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964 Involvement of Transcriptional and Post-Transcriptional Mechanisms in the Adaptive Regulation of the Human Intestinal Riboflavin (RF) Uptake Process
AGA Abstracts
signaling appears to be a major contributing mechanism to loss of IEC proliferation, EBF and migration. Overexpression of Slit2 may protect against TPN-associated mucosal atrophy.
conjunction with Fe. Nevertheless, our data point to the hazard of food processing and fortification regarding IBD and to the necessity of food safety testing in animal models thereof.
Slit2 and Robo1 mRNA expression were represent as ratio to beta actin (10-3) a, compared to sham group, p<0.05 b, compared to WT TPN, P<0.05
964 Involvement of Transcriptional and Post-Transcriptional Mechanisms in the Adaptive Regulation of the Human Intestinal Riboflavin (RF) Uptake Process Veedamali S. Subramanian, Abhisek Ghosal, Rubina Kapadia, Svetlana Nabokina, Hamid M. Said Background: The human intestine absorption process of the water-soluble vitamin RF is carrier-mediated, and all three recently cloned human RF transporters, i. e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. Expression of hRFVT-3 is restricted to the apical brush border membrane, that of hRFVT1 to the basolateral membrane (BLM), while expression of hRFVT-2 is mainly in intracellular vesicular structures with some at the BLM (BBA, 1808: 3016, 2011). Previous studies from our laboratory (AJP, 278: C270, 2000) have shown that the intestinal RF uptake process is adaptively regulated by the prevailing substrate level, but little is known about the molecular mechanism(s) involved. We addressed this issue in the present study. Methods: Human intestinal epithelial NCM460 cells were maintained in RF-deficiency and over-supplemented (approx.0 and 100 µM, respectively) growth media for 14 days. Uptake assays, western blotting, semi-quantitative RT-PCR, cell surface biotinylation, and promoter (firefly luciferase) activity assays were performed. Results: Uptake of 3H-RF by cells maintained under RF deficient condition was significantly higher than uptake of cells maintained under RF oversupplemented condition. This adaptive regulation was associated with a significantly higher level of expression of hRFVT-2 and -3 protein and mRNA (expression of hRFVT-1 was not affected) in the deficient state. In addition, an increase in cell surface expression (determined by biotinylation assay) of all RF transporters was observed in cells maintained under RFdeficient compared over-supplemented conditions. Focusing on hRFVT-3, we also found a higher SLC52A3 promoter activity in cells maintained under RF deficient compared to oversupplemented condition. By generating a serial of 5'-truncated SLC52A3 promoter-luciferase constructs, we identified the RF level-responsive region to be between -199 to +8 (TSS as +1) of the SLC52A3 promoter. Conclusions: These findings delineate, for the first time, important aspects of the molecular mechanisms involved in the adaptive regulation of intestinal RF absorption process and show the involvement of both transcriptional and post-transcriptional mechanisms. (Supported by DVA and the NIH grants DK56061 and DK 58057).
Overexpression of Slit2 signaling protected epithelial cell migration in TPN mice
963 Iron (III)-Sodium-EDTA As Used for Food Fortification Aggravates Intestinal Inflammation and Drives Tumorigenesis in Mouse Models of ColitisAssociated Cancer Rayko Evstatiev, Tina Austerlitz, Vineeta Khare, Kristine Jimenez, Sophie Klimscha, Michaela Lang, Anita Krnjic, Christoph Gasche
965 The Glucagon-Like Peptide 1 Agonist Liraglutide Reduces Jejunostomy Output and Improves Intestinal Absorption in Short Bowel Syndrome Patients With Intestinal Failure; A Pilot Study Mark Hvistendahl, Christopher F. Brandt, Siri Tribler, Rahim Naimi, Bolette Hartmann, Jens Juul . Holst, Jens F. Rehfeld, Mads Hornum, Jens R. Andersen, Per B. Mortensen, Palle B. Jeppesen
Background: Western lifestyle is associated with increasing prevalence of IBD, processed food and food fortification being a likely cause. Iron supplementation may increase gut inflammation and colitis-associated cancer (CAC). Novel Fe products which are chemically not Fe(II) salts and presumably better tolerated have become available. Methods: Here we compared various Fe compounds at 450mg elemental Fe/kg chow [Na-EDTA-Fe(III) (as used for food fortification), Fe(III) maltol (Iron Therapeutics Ltd), plant Fe (Biogena, Austria), and Fe(II)SO4)], in their potential to promote colitis and CAC in animal models of IBD (AOM/DSS and IL-10-/-). In addition, experiments were performed in vitro to evaluate their impact on ROS production (DCFD assay), DNA damage ( gH2AX) and activation of oncogenic pathways (in HT29, HCT116, Caco-2 and RKO, and the normal colonic cell line 1CT). Results: Unexpectedly, Na-EDTA-Fe(III)-treated mice developed the severest form of colitis and only the 1st and 4th DSS cycles were administered. Similarly, in IL-10-/- mice, the NaEDTA-Fe(III) group was euthanized 4 months before all other groups due to severe weight loss and disease activity (DAI). The mean DAI in the Na-EDTA-Fe(III) AOM/DSS mice was higher (1.82 vs. 0.94 (control), 0.76 (Fe(II)SO4), 0.67 (Fe(III) maltol), 0.54 (plant Fe), 0.80 (ID); p<0.001 by ANOVA). The total tumor area per mouse was higher with Na-EDTAFe(III) (Figure; p=0.001 by ANOVA), and there were more invasive tumors. ROS production was not elevated but there was an increase in gH2AX with Na-EDTA-Fe(III) and phosphorylation of Akt and Erk. Conclusion: Na-EDTA-Fe(III), as it is commonly used for food fortification in flour products and cereals, exacerbates intestinal inflammation and drives tumorigenesis in mouse models of IBD. It is currently unclear whether this toxic effect is caused by EDTA itself (which also is added to food as Na-EDTA or Na-EDTA-Ca(II)) or only in
