.4% versus 4.7±1.3%, p = 0.003). Conclusions: The bacterial fingerprint in the intestinal mucosa changed significantly with TPN administration. There was an associated increase in TLR-signaling in the LPL, suggesting that changes in the intestinal flora may lead to up-regulated TLR signaling and a pro-inflammatory state, potentially by depleting the Treg population. These results suggest that methods for controlling these bacterial population shifts may lead to safer administration of TPN to patients. TLR and cytokine mRNA expression
T1929 Role of the Cell Cycle Regulators in Both Intestinal Mucosal Hypertrophy and Atrophy Mouse Models Chaojun Zhang, Hua Yang, Hiroyuki Koga, Daniel H. Teitelbaum Background: Total parenteral nutrition (TPN) results in intestinal mucosal atrophy due to an absence of enteral nutrition, however short bowel syndrome (SBS) induces intestinal mucosal hypertrophy owing to an adaptive response to the loss of epithelium. The mechanisms of those morphological changes are not well understood. To our knowledge, it is currently unknown if the cell cycle regulators are involved in those mechanisms. This study examined the role of cell cycle regulators in both intestinal mucosal hypertrophy and atrophy models. Methods: Mouse model of villous atrophy was induced by the administration of TPN, oral feeding as control group. Villous hypertrophy was created by removal of 55% of the mid-small intestine (SBS), transaction without bowel resection served as sham-operated group. All mice were sacrificed at 7 days. Real time PCR was used to detect the cell cycle regulators mRNA expressions. Western blotting was used to determine the abundance expressions of cell cycle regulators. Intestinal mucosal proliferation was assessed by using
Expression normalized to β-actin. FOXP3 further normalized to stable portion of the T-cell receptor. Values are 10^-2.
AGA Abstracts
S-608