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594 Translocation of Transfected GLUT2 to the Apical Membrane in Rat Intestinal IEC-6 Cells
Values=Mean±SE;*p<0.05 vs WT 594 Translocation of Transfected GLUT2 to the Apical Membrane in Rat Intestinal IEC-6 Cells Ye Zheng, Judith A. Duenes, Hisham G. Qandeel, Michael G. Sarr
592
The mechanism of translocation of the glucose transporter GLUT2 in enterocytes is not fully understood. In this study, we transfected GFP-tagged Glut2 into IEC-6 cells which do not express GLUT2 constitutively. Utilizing this new GFP-GLUT2-IEC6 cell line, we investigated GLUT2 translocation indirectly. HYPOTHESIS: Uptake of glucose into enterocytes at high luminal glucose concentration is by activation of PKC and translocation of GLUT2 to the apical membrane via the microtubular system. METHODS: IEC-6 cells were seeded on a 24-well plate at a density of 4x104 cells/cm2 in cell culture media with 25 mM glucose and left to differentiate for 10 days after confluency. Glucose uptake was measured by incubation of the cell layer with 200 μl of Krebs buffer with different concentrations of glucose (0.550 mM), containing 14C-D-glucose to measure stereospecific transporter-mediated uptake and 3H-L-glucose to measure passive uptake with or without inhibitors for various incubation times (30 s to 10 min). The GLUT2 inhibitor (Phloretin; 1 mM), two different disrupters of microtubular integrity (nocodazole; 2 μM and cytochalasin B; 0.5 μM), and a PKC inhibitor (Calphostin C; 50 nM) were evaluated. Glucose uptake was stopped by adding ice-cold PBS, the cells were washed quickly with PBS twice and solubilized with 300 μl of 0.1N NaOH at 37°C for 30 min, and transporter-mediated (14C) and passive (3H) glucose uptake was measured using a β-scintillation counter. All experiments were repeated on 3 occasions in triplicate. RESULTS: In GFP-GLUT2 expressed cells, the Km and Vmax (20 mM and 1000 nmol/mg protein respectively) were increased greatly compared with that of non-transfected IEC-6 cells (5 mM and 300 nmol/mg protein), suggesting GLUT2 was responsible for the enhanced glucose uptake; this suggestion was supported by phloretin (the GLUT2 inhibitor) which significantly inhibited glucose uptake back to the level of IEC-6; 2 μM of nocodazole and 0.5 μM of cytochalasin B (microtubule disrupters) inhibited glucose uptake by 40-50% at high glucose concentrations 25 and 50 mM) during a 5 min incubation, but not so at lesser glucose concentrations during short incubations; 50 nM calphostin C, an inhibitor of PKC showed a similar inhibition pattern to that of nocodazole. CONCLUSION: Translocation of GLUT2 into the apical membrane of the IEC-6 cells occurs through glucose stimulation and a PKC-dependent signaling pathway and requires integrity of the microtubular system. (Support: NIH DK39337-MGS)
The Facilitative Glucose Transporter GLUT-2 Is Up-Regulated By Mucosal Leptin Through AMPK Involved Mechanisms in the Small Intestine Yassine Sakar, Amal Ait Omar, Corinne Nazaret, Arieh Gertler, Robert A. Ducroc, André Bado Absorption of carbohydrate-digested products by the small intestine constitutes one of the systems involved in delivering glucose to the blood. This glucose absorption is driven by SGLT-1 in preprandial state and by GLUT2 transporter upon ingestion of a sugar-rich meal. Leptin can inhibit SGLT-1 mediated glucose absorption. Here, we examined whether leptin can affect GLUT-2 operating pathway in the passive absorption of glucose and determined the intracellular mechanisms involved. Male Wistar rats and AMPKα2-/- mice were used. Rat jejunal trips and loops were used to quantify uptake and transepithelial apical to basolateral transport of 100 mM galactose added with 14C-galactose in absence and the presence of leptin (5nM) alone or in association with 10 nM L39A/D40A/F41A a leptinreceptor antagonist, or phloretin and cytochalasin B two inhibitors of GLUT-2 or compound C an inhibitor of AMPK. The GLUT-2 and SGLT-1 proteins on brush border membranes and the phosphorylation status of AMPK in mucosa extracts were analysed by Western Blot using specific antibodies. Acute and chronic