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Su1776 Activation of Rat Intestinal Mast Cells Depends on Types of Unsaturated Long-Chain Fatty Acids
AGA Abstracts
by downregulating folate uptake and folate transporters expression. However in another model of dietary folate deficiency, a marked upregulation in intestinal and renal folate uptake was observed along with increased expression of folate transporters i.e. reduced folate carrier (RFC), proton coupled folate transporter (PCFT) and folate receptor (FR). Keeping this in mind, the present study was designed to examine the effects of folate deficiency conditions and ethanol on regulation of folate transporters in human liver cell line. For experiments, HepG2 cells were grown under two different set of conditions. In the first set, cells were grown for 5 generations (7.5 days) in control and folate deficient medium. In another set of experiment, cells were grown in the medium containing 100mM ethanol for 96 hours. To study whether the effects of these folate deficiency conditions on the expression of transporters are reversible or not, two subgroups were made in folate deficient and alcohol treated group. Following the above mentioned treatments, one subgroup from each group was shifted on folate sufficient and alcohol free medium respectively for three generations. mRNA (real time RT-PCR) and protein (western blotting) expression of various folate transporters was studied. It was observed that in case of folate deficient group there was 14.8, 51.7 and 3.4 fold increase in RFC, PCFT and FR expression respectively. However, when cells were shifted to control medium mRNA expression of these genes restored to the control level. Similar results were observed in case of protein expression studies. In case of ethanol supplemented group there was 5.7 and 6.45 fold decrease in RFC and PCFT expression respectively. On shifting the cells to control medium, PCFT and RFC mRNA expression was restored to a significant extent. Similar trend was observed in protein expression studies in case of RFC however in case of PCFT there was complete restoration of the protein expression. No change was found in the expression of FR in ethanol treated group. Results of this investigation showed that there was marked up- and downregulation of folate transporters in folate deficient and ethanol treated conditions respectively, suggesting that both treatments leading to folate deficiency affect the folate transporters differently however such effects are reversible.
Su1776 Activation of Rat Intestinal Mast Cells Depends on Types of Unsaturated Long-Chain Fatty Acids Yasuhisa Sakata, Yong Ji, Qing Yang, Kazuma Fujimoto, Patrick Tso Background: In our previous study, using lymph fistula rat model, we have shown that fat ingestion stimulates release of mast cell mediators such as histamine, rat mucosal mast cell protease II (RMCP II), and prostaglandin D2 (PGD2) into intestinal lymph; however, it is unclear whether these responses are different depending on the type of fatty acid ingested. Aim: The purpose of this study was to determine the effects of dietary long-chain fatty acids with varying degrees of saturation on intestinal mast cell activation. Methods: We investigated lymphatic mast cell mediator concentrations after administration of three types of triacylglycerols, trilinolein (C18:2 n-6), trilinolenin (C18:3 n-3) or triolein (C18:1 n-9) by using the lymph fistula rat model. After cannulation of the major mesenteric lymph duct and duodenum of male Sprague-Dawley rat, a 3 ml bolus mixture of phosphate-buffered, taurocholatestabilized emulsion containing 120μmol of trilinolein or trilinolenin or triolein was provided to each animal through the intraduodenal feeding tube. The emulsion medium without triacylglycerol was used as control. Lymph was continuously collected for 6 hours and analyzed for RMCPII, histamine, PGD2, triglyceride and protein content. Results: Infusion of trilinolein and trilinolenin significantly increased the lymphatic release of mast cell mediators and influenced lymphatic protein concentration. In contrast, triolein did not induce the release of mast cell mediators. The peak RMCPII concentration after infusion of trilinolein (432.97 ± 77.53 ng/ml) and trilinolenin (317.50 ± 47.59 ng/ml) were significantly greater than triolein (141.46 ± 23.46 ng/ml) ( P ,0.01, P,0.05, respectively). Similar to the RMCPII response, the peak values of histamine were significantly greater than triolein (12.97 ± 0.62ng/ml) for trilinolein (34.2 ± 1.7 ng/ml, P ,0.01, n=3) and trilinolenin (19.85 ± 1.06ng/ ml, P,0.01, n=3). Infusion of trilinolein induced a significant increase of lymphatic PGD2 concentration peaked at 2 hour compared with the control (1461.23 ± 133.12 vs. 842.30 ± 129.02 pg/ml, P,0.05), but triolein did not induce (978.43 ± 153.74 pg/ml). Conclusion: These observations identify that the intestinal mast cell activation induced by dietary fat is dependent on the degree of unsaturation in fatty acid.
Fig.1 mRNA expression of folate transporters in HepG2 cells under conditions of folate deficiency and repletion. CFD-control for folate deficiency, FD- folate deficiency, CFDRcontrol for folate repletion, FDR- folate repletion. *p ,0.05, **p,0.01, ***p,0.001 vs. CFD
Su1777 GIP Activation of Signaling Pathways Traffick PepT1 Proteins to the Apical Membrane of Intestinal Epithelial Cells Steven D. Coon, John H. Schwartz, Satish K. Singh Background: GIP, an incretin hormone, is important in pathogenesis of obesity and diabetes. GIP increases both glucose and dipeptide transport in both murine jejunum and intestinal cells grown in vitro. GIP activates both the cAMP and PI3 kinase pathways but the mechanism and its affect on Pept1 regulation is not known. Objective: To elucidate the intracellular signaling pathways that regulate GIP-stimulated Pept1 trafficking to the apical membrane. Methods: Transport and signaling pathways for GIP were determined by growing IEC6 cells transfected with the CDX2 gene on filter inserts and measuring Pept1 uptake. 3H-GlySar (50 uM, a nonhydrolyzable dipeptide) and GIP (100 nM ) were added to the apical and basolateral sides, respectively. Trafficking of Pept1 was measured using biotinylation and western blotting techniques. Results: To investigate the cAMP pathway, we observed that the EPAC agonist (8-pCPT-2'-O-Me-cAMP-AM, 20 nM) increased Pept1 activity (520±41 pmol/min/mg-protein) while a PKA agonist (8-br-cAMP, 50 nM) failed to increase Pept1 activity (240±39 pmol/min/mg-protein) when compared to untreated (no GIP) control. Biotinylated apical membrane proteins run on Western blots showed significant similar increases in apical expression of Pept1 upon GIP and EPAC stimulation. when compared to control (n=3) Conclusion: GIP augmented Pept1 transport is consistent with a model where membrane targeting of the protein is induced via EPAC activation by cAMP by trafficking Pept1 to the apical membrane of epithelial cells.
Fig.2 mRNA expression of folate transporters in HepG2 cells under conditions of ethanol exposure and removal. CE- control for ethanol, E- ethanol, CER- control for ethanol removal, ER- ethanol removal. *p,0.05, **p,0.01 vs. respective controls, #p,0.05 vs. E Su1779 Association of Hgrhpr With the Human Sodium-Dependent Vitamin C Transporter-1 in Human Liver Cells Veedamali S. Subramanian, Svetlana Nabokina, Jonathan S. Marchant, Hamid M. Said
Su1778
Background: The human sodium-dependent vitamin C transporter-1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). While different aspects of hSVCT1 have been extensively studied, nothing is currently known about protein(s) that interact with hSVCT1 to modulate its role in physiology and cell biology. Method: Yeast two-hybrid (Y2H) screening was used to identify hSVCT1 interacting partners. Subsequently, 1-by-1 Y2H interaction assay, GST-pull-down, Co-immunoprecipitation, mammalian two-hybrid firefly luciferase assays and co-localization studies using live cell confocal imaging were used for confirming the protein-protein interactions.14C-AA uptake was performed to determine the functional impact of interactions together with gene silencing. Results: The human glyoxalate reductase/ hydroxypyruvate reductase (hGRHPR) protein was identified as an associated partner with
Regulation of Folate Transporters in Human Liver Cells Under Conditions of Folate Deficiency and Ethanol Exposure Shilpa Thakur, Deepti More, Jyotdeep Kaur Folic acid deficiency is considered to be one of the most common nutritional deficiencies in world. Deficiency of this micronutrient leads to a range of clinical abnormalities. The major contributors of folate deficiency are: deficient food supply and alcoholism. Previous studies conducted in rats in our lab have demonstrated that reduced folate status associated with chronic alcohol feeding results in folate malabsorption in intestine, liver and kidney
AGA Abstracts
S-474
by downregulating folate uptake and folate transporters expression. However in another model of dietary folate deficiency, a marked upregulation in intestinal and renal folate uptake was observed along with increased expression of folate transporters i.e. reduced folate carrier (RFC), proton coupled folate transporter (PCFT) and folate receptor (FR). Keeping this in mind, the present study was designed to examine the effects of folate deficiency conditions and ethanol on regulation of folate transporters in human liver cell line. For experiments, HepG2 cells were grown under two different set of conditions. In the first set, cells were grown for 5 generations (7.5 days) in control and folate deficient medium. In another set of experiment, cells were grown in the medium containing 100mM ethanol for 96 hours. To study whether the effects of these folate deficiency conditions on the expression of transporters are reversible or not, two subgroups were made in folate deficient and alcohol treated group. Following the above mentioned treatments, one subgroup from each group was shifted on folate sufficient and alcohol free medium respectively for three generations. mRNA (real time RT-PCR) and protein (western blotting) expression of various folate transporters was studied. It was observed that in case of folate deficient group there was 14.8, 51.7 and 3.4 fold increase in RFC, PCFT and FR expression respectively. However, when cells were shifted to control medium mRNA expression of these genes restored to the control level. Similar results were observed in case of protein expression studies. In case of ethanol supplemented group there was 5.7 and 6.45 fold decrease in RFC and PCFT expression respectively. On shifting the cells to control medium, PCFT and RFC mRNA expression was restored to a significant extent. Similar trend was observed in protein expression studies in case of RFC however in case of PCFT there was complete restoration of the protein expression. No change was found in the expression of FR in ethanol treated group. Results of this investigation showed that there was marked up- and downregulation of folate transporters in folate deficient and ethanol treated conditions respectively, suggesting that both treatments leading to folate deficiency affect the folate transporters differently however such effects are reversible.
Su1776 Activation of Rat Intestinal Mast Cells Depends on Types of Unsaturated Long-Chain Fatty Acids Yasuhisa Sakata, Yong Ji, Qing Yang, Kazuma Fujimoto, Patrick Tso Background: In our previous study, using lymph fistula rat model, we have shown that fat ingestion stimulates release of mast cell mediators such as histamine, rat mucosal mast cell protease II (RMCP II), and prostaglandin D2 (PGD2) into intestinal lymph; however, it is unclear whether these responses are different depending on the type of fatty acid ingested. Aim: The purpose of this study was to determine the effects of dietary long-chain fatty acids with varying degrees of saturation on intestinal mast cell activation. Methods: We investigated lymphatic mast cell mediator concentrations after administration of three types of triacylglycerols, trilinolein (C18:2 n-6), trilinolenin (C18:3 n-3) or triolein (C18:1 n-9) by using the lymph fistula rat model. After cannulation of the major mesenteric lymph duct and duodenum of male Sprague-Dawley rat, a 3 ml bolus mixture of phosphate-buffered, taurocholatestabilized emulsion containing 120μmol of trilinolein or trilinolenin or triolein was provided to each animal through the intraduodenal feeding tube. The emulsion medium without triacylglycerol was used as control. Lymph was continuously collected for 6 hours and analyzed for RMCPII, histamine, PGD2, triglyceride and protein content. Results: Infusion of trilinolein and trilinolenin significantly increased the lymphatic release of mast cell mediators and influenced lymphatic protein concentration. In contrast, triolein did not induce the release of mast cell mediators. The peak RMCPII concentration after infusion of trilinolein (432.97 ± 77.53 ng/ml) and trilinolenin (317.50 ± 47.59 ng/ml) were significantly greater than triolein (141.46 ± 23.46 ng/ml) ( P ,0.01, P,0.05, respectively). Similar to the RMCPII response, the peak values of histamine were significantly greater than triolein (12.97 ± 0.62ng/ml) for trilinolein (34.2 ± 1.7 ng/ml, P ,0.01, n=3) and trilinolenin (19.85 ± 1.06ng/ ml, P,0.01, n=3). Infusion of trilinolein induced a significant increase of lymphatic PGD2 concentration peaked at 2 hour compared with the control (1461.23 ± 133.12 vs. 842.30 ± 129.02 pg/ml, P,0.05), but triolein did not induce (978.43 ± 153.74 pg/ml). Conclusion: These observations identify that the intestinal mast cell activation induced by dietary fat is dependent on the degree of unsaturation in fatty acid.
Fig.1 mRNA expression of folate transporters in HepG2 cells under conditions of folate deficiency and repletion. CFD-control for folate deficiency, FD- folate deficiency, CFDRcontrol for folate repletion, FDR- folate repletion. *p ,0.05, **p,0.01, ***p,0.001 vs. CFD
Su1777 GIP Activation of Signaling Pathways Traffick PepT1 Proteins to the Apical Membrane of Intestinal Epithelial Cells Steven D. Coon, John H. Schwartz, Satish K. Singh Background: GIP, an incretin hormone, is important in pathogenesis of obesity and diabetes. GIP increases both glucose and dipeptide transport in both murine jejunum and intestinal cells grown in vitro. GIP activates both the cAMP and PI3 kinase pathways but the mechanism and its affect on Pept1 regulation is not known. Objective: To elucidate the intracellular signaling pathways that regulate GIP-stimulated Pept1 trafficking to the apical membrane. Methods: Transport and signaling pathways for GIP were determined by growing IEC6 cells transfected with the CDX2 gene on filter inserts and measuring Pept1 uptake. 3H-GlySar (50 uM, a nonhydrolyzable dipeptide) and GIP (100 nM ) were added to the apical and basolateral sides, respectively. Trafficking of Pept1 was measured using biotinylation and western blotting techniques. Results: To investigate the cAMP pathway, we observed that the EPAC agonist (8-pCPT-2'-O-Me-cAMP-AM, 20 nM) increased Pept1 activity (520±41 pmol/min/mg-protein) while a PKA agonist (8-br-cAMP, 50 nM) failed to increase Pept1 activity (240±39 pmol/min/mg-protein) when compared to untreated (no GIP) control. Biotinylated apical membrane proteins run on Western blots showed significant similar increases in apical expression of Pept1 upon GIP and EPAC stimulation. when compared to control (n=3) Conclusion: GIP augmented Pept1 transport is consistent with a model where membrane targeting of the protein is induced via EPAC activation by cAMP by trafficking Pept1 to the apical membrane of epithelial cells.
Fig.2 mRNA expression of folate transporters in HepG2 cells under conditions of ethanol exposure and removal. CE- control for ethanol, E- ethanol, CER- control for ethanol removal, ER- ethanol removal. *p,0.05, **p,0.01 vs. respective controls, #p,0.05 vs. E Su1779 Association of Hgrhpr With the Human Sodium-Dependent Vitamin C Transporter-1 in Human Liver Cells Veedamali S. Subramanian, Svetlana Nabokina, Jonathan S. Marchant, Hamid M. Said
Su1778
Background: The human sodium-dependent vitamin C transporter-1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). While different aspects of hSVCT1 have been extensively studied, nothing is currently known about protein(s) that interact with hSVCT1 to modulate its role in physiology and cell biology. Method: Yeast two-hybrid (Y2H) screening was used to identify hSVCT1 interacting partners. Subsequently, 1-by-1 Y2H interaction assay, GST-pull-down, Co-immunoprecipitation, mammalian two-hybrid firefly luciferase assays and co-localization studies using live cell confocal imaging were used for confirming the protein-protein interactions.14C-AA uptake was performed to determine the functional impact of interactions together with gene silencing. Results: The human glyoxalate reductase/ hydroxypyruvate reductase (hGRHPR) protein was identified as an associated partner with
Regulation of Folate Transporters in Human Liver Cells Under Conditions of Folate Deficiency and Ethanol Exposure Shilpa Thakur, Deepti More, Jyotdeep Kaur Folic acid deficiency is considered to be one of the most common nutritional deficiencies in world. Deficiency of this micronutrient leads to a range of clinical abnormalities. The major contributors of folate deficiency are: deficient food supply and alcoholism. Previous studies conducted in rats in our lab have demonstrated that reduced folate status associated with chronic alcohol feeding results in folate malabsorption in intestine, liver and kidney
AGA Abstracts
S-474