deternmled by Western blotting and mRNA expressinn by reverse transcriptase PCR, cor- rected tbr beta-actin expression. Results: Altering extracellular redox conditions for 24 did not afleet Gly-Sar transport; however, dipeptide transport function was decreased by 30% after 48 h in oxidizing culture medium (0 mV). Exposure to H202 (1 mM) for 24 h decreased Gly-Sar transport by 40% (control 202 +/- 6 versus I-I202 121 +/- 8); dipeptide transport remained decreased at this level with the higher I-I202 concentrations. Expression of PefFl protein and mRNA was diminished by H202 in a dose-dependent manner, Treat- ntent with GH, KGF or Ala-Gln did not alter Gly-Sar transport in the absence of t-1202. In contrast, both Gtt and Ala-Gln, but not KGF, prevented the decrease in dipeptide transport induced by 1 mM H202 (GH 207 +/- 21 and Ala-Gln 180 +/-14; NS vs control). Conclusion: Expression of PepT1 and PepTl-mediated dipeptide transport in Caco-2 cells is decreased by oxidative stress in a dose- and time-dependent manner, Both GH and Ala- Gln prevent the impairment in dipeptide transport induced by 1,1202 in this human colonic epifhelial cell/me,

M2186

Sodium Butyrate Mediated Sp3 Acetylation Represses IGFBP-3 Expression in Intestinal Epithelial Cells Nlcho/as R. Viq~ite, Peter Mulligan, lan R. Sanderson

Butylate induces histone acetyhtion, but this does not easily' explain the down-regulation of 1GFBP-3 by buwate, as histone ace@ation is associated with expansion of the nucleosome. Furtbermore, the down-regulation occurs in the absence of de novo protein synthesis, excluding the induction of a repressor as a possible mechanism, However, promoter analysis revealed the presence of a butyrate responsive element that included binding sites for p300 and Sp 1/5p3. Methods: We examined the acetylation and activity of Sp3 in bntyrate mediated IGFBP-3 regulation Results: Transfection of Caco-2 cells with EIA, an inhibitor of p300 aeetyhransferase activity., reversed the butyrate induced repmssinn of IGFBP-3. Sp3 is a kuown repressor of gene activation. Its potential for acetylation has recently been reported. We hypothesized that but~/~'ate might increase the aeetylation of Sp3 We, therefore, per- tbrmed EMSA and supersbift studies on nuclear extracts from butyrate treated and non- treated Caco-2 cells with the 1GFBP3 promoter. We also studied nuclear extracts by Western biotting analysis and imnmnopmcipitation 5mM butyrate retarded the Sp3-specific hand in EMSAs This band was further supershifted by an antbacetyl lysine-specific antibody. Immunoprecipitation indicated an increase in the amount of the acetylated form of Sp3, in the preserme of NaB, whereas the total amount of Sp3 protein was unchanged. Western blot analysis revealed the acetylation of an isoform of Sp3 in the presence of 5mM NaB. Transfec- tion with E1A abrogated the repression by NaB as evidenced by semi-quantitative PCR and Western blot analysiso Conclusions: Our evidence supports the hypothesis that butyrate down-regulates 1GFBP-3 through the acetylatinn of the repressor Sp3 and likely involves p300. We have shown for the first time that butyrate affects tile acetylation status of a non- histone DNA-binding prntein

M2187

Glucose Sensing and Signalling Mechanisms in the Small Intestine lane Dyer, Steve Vayro Soraya P, Shirazi-geecbey

Dietary sugars enhance the expression of tunctional Na+/glucose transporter (SGLT1), and metabolism of tbe sugar is not required tor the induction. Using sheep intestine as a model system, we have shown that luminal sugars regulate the expression of SGLT1 at both trs~scriptional and post-transcriptional levels Rumen development in sheep is a natural and efficient way of ensuring a virtual block in the delivery of monosaccharides into the small intestine. Associated with the decline in the levels of monosaccharides m the small intestine tbere is >50-Md decline in the abundance of functional SGLT1 protein and mRNA. Introduction of D-glucose, D-galactose, cr 3-O-methyl-D-glucose, D fructose and 2-deoxT-D-glucose into the luminal contents of ruminant sheep intestine results in > 50-fold enhancement in the expression of SGLT1. Transient tmnsiection reporter as~ys demonstrate activation of the ovine SGLTI promoter in response to media glucose /n order to determine if transport of the sugar into the imestinal cell is required for SGLT1 indnctkm, we have sya~tbesised a membrane impermeable glucose analogue, difglucos-&yl) polyethylene glycol 60~3 Introduction ot this compound to the luminal contents of the ruminant sheep led to enhancement in the expression of SGLT1. This glucose analogue did ~ot, however, inhibit Na%dependent glucose transport into ovine brust>border membrane vesicles. Furthermore, using an in v/tin assay system, we have shown that there was a 47% increase in the lewels of intracellular cAMP, in response to increased media glucose, kdditiralally, reporter assays indicate that 8 bromo cyclic AMP (8Br-cAMP), a protein kinase A (PKA) agonist, mimics ~he glucose-induced activation of the SGLT1 promoter. The glucose- induced SGLT1 promoter activity was inhibited by tile PKA inhibitor H-89. Pertnssis toxin, a G-protein (Gi) specific inhflntor, enhanced SGLTI abundance, mimicking the response to increased media glucose or 8B>cAMP. Collectively, these data suggest that monosaccha- rides in the lumen of the intestine are sensed by a sugar sensor, distinct from SGLT1, located on the external t~lce of the intestinal cell membrane, The sensor initiates a signalling event, invoMng a G-protein coupled receptor linked to a cAMP-PK~ pathway. This leads ultimately" to the activation ot SG1X1 gene transcription and an increase in the number of functional intestinal sugar transl:umer molecules All animal handling and experimental procedures were carried out under UK Home Office ticence.

M2188

The Importance of The Colonic Butyrate Transporter, Mctl, to Homeostasis of The Colonic Mucosa Mark A. Cul~, Soraya P, Shil~zi-Beechey

Butyrate is a naturalIy occurring monocarboxylate produced by microbial fermentation of dietary carbnhydrates in the lumen of the colon, It has protbund effects on proliferation, difterentiation and apoptosis of colonic epithelial cells, These effects are often associated

with changes in expression of genes important to these processes. In colonic cells, these genes include p21 (waf/cip~), intestinal alkaline phosphatasa, Cyckn D3, bcl2, and bak We have demonstrated previously that (i) uptake ofbutyrate across the colonic luminal membrane is predominantly mediated by the monocarboxylate transporter M c r ] , (ii) expression of human colonic MCT1 is adaptive to changes in butyrate concentration, and (iii) that this involves the dual control of MCTI transcription and stability of the MCT1 transcript. We have further reported that in colon carcinoma there is a dramatic decline in MCT1 expression compared to healthy colonic tissue. Given that many of the cellular eftects of butyrate are concentration dependent, it is reasonable to suggest that the ability of butyrate to exert these effects may be dependent upon its intracellular concentration. By extension, factors affecting the mtracellular accumulation of butyrate have the potemial to influence its ability" to modulate processes such as prolii~:ration and apoptosis. Accordingly, we have suggested that upregulation of MCTI may serve to maximise the intracellular availability of butyrate to influence the processes maintaining homeostasis in the colonic epithelium. In this study, we sought to test this hypothesis by investigation of the effect ot inhibition of both MCT1 expression and function on the ability of butymte to exert its downstream effects on gene expression in vitro. To this end, cuhured human colonic epitheliai cells, were transiected with small inhibitory RNA duplexes directed against the MCT1 coding region. This led to a considerable (70%) specific decline in MCT1 abundance, which was reflected in a corresponding decrease in butyrate uptake into luminal membrane vesicks isolated from the colonic cells. Furthermore, this effect inhibited subsequent butyrate-induced changes in expression of a number of well-recognised target genes. Simdar msuhs were obtained by inhibition of MCT1 [unction using a-cyano-4-hydroxycinnanlate, Tbese resuhs represent the first direct indication of the importance of the modulation of MCT1 expression, and hence butyrate transport to the maintenance of colonic tissue homeostasis.

M2189

Glutamine Decreases Intestinal Epithelial lnterleulon-8: Relationship with Nuclear factor kappa B and the Mitogen Activated Protein Kinase Pathway Kellym Liboni, Nan Li, Ying Huang, Josaf Neu

Background: Pro-mtlarumatory cytokines produced by the intestine play an important role in the pathogenesis of intestinal and distal organ disease. Glutamine (Gin) has been shown to diminish pro-inflammatory c71okines m the intestine. The interaction of Gin with inflam- matory mediator signaling pathways and transcription factors is poorly understood We previously denmnstrated that the stimulation of E. coli lipopolysaccharide (LPS)-mduced pro-inflammatory interleukin 8 (IL-8) production by human intestinal epithelial cells (Caco- 2) and its expression is decreased by Gin. However, the down-regulation of IL-8 production with Gin does not appear to occur via NF-kB. It is not known whether the effect of Gin is via activation of the mitogen-actwated protein kinases (MAPK) or whether Gin affects activator protein 1 regulatory element (AP-1) binding, which could, in turn, inhibit IL-8 production. Objective: We hypothesize that Gin modulates lL-8 production via the p38 MAPK pathway in Caco-2. Methods: Caco-2 ceils were incubated with dift~rent concentrations of Gin with or without methlonine sulfbximine (MS), an inhibitor of glutamine synthetase for 24hours. Then, O. 1 I~M, 1 ~M and 10 IxM of the pyridinyl imidazole SB 203580 (a selective inhibitor of p38 MAPK activity) was added for 1.5 hours fbllowmg stimulation by LPS (100 p.g/ml for 24hs). IL-8 protein and m-RNA levels were studied by ELISA and reverse transcription-PCR (RT-PCP.), respectively. NF-kB activity was deternrined rising an ELISA- based kit and immunofluorescent staining. Results: LPS increased IL-8 production and cell nucleus NF-kB expression in the presence or absence of MS (p<O.00 i). SB 203580 inhibit ed LPg-induced IL-8 peptide production in the absence of MS (p<0.001) and in the presence of MS (p<O.O1) in a dose dependent manner ( b l 0 IxM) and also inhibited LPS-induced IL-8 mRNA expression (p<O.05). Diiterent doses of Gin in the SB treated group did not alter the IL-8 production. Conclusions: These results indicate that p38 MAPK regulates LPS- induced IL-8 expression in Caco-2 cells. LPS stimulates IL-8 production in Caco-2 ceils through NF-kB signal pathway and p38 MAPK pathway. The glutamine-mediated inhibition of IL-8 production in Caco-2 cells does not appear to be mediated via the p38 bL4PK or NF-kB pathway. Acknowledgement: This study was supported by NIH grant RO1HD38954.

M2190

Glutamine Potentiation of Hsp72 Expression is Independent of HSF-1 Phosphorylation and its Transcriptional Activation Jacob Riehm, Mark J, Ropeleski, Mark W, Musch, Eugene B. Chang

Hypothesis and Aims: l--glutamine's(L-gln) protection of the intestinal mucosa during penods of physiological stress may reflect modulation of endogenous heat shock protein (hsp) expression. The potentiation of intestinal epithelial hsp72 expression by L@n during heat shock(hs) is described, yet the mechanisms remain unclear, We hypothesized that bg ln modulates the transcriptional acti~aty and nuclear localization of heat shock factor-I (HSF- i) in heat-shocked IEC-18 cells Methods: Cells were exposed to 0raM, 4 mM I_-gln or 4mM D-gin for 6 hours in serum-free, +/-phosphate-free DMEM, followed by hs at 42 degrees C for 23 mill. 32Pi labeling was tollowed by immunoprecipitation of HSF-1 and SDS PAGE. Shifts in HSF-1 pKi were determined by- 2-D isoelectric focnsing(IEF) gel electrophoresis. Cells grown on coverslips were hs and HSE-1 was locahzed by" laser contdeal microscopy. Parallel luciferase reporter experiments were perldemed using tandem HSE's upstream of a minimal promoter and compared to 1500bp hsp 72 promoter data. Results: By confocal microscopy, the absence of L-gin for 6 flours abolishes nuclear localization ot HSFq during hs, while nuclear localization is preserved by 4raM Dgln. While endogenous isomerase activity is unknown, D-gin restores so m e but significantly, less nuclear locaIization, Treatment with 5% FBS + 0raM L-gln was insufficient to restore nuclear localization of HSF-I during hs. Compared to 0raM L-gin, 4mM L-gin led to no significant difterence in HSF-1 phosphorylation during hs as d etemlined by IEF and HSF-1 32Pidabekng. Despite L-gln mediated increases in luciferase activity using a full-length hsp72 promoter, no etfect on a minimal heat shock element (HSE) promoter was seen. Conclusions:The potentiating etfect of L-gin on h eat-induced hsp72 transcript abundance and wild-type promoter activity have been described. Our data argue against direct L-gin mediated aherations in HSF-1 transcriptional activation during hs, The absence of HSF-1 in nuclei of 1.-gin depleted cells

A-433 AGA Abstracts

su gg est s that L-gin may modulate processes related to the trimerization and nuclear localization of HSF-1 during ha in irttesnnal epithdial cells, therefore underscoring the importance of L-gln as a detemlinant hap expression in the intestinal mucosa during physio- logical stress. (Work supported by" a 2002 CAG/Novartis/CIHR Research FeIlowshlp)

M2191

Gene Expression in The Human Small Intestine in Vivo, Effects of Iron-Inducod Lipid Peroxidation Freddy J, Troost, Wim H Saris, Robert-Jan M Brummer

Imroduction Iron induces lipid peroxidation and the subsequent release of antioxidants in the lumen of the small intestine. This may mediate geue expression in the intestinal waft. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine Methods Ten volunteers (22 +/-2 y) were tested on two occasions. Atter an overnight fast, duodenal biopsies, taken in the horizontal part of the duodenum (15 cm distally from the pylorus), were Obtained by duodenoseopy Subsequently, a catheter was inserted into the proximal small intestine ~,ath an injection port positioned in the proximal descending duodenum. After positioning, saline was injected at 10 ml/min and, after reaching steady state, either 80 or 400 mg iron as terrous gluconate was injected for 30 min. Finally, saline was injected for another 60 rain. A second duodenoscopy was pertbrnled to obtain tissue samples after the iron challenges. In the tissue samples, gene expression was measured using Affimetrix U 133A microarray chips (> 20.000 known genes). Resuhs A total number of 9880 genes were expressed in the small intestine. The expression of 91 genes was significantly changed by both the low and the high iron perfusion, whereas 293 genes tended to be regulated by the two iron challenges. Twenty-eight genes were statistically upregulated, whereas 63 genes were significantly dmvnregulated by both iron intervention_s Two of the upregnlated genes serve functions in cell growth and tumor suppression, one gene encodes for sdenoprotein P, which has antioxidant properties, haterest- ingly, two genes downregnlated by iron are also associated with tumor suppression and negative control of cell proliferation. Conclusion A large number of genes is expressed in the human proximal small intestine. Iron administration, resulting in lipid peroxidation, mediates expression of at least 91 genes in the small bowel.

M2192

Resveratrol Modulates the Polyamine Metabolism Of Colorectal Carcinoma Cells Freya Woher, Lyudmila Turehanowa, Juergen Stein

Background and Aims: Polyamines are essential tot proliferation and polyamine content is high in colon cancer cells. Because metabolism of polyamines is an interesting target in cbemoprevention, the aim of the present study was to examine the effect of the red wine polypbenol resveratrol on polyamine homeostasis in Caco-2 colon cancer calls. Methods: Caco-2 cells were incubated with resveratrol vs DMSO as control (<0.1%) for 24 h. Activity of ornithine-decarboxylase (ODC) and S-adenosylmethionine-decarboxTlase (SAMDC) were measured by quantification of t+{ClO2-release from ~4[C]-labdled omithine and S-adenosyl- methionine. SSkT-actwity was determined by quantification of 3[H]-acetylspermidine after addition of [acetyl-3Hl-CoA. Intracellular polyamine concentrations were measured with HPLC. Proteins were detected with Western blot, mRNA with PCR, and DNA-binding activity was quantified wittr a kit. Results: Resvemtrnl reduced ODC-activity and SAMDC-actMty at a concentration of 50 txM (543% and 599% of contrnl, respectively, p<0.05, n = 4), At the same tune dimimsbed expression of ODC mRNA and protein and c-myc protein were detected. SSAT-activity was enhanced (143.7% of control with 50 btM, p<0.05, n = 4 ) and levels of N -acetylspermidine and putreseine, markers of polyamine degradation, were elevated (200 btM; 242% and 651% of control, respectively). Resveratrol augmented protein levels of c-los and c-jun, but only" induced DNA-binding activity of c-los (362% of control with 100 ~tM). Conclusions: Resvemtrol perturbs polyamine synthesis by inhibiting ODC and SAMDC activity and enhancing degradation of polyanrines by SSAT. The effect on ODC- actMty is probably mediated by reduced ODC-expression, which might be caused by' diminished levels of its transcription factor c-myc. We speculate that increased putrescine levels might be responsible |or induction of orbs, which is also induced by the cbemopreven- tire sbm't chain fatty acid butyrate Modulation of polyamine metabolism seems to be another promising mechanism by which resveratroI exerts its chemopreventive effects on colon cancer cells. C-tos might ptay a role in difterentiation and growth arrest.

M2193

Identification of Mutations in the Human Reduced Folate Carrier in Patients with Folate Malabsorption Syndrome Bdamurngan Krisbuaswamy, Claudio Sandoval, Harold M. Said

Background: Tile intestinal absorption process of folate in human involves the reduced- tblate carrier ~RFC), A variety of conditions have been shmvn to interfere with this process, and recently two siblings were identified with a defect(s) in intestinal folate absorption which were linked with the so-called hereditary folate malabsorption (HFM) syndrome. Since the affected siblings responded favorably to oral pharmacologmal doses of folinic acid, it was hypothesized that mutations in the human reduced rotate carrier gene (hRFC or SLC19A1) may be involved. The aim of the present study was, therefore, to test this hypothesis. Methods: We evaluated the genomic DNA of the two sisters for mutations in the gene encoding the human reduced folate carrier using polymerase chain reaction (PCR) amplification tbllowed by' direct sequencing. Site-directed mutagenesis and transfection analyses were used to deternliue folate absorption, Western blotting and immunoflnorescence were used to detemline the localization of wild-type and mutated hRFC protein. Results: Tile mutations are located in well-conserved positions in human, mouse, rat, and hamster RFC, To determine the hmctional consequences of these mutations on the activity of the hRFC protein, we examined the eftect of introducing these mutations experimentally into

the open reading frame of hRFC on functional activity- aud membrane expression of the protein following its expression in HeLa cells. The resuhs showed that all the identified mutations to lead to real-function of hRFC manitesting as decreased folate uptake. We.stem blot analysis and immnnofluorescence studies revealed that all the mutant proteins were expressed at the plasma membrane. Conclusions: These findings report the identification of mutations in the hRFC that cause malfunctioning of the carrier protein but have no effect on its expression at the cell membrane. [Supported by grants from the Department of Veterans Affairs and the National Institutes of Health (DK56061 and DK58057)].

M2194

Fat Absorption and Enterohepatic Circulation of Bile Salts in Cystic Fibrosis Mice Marcel Bijvelds, Christian Hulzebos, Inez Bronsveld, Rick Havinga, Frans Stellaard, Maarten Sinaasappel, Hugo De Jonge, Henkjan J. Verkade

Background & Aims: Pancreatm lipase supplementation reduces fecal fat excretion in patients with cystic fibrosis (CF). We recently demoustrated, however, that fat malahsorption does not disappear completely, indicating that factors other than lipase deficiency contribute to fat malabsorption in CF (AJCN 1999;69:127). We now aimed to delineate the role of (post)lipolytic processes in CF-related fat malahsocption. Methods: Fat absorption in two murine CF models was determined by measuring fecal fat excretion. Lipolysis and fatty" acid uptake were assessed by comparing uptake of 3H-triolein-derived lipid and ~C-oleate, Pancreatic and bdiary function was investigated by, assessing fecal chymotrypsin excretion, bdiary bile salt (BS) composition, and kinetics of the enterohepatic BS circulation, using a stable isotope dilution method. Results: Fecal fat excretion was increased in Cftr null mice, compared with controls (17_+7 vs. 6_+3% of dietary fat intake, resp. p<0.01), but was not affected in homozygous AF508 mice. At 4 h after administration, uptake of 3H- and J~C-labeled dmtary lipid in intestinal mucosa and their appearance in plasma were 40-60% reduced in Cftr null mice, cmnpared with controls (p<0.01). The uptake of 3H-triolein- derived lipid was specifically reduced, compared to uptake of 14C-oleate. No indication was tound for a reduced pancreatic enzyme secretion. Fecal BS loss was increased m both CF models (Cftr null, +85%, p<0.01; AF508, +85%; p<0.001). Compared with controls, the synthesis rate of the primary bile salt cholate was increased in cftr null mice (10 -+ 2 vs. 17+_2 ~mol.100g*.day *, resp., p<0.01), in agreement with compensation for increased fecal losses. The size of the BS pool and the bihary BS secretion rates were similar between the CF mice and their controls Analysis of intestinal BS transport and expression of the principal ileal BS transporter (Asbt) suggested involvement of epithelial remodeling in BS wasting. Conclusions: Fat malahsorption in Cftr null mice is caused by impairnaeut of lipolysis and by a post-lipolytic process, but is unrelated to decreased pancreatic enzyme secretion or biliary pathology. We propose CFTR inactivation impedes micellar solubilization of lipids, iipase activity and epithelial transport of both fatty acids and bile salts.

M2195

Fructose Free Diet Does Not Improve GI Symptoms in Patients With Fructose Malabsorption If Irritable Bowel Syndrome Is Present Eva Fritz, Johann Hammer, Harald Vogelsang

Background: Fructose malabsorption (FM) has been identified as a cause of GI symptoms, The relation of FM and irritable bowel syndrome (1BS) is not clear, Hypothesis: Fructose reduced diet improves sympmms only m FM patients that do not suffer from IBS. Aim: 1) To evaluate the effect of diet in fi-uctose malabsorbers depending o u a diagnosis of IBS 2) To evaluate the influence of GI symptoms in FM patients on psychologmal status. Methods: 62 confirmed FM patients received a validated questionnaire evaluating frequency and severity of gastrointestinal synaptoms and effect of diet 12 months after diagnosis. Quality of Lite (QoL) and psychological status were assessed with SF 12, Hospital-Anxiety-Depression- Scale and the Eysenck Short Neuroticism Scale. Results: 49 questionnaires (79%) were returned, 30 patients were on diet for >- 12months. 50% of patients who were on diet for more than 12 months had IBS according to Rome I1. There was no significant difference in age, BMI, quality of life, and psychological status between IBS positive( + ) and negative(-) patients. Diet improved abdominal symptoms significantly more ofien in IBS- patients than in IBS+ patients (63% vs 28%; p=0.002). The compliance in keeping to the diet was comparable (81% vs 73%; p =0.2). hrrprnvement of GI symptoms by fructose reduced diet was not sigmficantly different between the 25 gram positive tested patients (n = 8) compared to those who tested 25 gram negative/50 gram positive (n = 19), The number of GI-symptoms that were classified as severe correlated negatwely with physical component (>0.362, p=0.022) and mental component (>0.533, p<0.001) of QoL Furthermore the number of severe G1 symptoms correlated positively with level of anxiety (r = 0.463, p = 0.003), depres- sion (r=0.498, p < 0 001) and neumticism (r=0.441, p=0.005). Conclusion: In FM the positive effect of diet on abdominal symptoms is markedly reduced if tire patient is suflenng of IBS. Severe Gl-symptoms significantly decrease quality of life and influence negatively the psychological status in fructose malabsorbers.

M2196

Evaluation of Pectin Digestion and Absorption in the Small Intestine and of Fermentation in the Large Intestine in the Same Subject Daisuke Chinda, Shigeyuki Nakali, Shinsaku Fukuda, Juichi Sakamoto, Tadashi Shimoyama, Kazuma Danjo, Daisuke Smm, Teruo Nakamura, Akihiro Muuakata, Kazuo Sugawara

BACKGROUND: Pectin is a form of dietary fiber that in principle is not digested m the small intestine, but which on entering the colon is fermented by conmrensal bacteria, with the production of gases and short chain fatty acids as potential energy sources. However, it is not known what proportion of pectin is actually digested by enzymes in the small intestine and is fermented in the large intestine of the same subject. It is therefore important

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