effects of oral leptin were determined on GLUT2 mRNA levels by qRT-PCR. Finally, jejunal GLUT-2 and SGLT-1 proteins were determined in wild-type (WT) and AMPKα2-/- mice an the effect of leptin on SGLT-1 activity was analysed in jejunum trips mounted in Ussing Chamber and challenge with 10mM glucose. Leptin concentration-dependently increased galactose uptake in jejunal rings, with a maximal effect. Addition of luminal leptin (5nM) had no effect on transport of 14C-mannitol but induced a two-fold increase (P<0.01) in galactose transport in jejunum loops In Vitro. The leptin stimulation was inhibited by a leptin-receptor specific L39A/D40A/F41A, suppressed by phloretin and 20μM cytochalasin B. It was also reduced by 40μM compound C consistent with the leptin stimulation of AMPK phosphorylation. Leptin-stimulated galactose transport was paralleled with increased GLUT-2 and reduced SGLT-1 abundance at the BBM. Acute and chronic oral leptin induced a 2-fold increase in GLUT2 mRNA levels (P<0.01). AMPKα2-/-mice exhibited a 3-fold reduced GLUT2 and a 2.5-fold increased SGLT-1 proteins in jejunum. The increase in SGLT-1 was consistent with the increase in glucose-induced Isc compared to WT (P<0.01). Interestingly, glucose-induced Isc in AMPKα2-/- jejunum was unsensitive to mucosal leptin in comparison to WT jejunum. We conclude that the facilitative GLUT-2 transporter is a direct target of leptin operating through its apical receptor coupled in a critically manner to AMPK pathways. These data may have physiological relevance in the context of increased consumption of sugars.
595 The Role of Luminal Tastants and Olfactants in the Regulation of Gut Neuroendocrine Cell Secretion and the Implications for Gastrointestinal Health and Disease Irvin M. Modlin, Bjorn I. Gustafsson, Zakiya-Luna Siddique, Ignat Drozdov, Roswitha Pfragner, Mark Kidd Background: Neuroendocrine cells of the diffuse neuroendocrine cell system express luminal G-protein coupled receptors and transporters for amines and salts and are considered to function as luminal-sensors. We postulated that “open” neuroendocrine cells would respond to agents in the diet thus regulating GI physiology, particularly gastric acid secretion and motility in the GI tract. Methods: For gastric studies, antral mucosa and a pure (>97%) FACS-sorted gastrin cell preparation was studied. For intestinal studies, a perfusion model, pure EC cells and a neoplastic cell line (KRJ-I) were studied. Gastrin and serotonin secretion in response to aromatic, sweet-tasting amino acids, bitter ligands (denatonium) and bile salts (sodium deoxycholate - SDC) were assessed and signaling pathways defined. Results: In the stomach, uM concentrations of sweet and bitter ligands as well as SDC stimulated gastrin release (>2-fold) and gastrin transcription (after 30 mins) from antral mucosa. Isolated G cell preparations were ~1,000 fold more sensitive (EC50=1.2-3.9nM) for gastrin release and transcription. In the small intestine, perfusion with 10uM concentrations of these ligands stimulated serotonin release (2-3-fold) and transcription of the rate-limiting serotonin synthesis enzyme, Tph1 (1.8-2.1-fold). Similarly to the stomach, both isolated EC cell preparations and KRJ-I were 500-1,200x more sensitive to these agents. In addition, stimulation of KRJ-I cells with beta-casomorphin, a milk-derived product that mimics opioid receptor ligands, inhibited serotonin release (IC50=22nM). These effects could be reversed (40-90%) by the PKA inhibitor H-89 (10uM) and by PD98059 (a MEK1 inhibitor: 10uM). Conclusion: Gastrin and serotonin secreting neuroendocrine cells in the antrum and intestine are sensitive to diet-associated ligands and exhibit transcription and release of their protein and amine products. These effects are PKA- and MAPK-dependent and can be reversed by beta-casomorphin. The delineation of dietary factors that modulate GI neuroendocrine luminal-sensory function provides a novel opportunity to define and evaluate the role of ingested agents on normal gut physiology as well as their role in abnormal gut function including irritable bowel syndrome and obesity related disorders.
593 Enteric Nematode Infection Induces a “Lean” Epithelial Cell Phenotype Through Differential Regulation of Glucose Transporter Expression Kathleen B. Madden, Diana C. Riera, Aiping Zhao, Jennifer A. Stiltz, Ildy M. Katona, Joseph F. Urban, Terez Shea-Donohue Enteric nematode infection upregulates Th2 cytokines, such as IL-4 and IL-13, as well as alternatively activated macrophages (AAMΦ). This is associated with stereotypic STAT6dependent changes in intestinal epithelial cell responses including increased permeability, and decreased glucose absorption. Glucose transport in the small intestine is coordinated through several transporters, including SGLT1 and GLUT-2. Aim: To investigate the mechanisms involved in the regulation of glucose transporter expression that contribute to infectioninduced alterations in glucose absorption. Methods: BALB/c or STAT6-/- mice were either infected with the rodent nematode Nippostrongylus brasiliensis (Nb) or remained uninfected (WT), and were studied 9 days later. Jejunal samples were taken for both real-time PCR and laser capture microdissection (LCM). Results: Nb infection did not alter SGLT1 expression. GLUT-2 expression was decreased by approximately 90% in Nb-infected mice, compared to WT controls, and was STAT6-independent (Table 1). This result may be attributed to a reduction in taste receptor expression by approximately 90% (data not shown), which in turn regulates GLUT-2 expression. In contrast, there was a STAT6-dependent, 11-fold increase in GLUT-1 expression, a transporter that is upregulated by stress (Table 1). To further investigate the cell populations responsible for the observed increase in GLUT-1 expression, real-time PCR was performed on LCM samples of jejunal epithelium and lamina propria from WT and Nb-infected mice. GLUT-1 expression revealed a 46-fold increase in the epithelium, and a 15-fold increase in the immune cells in the lamina propria. Nb infection also induced 7-fold increase in insulin growth factor (IGF)-1α. IGF-1α is produced by macrophages, binds to epithelial cells and stimulates hypoxia inducible factor (HIF)-1α. Infection with Nb increased expression of HIF-1α, which in turn upregulated GLUT-1 expression. Conclusions: Enteric nematode infection induces a “lean” epithelial cell phenotype, with components that are non-immune (STAT6-independent downregulation of GLUT2) and immune, which involve AAMΦ (STAT6-dependent upregulation of GLUT-1 expression). Such changes in the expression of glucose transporters induced by enteric nematode infection may explain, in part, the ability of these infectious agents to prevent the development of autoimmune conditions such as diabetes and IBD.
A-95
AGA Abstracts
AGA Abstracts
CD40 when compared to CD11b+ and CD11b-Ly6c- DCs. FACS sorted colonic pDCs produce IL-10 in response to TLR signals and regulate IL-10 secretion during antigenspecific activation of naïve CD4+ T cells. T cells expressing IL-10 were found to be a subset of Foxp3+ Tregs which emerged in the lamina propria during the recovery from colitis. Depletion of pDCs did not impact the recruitment of Foxp3+ Tregs, but impaired IL-10 secretion by Foxp3+ Tregs and delayed mucosal repair. The regulatory function of pDCs during mucosal inflammation also included the control of intestine-specific secretion of IL17 but not IFN-γ by CD4+Foxp3- T cells. CONCLUSION: During colitis, pDCs become a major DC subset in the lamina propria and direct mucosa-specific T cell functions essential for the resolution of inflammation. Our results demonstrate that pDCs display a distinct function in the colon in defining intestine-specific adaptations of T effectors and Foxp3+ Tregs to mucosal inflammation.
592
The mechanism of translocation of the glucose transporter GLUT2 in enterocytes is not fully understood. In this study, we transfected GFP-tagged Glut2 into IEC-6 cells which do not express GLUT2 constitutively. Utilizing this new GFP-GLUT2-IEC6 cell line, we investigated GLUT2 translocation indirectly. HYPOTHESIS: Uptake of glucose into enterocytes at high luminal glucose concentration is by activation of PKC and translocation of GLUT2 to the apical membrane via the microtubular system. METHODS: IEC-6 cells were seeded on a 24-well plate at a density of 4x104 cells/cm2 in cell culture media with 25 mM glucose and left to differentiate for 10 days after confluency. Glucose uptake was measured by incubation of the cell layer with 200 μl of Krebs buffer with different concentrations of glucose (0.550 mM), containing 14C-D-glucose to measure stereospecific transporter-mediated uptake and 3H-L-glucose to measure passive uptake with or without inhibitors for various incubation times (30 s to 10 min). The GLUT2 inhibitor (Phloretin; 1 mM), two different disrupters of microtubular integrity (nocodazole; 2 μM and cytochalasin B; 0.5 μM), and a PKC inhibitor (Calphostin C; 50 nM) were evaluated. Glucose uptake was stopped by adding ice-cold PBS, the cells were washed quickly with PBS twice and solubilized with 300 μl of 0.1N NaOH at 37°C for 30 min, and transporter-mediated (14C) and passive (3H) glucose uptake was measured using a β-scintillation counter. All experiments were repeated on 3 occasions in triplicate. RESULTS: In GFP-GLUT2 expressed cells, the Km and Vmax (20 mM and 1000 nmol/mg protein respectively) were increased greatly compared with that of non-transfected IEC-6 cells (5 mM and 300 nmol/mg protein), suggesting GLUT2 was responsible for the enhanced glucose uptake; this suggestion was supported by phloretin (the GLUT2 inhibitor) which significantly inhibited glucose uptake back to the level of IEC-6; 2 μM of nocodazole and 0.5 μM of cytochalasin B (microtubule disrupters) inhibited glucose uptake by 40-50% at high glucose concentrations 25 and 50 mM) during a 5 min incubation, but not so at lesser glucose concentrations during short incubations; 50 nM calphostin C, an inhibitor of PKC showed a similar inhibition pattern to that of nocodazole. CONCLUSION: Translocation of GLUT2 into the apical membrane of the IEC-6 cells occurs through glucose stimulation and a PKC-dependent signaling pathway and requires integrity of the microtubular system. (Support: NIH DK39337-MGS)
The Facilitative Glucose Transporter GLUT-2 Is Up-Regulated By Mucosal Leptin Through AMPK Involved Mechanisms in the Small Intestine Yassine Sakar, Amal Ait Omar, Corinne Nazaret, Arieh Gertler, Robert A. Ducroc, André Bado Absorption of carbohydrate-digested products by the small intestine constitutes one of the systems involved in delivering glucose to the blood. This glucose absorption is driven by SGLT-1 in preprandial state and by GLUT2 transporter upon ingestion of a sugar-rich meal. Leptin can inhibit SGLT-1 mediated glucose absorption. Here, we examined whether leptin can affect GLUT-2 operating pathway in the passive absorption of glucose and determined the intracellular mechanisms involved. Male Wistar rats and AMPKα2-/- mice were used. Rat jejunal trips and loops were used to quantify uptake and transepithelial apical to basolateral transport of 100 mM galactose added with 14C-galactose in absence and the presence of leptin (5nM) alone or in association with 10 nM L39A/D40A/F41A a leptinreceptor antagonist, or phloretin and cytochalasin B two inhibitors of GLUT-2 or compound C an inhibitor of AMPK. The GLUT-2 and SGLT-1 proteins on brush border membranes and the phosphorylation status of AMPK in mucosa extracts were analysed by Western Blot using specific antibodies. Acute and chronic effects of oral leptin were determined on GLUT2 mRNA levels by qRT-PCR. Finally, jejunal GLUT-2 and SGLT-1 proteins were determined in wild-type (WT) and AMPKα2-/- mice an the effect of leptin on SGLT-1 activity was analysed in jejunum trips mounted in Ussing Chamber and challenge with 10mM glucose. Leptin concentration-dependently increased galactose uptake in jejunal rings, with a maximal effect. Addition of luminal leptin (5nM) had no effect on transport of 14C-mannitol but induced a two-fold increase (P<0.01) in galactose transport in jejunum loops In Vitro. The leptin stimulation was inhibited by a leptin-receptor specific L39A/D40A/F41A, suppressed by phloretin and 20μM cytochalasin B. It was also reduced by 40μM compound C consistent with the leptin stimulation of AMPK phosphorylation. Leptin-stimulated galactose transport was paralleled with increased GLUT-2 and reduced SGLT-1 abundance at the BBM. Acute and chronic oral leptin induced a 2-fold increase in GLUT2 mRNA levels (P<0.01). AMPKα2-/-mice exhibited a 3-fold reduced GLUT2 and a 2.5-fold increased SGLT-1 proteins in jejunum. The increase in SGLT-1 was consistent with the increase in glucose-induced Isc compared to WT (P<0.01). Interestingly, glucose-induced Isc in AMPKα2-/- jejunum was unsensitive to mucosal leptin in comparison to WT jejunum. We conclude that the facilitative GLUT-2 transporter is a direct target of leptin operating through its apical receptor coupled in a critically manner to AMPK pathways. These data may have physiological relevance in the context of increased consumption of sugars.
595 The Role of Luminal Tastants and Olfactants in the Regulation of Gut Neuroendocrine Cell Secretion and the Implications for Gastrointestinal Health and Disease Irvin M. Modlin, Bjorn I. Gustafsson, Zakiya-Luna Siddique, Ignat Drozdov, Roswitha Pfragner, Mark Kidd Background: Neuroendocrine cells of the diffuse neuroendocrine cell system express luminal G-protein coupled receptors and transporters for amines and salts and are considered to function as luminal-sensors. We postulated that “open” neuroendocrine cells would respond to agents in the diet thus regulating GI physiology, particularly gastric acid secretion and motility in the GI tract. Methods: For gastric studies, antral mucosa and a pure (>97%) FACS-sorted gastrin cell preparation was studied. For intestinal studies, a perfusion model, pure EC cells and a neoplastic cell line (KRJ-I) were studied. Gastrin and serotonin secretion in response to aromatic, sweet-tasting amino acids, bitter ligands (denatonium) and bile salts (sodium deoxycholate - SDC) were assessed and signaling pathways defined. Results: In the stomach, uM concentrations of sweet and bitter ligands as well as SDC stimulated gastrin release (>2-fold) and gastrin transcription (after 30 mins) from antral mucosa. Isolated G cell preparations were ~1,000 fold more sensitive (EC50=1.2-3.9nM) for gastrin release and transcription. In the small intestine, perfusion with 10uM concentrations of these ligands stimulated serotonin release (2-3-fold) and transcription of the rate-limiting serotonin synthesis enzyme, Tph1 (1.8-2.1-fold). Similarly to the stomach, both isolated EC cell preparations and KRJ-I were 500-1,200x more sensitive to these agents. In addition, stimulation of KRJ-I cells with beta-casomorphin, a milk-derived product that mimics opioid receptor ligands, inhibited serotonin release (IC50=22nM). These effects could be reversed (40-90%) by the PKA inhibitor H-89 (10uM) and by PD98059 (a MEK1 inhibitor: 10uM). Conclusion: Gastrin and serotonin secreting neuroendocrine cells in the antrum and intestine are sensitive to diet-associated ligands and exhibit transcription and release of their protein and amine products. These effects are PKA- and MAPK-dependent and can be reversed by beta-casomorphin. The delineation of dietary factors that modulate GI neuroendocrine luminal-sensory function provides a novel opportunity to define and evaluate the role of ingested agents on normal gut physiology as well as their role in abnormal gut function including irritable bowel syndrome and obesity related disorders.
593 Enteric Nematode Infection Induces a “Lean” Epithelial Cell Phenotype Through Differential Regulation of Glucose Transporter Expression Kathleen B. Madden, Diana C. Riera, Aiping Zhao, Jennifer A. Stiltz, Ildy M. Katona, Joseph F. Urban, Terez Shea-Donohue Enteric nematode infection upregulates Th2 cytokines, such as IL-4 and IL-13, as well as alternatively activated macrophages (AAMΦ). This is associated with stereotypic STAT6dependent changes in intestinal epithelial cell responses including increased permeability, and decreased glucose absorption. Glucose transport in the small intestine is coordinated through several transporters, including SGLT1 and GLUT-2. Aim: To investigate the mechanisms involved in the regulation of glucose transporter expression that contribute to infectioninduced alterations in glucose absorption. Methods: BALB/c or STAT6-/- mice were either infected with the rodent nematode Nippostrongylus brasiliensis (Nb) or remained uninfected (WT), and were studied 9 days later. Jejunal samples were taken for both real-time PCR and laser capture microdissection (LCM). Results: Nb infection did not alter SGLT1 expression. GLUT-2 expression was decreased by approximately 90% in Nb-infected mice, compared to WT controls, and was STAT6-independent (Table 1). This result may be attributed to a reduction in taste receptor expression by approximately 90% (data not shown), which in turn regulates GLUT-2 expression. In contrast, there was a STAT6-dependent, 11-fold increase in GLUT-1 expression, a transporter that is upregulated by stress (Table 1). To further investigate the cell populations responsible for the observed increase in GLUT-1 expression, real-time PCR was performed on LCM samples of jejunal epithelium and lamina propria from WT and Nb-infected mice. GLUT-1 expression revealed a 46-fold increase in the epithelium, and a 15-fold increase in the immune cells in the lamina propria. Nb infection also induced 7-fold increase in insulin growth factor (IGF)-1α. IGF-1α is produced by macrophages, binds to epithelial cells and stimulates hypoxia inducible factor (HIF)-1α. Infection with Nb increased expression of HIF-1α, which in turn upregulated GLUT-1 expression. Conclusions: Enteric nematode infection induces a “lean” epithelial cell phenotype, with components that are non-immune (STAT6-independent downregulation of GLUT2) and immune, which involve AAMΦ (STAT6-dependent upregulation of GLUT-1 expression). Such changes in the expression of glucose transporters induced by enteric nematode infection may explain, in part, the ability of these infectious agents to prevent the development of autoimmune conditions such as diabetes and IBD.
A-95
AGA Abstracts
AGA Abstracts
CD40 when compared to CD11b+ and CD11b-Ly6c- DCs. FACS sorted colonic pDCs produce IL-10 in response to TLR signals and regulate IL-10 secretion during antigenspecific activation of naïve CD4+ T cells. T cells expressing IL-10 were found to be a subset of Foxp3+ Tregs which emerged in the lamina propria during the recovery from colitis. Depletion of pDCs did not impact the recruitment of Foxp3+ Tregs, but impaired IL-10 secretion by Foxp3+ Tregs and delayed mucosal repair. The regulatory function of pDCs during mucosal inflammation also included the control of intestine-specific secretion of IL17 but not IFN-γ by CD4+Foxp3- T cells. CONCLUSION: During colitis, pDCs become a major DC subset in the lamina propria and direct mucosa-specific T cell functions essential for the resolution of inflammation. Our results demonstrate that pDCs display a distinct function in the colon in defining intestine-specific adaptations of T effectors and Foxp3+ Tregs to mucosal inflammation.