AGA Abstracts
X : 55624$1AGA 03-28-15 04:55:56 PDFd : 55624B : e
Background & Aim: Short bowel syndrome patients with intestinal failure (SBS-IF) and end-jejunostomy have rapid gastric emptying and gastric hypersecretion contributing to a high ostomy output and the need for parenteral support (PS). By restoring the ‘ileal-colonicbrake' mechanism, the glucagon-like peptide-1 (GLP-1) agonist, liraglutide, may prolong intestinal transit time and reduce the gastric hypersecretion, thus reducing ostomy output and increasing intestinal absorption. Methods: In an 8-week, open-label, "proof-of-concept", pilot study, 8 jejunostomy patients, mean (range); age 63.4 y (42.5-74.8); small bowel length 110 cm (30-200), were admitted for two 72h metabolic balances and were given subcutaneous liraglutide (titration from 0.6 to 1.8 mg/d) after the first balance studies for 56 days. During balance studies, the patients were allowed flexible diet intake of solids, whereas oral fluid intake and PS was intended to be constant. The wet weight, electrolyte, energy and macronutrient content of the oral intake, and the ostomy and urine outputs were analysed. If patients had PS volume reductions during treatment, e.g. due to oedema, they resumed their original baseline PS regime at the beginning of week 8 and during the second balance study. The primary endpoint was a reduction in the ostomy wet weight output. Results: Treatment with liraglutide reduced ostomy wet weight output by 474 ± 563 g/d (p=0.02, student t test) from 3249 ± 1352 to 2775 ± 1187 g/d (Figure 1). Oral wet weight and energy intakes (bomb calorimetry) were equivalent during both balance periods. Intestinal wet weight absorption increased by 464 ± 557 g/d (p=0.03), as did urine
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signaling appears to be a major contributing mechanism to loss of IEC proliferation, EBF and migration. Overexpression of Slit2 may protect against TPN-associated mucosal atrophy.
conjunction with Fe. Nevertheless, our data point to the hazard of food processing and fortification regarding IBD and to the necessity of food safety testing in animal models thereof.
Slit2 and Robo1 mRNA expression were represent as ratio to beta actin (10-3) a, compared to sham group, p<0.05 b, compared to WT TPN, P<0.05
964 Involvement of Transcriptional and Post-Transcriptional Mechanisms in the Adaptive Regulation of the Human Intestinal Riboflavin (RF) Uptake Process Veedamali S. Subramanian, Abhisek Ghosal, Rubina Kapadia, Svetlana Nabokina, Hamid M. Said Background: The human intestine absorption process of the water-soluble vitamin RF is carrier-mediated, and all three recently cloned human RF transporters, i. e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. Expression of hRFVT-3 is restricted to the apical brush border membrane, that of hRFVT1 to the basolateral membrane (BLM), while expression of hRFVT-2 is mainly in intracellular vesicular structures with some at the BLM (BBA, 1808: 3016, 2011). Previous studies from our laboratory (AJP, 278: C270, 2000) have shown that the intestinal RF uptake process is adaptively regulated by the prevailing substrate level, but little is known about the molecular mechanism(s) involved. We addressed this issue in the present study. Methods: Human intestinal epithelial NCM460 cells were maintained in RF-deficiency and over-supplemented (approx.0 and 100 µM, respectively) growth media for 14 days. Uptake assays, western blotting, semi-quantitative RT-PCR, cell surface biotinylation, and promoter (firefly luciferase) activity assays were performed. Results: Uptake of 3H-RF by cells maintained under RF deficient condition was significantly higher than uptake of cells maintained under RF oversupplemented condition. This adaptive regulation was associated with a significantly higher level of expression of hRFVT-2 and -3 protein and mRNA (expression of hRFVT-1 was not affected) in the deficient state. In addition, an increase in cell surface expression (determined by biotinylation assay) of all RF transporters was observed in cells maintained under RFdeficient compared over-supplemented conditions. Focusing on hRFVT-3, we also found a higher SLC52A3 promoter activity in cells maintained under RF deficient compared to oversupplemented condition. By generating a serial of 5'-truncated SLC52A3 promoter-luciferase constructs, we identified the RF level-responsive region to be between -199 to +8 (TSS as +1) of the SLC52A3 promoter. Conclusions: These findings delineate, for the first time, important aspects of the molecular mechanisms involved in the adaptive regulation of intestinal RF absorption process and show the involvement of both transcriptional and post-transcriptional mechanisms. (Supported by DVA and the NIH grants DK56061 and DK 58057).
Overexpression of Slit2 signaling protected epithelial cell migration in TPN mice
963 Iron (III)-Sodium-EDTA As Used for Food Fortification Aggravates Intestinal Inflammation and Drives Tumorigenesis in Mouse Models of ColitisAssociated Cancer Rayko Evstatiev, Tina Austerlitz, Vineeta Khare, Kristine Jimenez, Sophie Klimscha, Michaela Lang, Anita Krnjic, Christoph Gasche
965 The Glucagon-Like Peptide 1 Agonist Liraglutide Reduces Jejunostomy Output and Improves Intestinal Absorption in Short Bowel Syndrome Patients With Intestinal Failure; A Pilot Study Mark Hvistendahl, Christopher F. Brandt, Siri Tribler, Rahim Naimi, Bolette Hartmann, Jens Juul . Holst, Jens F. Rehfeld, Mads Hornum, Jens R. Andersen, Per B. Mortensen, Palle B. Jeppesen
Background: Western lifestyle is associated with increasing prevalence of IBD, processed food and food fortification being a likely cause. Iron supplementation may increase gut inflammation and colitis-associated cancer (CAC). Novel Fe products which are chemically not Fe(II) salts and presumably better tolerated have become available. Methods: Here we compared various Fe compounds at 450mg elemental Fe/kg chow [Na-EDTA-Fe(III) (as used for food fortification), Fe(III) maltol (Iron Therapeutics Ltd), plant Fe (Biogena, Austria), and Fe(II)SO4)], in their potential to promote colitis and CAC in animal models of IBD (AOM/DSS and IL-10-/-). In addition, experiments were performed in vitro to evaluate their impact on ROS production (DCFD assay), DNA damage ( gH2AX) and activation of oncogenic pathways (in HT29, HCT116, Caco-2 and RKO, and the normal colonic cell line 1CT). Results: Unexpectedly, Na-EDTA-Fe(III)-treated mice developed the severest form of colitis and only the 1st and 4th DSS cycles were administered. Similarly, in IL-10-/- mice, the NaEDTA-Fe(III) group was euthanized 4 months before all other groups due to severe weight loss and disease activity (DAI). The mean DAI in the Na-EDTA-Fe(III) AOM/DSS mice was higher (1.82 vs. 0.94 (control), 0.76 (Fe(II)SO4), 0.67 (Fe(III) maltol), 0.54 (plant Fe), 0.80 (ID); p<0.001 by ANOVA). The total tumor area per mouse was higher with Na-EDTAFe(III) (Figure; p=0.001 by ANOVA), and there were more invasive tumors. ROS production was not elevated but there was an increase in gH2AX with Na-EDTA-Fe(III) and phosphorylation of Akt and Erk. Conclusion: Na-EDTA-Fe(III), as it is commonly used for food fortification in flour products and cereals, exacerbates intestinal inflammation and drives tumorigenesis in mouse models of IBD. It is currently unclear whether this toxic effect is caused by EDTA itself (which also is added to food as Na-EDTA or Na-EDTA-Ca(II)) or only in
AGA Abstracts
X : 55624$1AGA 03-28-15 04:55:56 PDFd : 55624B : e
Background & Aim: Short bowel syndrome patients with intestinal failure (SBS-IF) and end-jejunostomy have rapid gastric emptying and gastric hypersecretion contributing to a high ostomy output and the need for parenteral support (PS). By restoring the ‘ileal-colonicbrake' mechanism, the glucagon-like peptide-1 (GLP-1) agonist, liraglutide, may prolong intestinal transit time and reduce the gastric hypersecretion, thus reducing ostomy output and increasing intestinal absorption. Methods: In an 8-week, open-label, "proof-of-concept", pilot study, 8 jejunostomy patients, mean (range); age 63.4 y (42.5-74.8); small bowel length 110 cm (30-200), were admitted for two 72h metabolic balances and were given subcutaneous liraglutide (titration from 0.6 to 1.8 mg/d) after the first balance studies for 56 days. During balance studies, the patients were allowed flexible diet intake of solids, whereas oral fluid intake and PS was intended to be constant. The wet weight, electrolyte, energy and macronutrient content of the oral intake, and the ostomy and urine outputs were analysed. If patients had PS volume reductions during treatment, e.g. due to oedema, they resumed their original baseline PS regime at the beginning of week 8 and during the second balance study. The primary endpoint was a reduction in the ostomy wet weight output. Results: Treatment with liraglutide reduced ostomy wet weight output by 474 ± 563 g/d (p=0.02, student t test) from 3249 ± 1352 to 2775 ± 1187 g/d (Figure 1). Oral wet weight and energy intakes (bomb calorimetry) were equivalent during both balance periods. Intestinal wet weight absorption increased by 464 ± 557 g/d (p=0.03), as did urine
S-188
Page 188