A72 AGA ABSTRACTS

557

EFFECT OF FATTY ACIDS OF DIFFERENT CHAIN LENGTH ONINTERFERON-f PRODUCTION FROM INTRAEPITHELIALLYMPHOCYTES.Yuriko Hara, Soichiro Miura, Shunsuke Komoto, Takashi Ogino, SeiichiroKoseki, Hitoshi Fujimori, Ryota Hokari, Hiroshi Nagata, SatoshiHachimura, Shuichi Kaminogawa, Hiromasa Ishii, Keio Univ Sch ofMedicine, Tokyo, Japan; National Defense Med Coli, Saitama, Japan; TheUniv of Tokyo, Tokyo, Japan.

Background: It is known that intraepitheliailymphocytes(lELs) play im­portant roles in intestinal mucosal immune response. We have previouslyreported that the release of several cytokines is enhanced in intestinalepithelial cells by long-chain fatty acids (Gastroenterology 116: A 588,1999). In this study, we examined how fatty acids of different chain lengthaffects the interferontlf'Ni-yproduction of intestinal intraepithelial cell­s(IELs) in murine small intestine after stimulation with anti-CD3 mono­clonal antibody(mAb) or interleukin (IL)-12/IL-18. Methods: IELs wereisolated from small intestine of BALB/c mice. IELs were stimulated invitro with plate-coated anti-CD3 mAb or IL-12/1L-18. IELs (200p.l,4X 105

) were coincubated in microtiter plates for 3 days with fatty acidmicelles of various concentrations. We used arachidonic acid, linoleic acidand oleic acid as long-chain fatty acids, octanoic acid as a middle-chainfatty acid and sodium butyrate as a short-chain fatty acid. IFN-yin thesupernatants was measured by ELISA. Expression of IFN-')IlIlRNAin IELswas determined by RT-PCR.Cell proliferation was assayed by 3H-thymi­dine uptake. Results: Significant production of IFN-yfrom IELs was ob­served after anti-CD3 mAb stimulation. IL-12 and IL-18 treatment alonedid not show any detectable induction of IFN-y, however, the combinationof IL-12 and IL-18 induced significant levels of IFN-Wroduction withoutTCR stimulation. Increased IFN-')IlIlRNAwas also observed after anti-CD3mAb or IL-12/1L-18 stimulation. Long-chain fatty acids inhibited dose­dependently the stimulated IFN-Wroduction and increased mRNA ofIFN-yat the doses more than 10JLM. Long-chain fatty acids also inhibitedthe anti-CD3 mAb stimulated cell proliferation. On the other hand, middle­chain fatty acid did not show any significant changes in IFN-Wroduction.Sodium butyrate at the high concentrations (~lmM) significantly inhibitedthe anti-CD3 mAb- or IL-12/1L-18- induced IFN-wroduction and theanti-CD3 mAb stimulated cell proliferation. In contrast, sodium butyrate atthe lower concentrations (l0-l00JLM) rather augmented the IFN-yproduc­tion. Conclusion: Administration of fatty acids differently modulates IFN­Wroduction from IELs depending upon their chain length, suggesting thatexposure of IELs to intraluminal fatty acids significantly modifies theimmune function of intestinal mucosa.

558

INVESTIGATION INTO THE PHYSICAL PROPERTIES OF AVISCOUS FIBRE IN RELATION TO ITS FUNCTION IN THELARGE BOWEL.Saphwan Al-Assaf, Glyn O. Phillips, Peter A. Williams, Michael E. Hav­ler, Peter W. Dettmar, The North East Wales Institute, Wrexham, Wales,United Kingdom; The North East Wales Institute, Wrexham, United King­dom; Reckitt & Colman Products Ltd, Hull, United Kingdom.

BACKGROUND: Plantago ovata (Po, Psyllium) is a natural fibre pos­sessing the properties required for it to have a range of beneficial gastro­intestinal effects. A major factor in its physiological behaviour is itsgel-like nature which is related to molecular parameters associated with ahigh molecular weight polysaccharide. Evidence indicates that the gelstructure is robust enough in-vivo to survive passage through the gastro­intestinal tract even though it acts as a substrate for bacterial fermentationin the colon. To investigate this further, the molecular structure has beendegraded in controlled steps and the effects on physicochemical parametershave been determined. METHODS: Molecular parameters were investi­gated principally in alkaline extracts using a GPC-MALLS system toseparate and quantitate various fractions. Rheological measurements werecarried out using a Stress Rheometer in oscillatory mode. Water holdingcapacity was a simple measure of gel volume produced after aqueoushydration. In-vitro fermentation was conducted in anaerobic batch cultures,whilst non-starch polysaccharides were determined as described by En­glyst. RESULTS: The alkali-soluble fraction of Po retains its viscoelasticproperties in the presence of substantial reduction (60%) in molecularweight. On rheological evaluation, the behaviour changes from a gel-likematrix to a viscous solution. In contrast, whole Po on hydration retains G'dominance (elastic component) throughout the series of degraded samples.Total dietary fibre content and fermentation profiles remained unchangedin samples with decreasing molecular weight whilst water holding capacitywas only slightly reduced. CONCLUSION: In the presence of substantialmolecular disruption, the principal properties of Po appear to remain intact.Although the isolation of soluble fractions allow the determination ofmolecular weight parameters of the active polysaccharide, in-vivo behav­iour of Po is more likely to be dependent on the macrostructure of thewhole husk.

GASTROENTEROLOGY Vol. 118, No.4

559BOTH BILE SALT TYPE AND PRESENCE OF CHOLESTEROLMARKEDLY INFLUENCE FATTY ACID ACTIVITY IN A SIMU­LATED MICELLAR PHASE.James E. Heubi, Samantha Gee, Xiaojian Cao, David Y. Hui, Patrick Tso,Acad Hosp Med Ctr, Cincinnati, OH; Univ of Cincinnati Coli of Medicine,Cincinnati, OH.

Background. In the absorption of triglyceride, fatty acid is dispersed inmixed micelles and taken up as monomers by the enterocyte. Neither theconjugated bile acid type nor the presence of cholesterol has been consid­ered to influence fatty acid monomeric activity. Aim. Experiments wereperformed to test the validity of this concept. Methods. A micellar solutioncontaining 15 mM conjugated bile acid, 1.3 mM mono-olein, and 2.6 mMfatty acid (labeled with 14C-oleate) was adjusted to pH 6.4. Fatty acidmonomeric activity was assessed by measuring % extracted into petroleumhydrocarbon (four volumes). Results. When the bile acid was taurocholate(TC), only 0.57::':0.08% (MeanzSl), n= 14) was extracted; in contrast,when the bile acid was tauroursodeoxycholate (TUDC), extraction was fargreater (34.4::':6%)(n=20). To verify this astonishing finding, mixtures ofTC and TUDC (total 15 mM) were used. These experiments showed thatas the TC/TUDC proportion increased, fatty acid extraction decreased in anexponential relationship. In a second set of experiments, the effect ofadding cholesterol (0.6 mM) was assessed. In the TC containing solution,fatty acid extraction increased sixty-fold to 36.0::':0.8% (n =4); in theTUDC solution, extraction also increased (from 36% to 45%). Conclu­sions. The presence of TC in a mixed micelle results in fatty acid bindingthat inhibits its transfer to a hydrophobic phase to a much greater extentthan when TUDC is present. The addition of cholesterol decreases fattyacid binding, perhaps by competing for binding sites in the mixed micelle.These in vitro experiments provide the first evidence that the compositionof the mixed micelle with respect to conjugated bile salt type and thepresence of cholesterol influence fatty acid monomeric activity. Theseeffects could influence the rate of fatty acid uptake from the micellar phasein vivo.

560REDUCTION OF PHORBOL ESTER INDUCED HYPERPERME·ABILITY BY GLUTAMINE IN HT-29CL.19A INTESTINALCELLS: THE ROLE OF GLUTAMATE.Hong Luo, Jack A. Groot, Paul A. van Leeuwen, Alexander P. Houdijk,Institute of Neurobiology, Univ of Amsterdam, Amsterdam, Netherlands;Dept of Surg, Vrije Univ Ziekenhuis, Amsterdam, Netherlands; Dept ofSurg, Slotervaart Ziekenhuis, Amsterdam, Netherlands.

Introduction Mucosal atrophy is associated with intestinal hyperpermeabil­ity. Glutamine reduces intestinal hyperpermeability in experimental mod­els of gut injury. We have shown that glutamine reduces phorbol estermediated hyperpermeability in monolayers of HT-29C1.l9A intestinalcells. The underlying mechanism for this effect is not known. Here wepresent data on the role of glutamate, the most important intestinal metab­olite of glutamine, in this proces. Methods Confluent monolayers of HT­29C1.l9A cells were cultured on permeable filters. After 14-17 days thecells were grown for another two days in glutamine free medium. Apicalto basolateral transepithelial permeability for horseradish peroxidase(HRP) was assessed by enzymatic assay. Phorbol-12,13-dibutyrate (PDB,IJLmollL) was used to increase HRP permeability. We investigated theeffect of glutamine, glutamate and the y-glutamyltransferase inhibitoracivicin. All the agents were added to the apical compartment. ResultsPDB increased HRP flux 3 times compared to control after 150 to 270 minof stimulation (p<O.OI). Glutamine (0.6 mmollL) significantly decreasedthis hyperpermeability confirming earlier results (p<0.05). Glutamate (0.6mmollL)had the same effect (p<O.OOl). Acivicin prevented the glutaminemediated decrease in PDB induced HRP hyperpermeability (p< 0.01). Theeffect of acivicin was not apparent in the presence of glutamate. Conclu­sion The results suggest that the inhibitory effect of glutamine on PDBinduced hyperpermeability in Ht-29C1.l9A intestinal cells is dependent onthe conversion of glutamine to glutamate by y-glutamyltransferase.

561

CHANGES IN EXPRESSION OF INTESTINAL PEPTIDE ANDAMINO ACID TRANSPORTERS IN RATS FED BY TOTAL PAR­ENTERAL NUTRITION.Alison Howard, Mark Jordinson, Robert A. Goodlad, Julian Rf Walters,Dianne Ford, Barry H. Hirst, Univ of Newcastle upon Tyne, Newcastle,United Kingdom; Imperial Coli Sch of Medicine, London, United King­dom; Imperial Cancer Research Fund, London, United Kingdom.

Expression of transporters involved in absorption of peptides and aminoacids across the small intestinal epithelium is regulated by dietary substrateavailability and by other factors such as hormones. In the present study, wehave examined mRNA expression for three of these transporters, thedipeptide transporter, PepTl, cationic amino acid transporter, CAT-I, andneutral amino acid transporter, ASCT2, in the intestine of rats fed by totalparenteral nutrition (TPN). Rats were established on TPN given into thejugular vein and received no oral intake for seven days. Control animalsreceived saline infusions and had free access to oral diets. Total RNA wasextracted from the mucosa of duodenal and ileal regions of the smallintestine. cDNA was prepared and semi-quantitative polymerase-chainreaction (PCR) performed. Products were analysed by agarose gel electro-

April 2000

phoresis. The signals were quantified by spot-densitometry, relative to 18srRNA values. Both CAT-l and Pep'I'l were expressed at equivalent levelsin the duodenum and ileum of control animals. ASCT2 expression wasapproximately two-fold higher in the ileum than in the duodenum. CAT-lexpression was not affected by TPN in either duodenum or ileum. Expres­sion of both Pep'TI and ASCT2 was significantly increased in the ileum ofTPN fed animals. Pep'I'l transcripts were increased to 296% of controlvalues (p<0.OO5) and ASCT2 transcripts to 220% (p<0.OO5). Expressionof both transcripts was not significantly altered in the duodenum (Pep'I'l:145%, p>0.05; ASCT2: 84%, p>O.4). The results demonstrate that ex­pression of these transporters is not solely dependent on the presence ofluminal contents. In these calorifically-maintained animals, up-regulationof Pep'I'l and ASCT2 may reflect adaptational response to maintaining themetabolic requirements of the enterocytes in the absence of luminal nutri­tion. Supported by BBSRC

562A NOVEL VITAMIN E DERIVATIVE (TMG) PROTECTS GAS­TRIC MUCOSAL DAMAGE INDUCED BY ISCHEMIA ANDREPERFUSION.Hiroshi Ichikawa, Hirohisa Takano, Yuji Naito, Norimasa Yoshida,Toshikazu Yoshikawa, Motoharu Kondo, Kyoto Prefectural Univ of Med­icine, Kyoto, Japan.

The antioxidative defense effect of a novel vitamin E derivative, 2-(a-D­glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-01 (TMG), whichhas excellent water-solubility (> 1 x 103 mglml), was investigated using thereperfusion-induced rat gastric mucosal injury model in vivo. It has beenreported that TMG is somewhat located within the membrane surface andacts as a powerful antioxidant by scavenging radicals generated either inthe aqueous or in the lipid phase. Clamping the celiac artery for 30 mininduced ischemia, and reoxygenation was produced by removal of theclamp 60 min after ischemia. Just before removal of the vascular clamp,TMG at a dose of 0.4 - 4 mglkg dissolved in physiological saline wereinjected intravenously. The area of gastric mucosal erosion or ulcerativelesion (ulcer index) significantly increased from mean basal levels after 60min of reperfusion. This ulcer index was significantly inhibited by pre­treatment with TMG. The concentration of thiobarbituric acid-reactivesubstances (TBA-RS) in the gastric mucosa, an index of lipid peroxidation,were also significantly higher than basal level after 60 min of reperfusion.This increase in TBA-RS in the gastric mucosa after ischemia-reperfusionwas inhibited by the pretreatment with TMG. Furthermore, myeloperoxi­dase (MPO) activity in the gastric mucosa, an index of neutrophil infiltra­tion, significantly increased by ischemia reperfusion, and pretreatment ofTMG significantly reduced this increase of MPO activity in gastric mucosa.These results suggest that TMG may have protective effects against theoxidative stress in the reperfusion-induced injury models.

563THE MECHANISM OF GERMINATED BARLEY FOODSTUFF INATTENUATING INTESTINAL INFLAMMATION IN COLITIS.Osamu Kanauchi, Akira Andoh, Yoshio Araki, Keiichi Mitsuyama, At­sushi Toyonaga, Michio Sata, Toshifumi Hibi, Toshihiko Iwanaga, TadaoBamba, Kirin Brewery Co, Takasaki, Japan; Shiga Univ of Med Sci, Ohtsu,Japan; Kurume Univ Sch of Medicine, Kurume, