Km (≈20 mM) and Vmax (≈1200 nmol/mg protein), suggesting recruitment of anothercarrier system in apical membrane. Phloretin, a GLUT2 inhibitor, significantly inhibitedglucose uptake, especially at high glucose concentration and in incubation durations of >1min raising the possibility that the increased Km and Vmax were due to translocation ofGLUT2. Western blotting of the apical membrane using antibodies against SGLT-1 andGLUT2 suggested that GLUT2 rather than SGLT-1 translocated to apical membrane. Nocoda-zole inhibited carrier-mediated glucose uptake at high glucose concentrations (25 and 50mM) during the 5 and 10 min incubation, but not at low glucose concentrations (0.5-10mM) or with short durations of exposure (30 s and 1 min). CONCLUSION: GLUT2 istranslocated to the intestinal apical membrane of Caco-2 cells via a glucose-dependentmechanism acting via the microtubular system. (Support: NIH DK39337-MGS)

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Both NH2 and COOH Terminals Are Essential for Cell Membrane Targeting ofHuman Sodium-Dependent Vitamin C Transporter-2 (hSVCT2) in HumanLiver CellsVeedamali S. Subramanian, Jonathan S. Marchant, Hamid M. Said

Background: The human sodium-dependent vitamin C transporter-2 (hSVCT2) is involvedin cellular accumulation of ascorbic acid in liver cells. Most mammals synthesize vitaminC in the liver, however, humans lost the ability to synthesize endogenous vitamin C duringthe course of evolution and obtain it from dietary sources. Structurally hSVCT2 is predictedto possess twelve membrane spanning helices with cytoplasmic NH2 and COOH termini.To date, however, little is known about the molecular determinants that direct hSVCT2 tothe cell surface and the mechanisms of intracellular trafficking in hepatocytes. The aim ofthis study was to address these issues. Methods: Live cell (HepG2) confocal imaging wasused to resolve the cell surface expression of the full-length hSVCT2 and truncated constructsfused to yellow fluorescence protein (YFP). Trafficking vesicles were monitored using videorate confocal microscopy. Uptake assays were performed using 14C-ascorbic acid to deter-mine the functionality of overexpressed hSVCT2-YFP constructs. Results: The full-lengthhSVCT2-YFP protein was found to be expressed at the cell membrane and functional inHepG2 cells. Serial truncation analysis demonstrated that an essential role for both the NH2andCOOH terminal sequence for cell surface expression. Video-rate confocal imaging showedevidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles. Nocodazole (amicrotubule disrupting agent) treatment of hSVCT2-YFP expressing cells impaired traffickingvesicle movement. Conclusion: These results show that both NH2 and COOH-terminals areessential for proper targeting of hSVCT2 to the cell membrane and that cell surface deliveryis dependent on intact microtubules [Supported by grants from the DVA and the NIH].

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Brief Exposure to Crude Red Kidney Beans Increases Intestinal PermeabilityYatin Patel, Roberto Aguero, Wen-Huan S. Ho, Michel Boivin, Henry C. Lin

Background and Aim: Lectins are sugar-binding proteins that are present in many commonfoods. They exist in particularly high concentrations in legumes (e.g, red kidney beans) andnuts. Dietary lectins can be toxic when consumed without adequate cooking, occasionallyleading to an acute gastroenteritis. This toxicity may be secondary to increased intestinalpermeability, since lectins have been shown to decrease trans-epithelial resistance in cellcultures. However, the length of exposure to a diet rich in lectins required to produceincreased intestinal permeability is not known. Our aim was to test the hypothesis that evenbrief exposure to crude red kidney beans in whole animals may increase intestinal permeabil-ity and decrease animal weight gain. Methods: Fourteen rats were randomized to an ad libdiet of either standard rat chow (control, n=7) or a chow containing 26% crude red kidneybeans (RKB, n=7) for 24 hours. After an 8-hour fast, the rats were gavaged with a 1mlsolution containing 15mg/ml lactulose and 10mg/ml mannitol. Urine was collected for 6hours, and the concentration of lactulose and mannitol was measured by an enzymaticmethod. Intestinal permeability was represented by the lactulose recovery since mannitolrecovery was minimal. Results: Rats fed crude red kidney beans for 24h had greater urinaryrecovery of lactulose in comparison to control (0.88±0.25 vs. 0.24±0.08; p<0.05) and lessweight gain when compared to controls(-1.29±0.16 vs. 3.21±0.44; p<0.0001). Conclusions:1) Even a brief exposure (24hours) to crude red kidney beans increased intestinal permeabil-ity. 2) Rats exposed to crude red kidney beans in their diet showed less weight gain comparedto control animals. 3) Since red kidney beans and other legumes are an important sourceof dietary protein in the world, their potential toxicity via increasing intestinal permeabilitymay have important implications for many people.

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Mechanisms of Human Hepatic Vitamin C Uptake: Promoter Analysis andAdaptive Regulation of the Hsvct SystemsJack Reidling, Hamid M. Said

Vitamin C is a vital antioxidant required for cellular functions including growth and develop-ment. The human liver plays a pivotal role in regulating and maintaining body vitamin Chomeostasis, yet the molecular mechanisms involved in the regulation of the vitamin Ctransporters (hSVCT1 and hSVCT2) in the human liver are poorly understood. To furtherour understanding of the regulation of these transporters we first characterized the minimalpromoter regions and investigated the role of putative cis-elements in regulating the activityof SVCT1 & 2 promoters in human liver epithelial cells (HepG2). Then we examined theeffects of vitamin C supplementation and deprivation on the activity of the transport systemsand determined if transcriptional mechanisms are involved in any observed regulatory events.Using the cloned 5'-regulatory regions of the genes we confirmed activity of the promotersfollowing transfection into HepG2 cells and identified the minimal promoter regions requiredfor basal activity. Both regions were found to include multiple cis-regulatory elements andupon mutational analysis we demonstrated that mutation of Hepatic Nuclear Factor sites inthe hSVCT1 promoter and Sp1/KLF sites in the hSVCT2 promoter were essential for activities.

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We also found that maintaining the cells in vitamin C-deficient medium led to a significant(P < 0.01) and specific up-regulation in [14C]-Ascorbic acid uptake, which was associatedwith an increase in hSVCT-1 and hSVCT-2 mRNA levels as well as promoter activities.Interestingly, the exact opposite result occurred when HepG2 cells were over-supplementedwith vitamin C. We have mapped the vitamin C responsive region in the promoters andare determining the specific elements involved in the adaptive regulatory response. Theresults reported here demonstrate that substrate availability plays an important role in self-regulatory events of hSVCT1 and 2 message levels in human liver epithelial cells ultimatelyparticipating in the physiological amount of vitamin C transported.

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Intestinal Fat Absorption Is Associated with the Release of Histamine and ItsDegrading Enzyme Diamine OxidaseYong Ji, Xiaoming Li, Qing Yang, Min Xu, Armin Wollin, Patrick Tso

Background. Intestinal fat absorption is associated with the formation and secretion ofchylomicrons into lymph. Lipid absorption is also associated with an increase in lymphaticsecretion of diamine oxidase. Aim. The aim of this study was to determine the mechanismby which fat absorption stimulates diamine oxidase (DAO) secretion into lymph. Methods.Intestinal lymph fistula rats were used. The mesenteric lymph duct was cannulated inanesthetized rats for the collection of intestinal lymph. In addition, intraduodenal cannulationwas installed for the infusion of nutrients. Lymphatic histamine and rat mast cell proteaseII (RMCPII) concentration were measured by ELISA. DAO activity was measured by radiomet-ric assay using [3H] putrecine as substrate. Results. Intraduodenal infusion of Lyposin (4.43kcal/3ml) induced a significant (3.7 fold) increase in lymphatic DAO output compared withsaline control at the peak of 1 hr, whereas isocaloric and isovolumetric treatment withdextrin, a glucose polymer, did not increase DAO output in the intestinal lymph. Similarly,lymphatic histamine level doubled by 1 hour after Lyposin infusion, raising the questionof whether histamine could be the mediator in DAO secretion during fat absorption. Toaddress this question, we intraperitoneally (i.p.) infused histamine (5mg/kg and 10 mg/kg)in fasting lymph fistula rats and this induced an increase in lymphatic release of DAO dose-dependently. I.p. injection of a histamine H1-receptor antagonist (chlorpheniramine maleate,10 mg/kg) or an H2 antagonist (ranitidine raditine, 8 mg/kg) inhibited peak lipid-inducedDAO activity by 38.6% and 21.7 %, respectively, suggesting that the release of DAO byhistamine is complex and may involve more than one receptor. There was also an 11-foldincrease in lymphatic RMCPII concentration after lyposin infusion indicating that lipidinduced histamine release may be mucosal mast cell mediated. Conclusion. We concludedthat intestinal secretion of DAO stimulated by lipid absorption is likely histamine mediated.The increase in histamine release probably originates from the mucosal mast cells. Weconcluded that fat absorption induces the mucosal mast cells to degranulate to releasehistamine and that the histamine in turn stimulates the release of mucosal DAO. Thephysiological and pathophysiological importance of this process warrants further investi-gation.

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Acyl Chain Length and Saturation Determines the Efficiency of IntestinalDietary Fatty Acid AbsorptionRyan L. McKimmie, Linda Easter, Richard B Weinberg

Background: Absorption of dietary fat by the small intestine is, overall, highly efficient, butthe processes involved may be sensitive to the physicochemical properties of individual fattyacids (FA). Hypothesis: Dietary FA's are absorbed with different efficiencies. Methods: FAabsorption efficiency was measured in 13 healthy subjects by the sucrose polybehenate(SPB) method, in which SPB (a component of Olestra®) serves as a non-absorbable marker.Subjects were fed diets prepared in a metabolic kitchen (15% protein, 50% carbohydrate,and 35% total fat), supplemented with 1000 mg of fish oil at lunch and supper. Each mealincluded an item (muffins, cookies) that contained 5% SPB. Samples of homogenized com-plete daily diets and day 2 and 3 stool samples were analyzed by GC-mass spectroscopy toquantitate behenic acid (BA) and FA. The coefficient of absorption of each FA was calculatedas {1-(FA/BA)feces/(FA/BA)diet}. Results: For saturated FA's, absorption coefficients decreasedwith increasing hydrophobicity and melting point (mean ± SE): 0.94 ± 0.02 for myristate(14:0); 0.85 ± 0.03 for palmitate (16:0); 0.70 ± 0.08 for stearate (18:0); and 0.13 ±0.03 for 20:0. For unsaturated C18 FA's, absorption coefficients increased with increasingdesaturation: 0.70 ± 0.08 for 18:1(trans); 0.90 ± 0.03 for oleate (18:1); 0.95 ± 0.01 forlinoleate (18:2); and 0.94 ± 0.02 for linolenate (18:3). Complete absorption was observedfor palmitoleate (14:1), arachidonate (20:4) and the ω3 FA's EPA (20:5) and DHA (22:6).Conclusions: Acyl chain length, hydrophobicity, and degree of desaturation are major deter-minants of the efficiency with which FA's present in dietary fat are absorbed by the smallintestine. This raises the possibility that digestive disorders that cause fat malabsorption maydifferentially affect individual FA's.

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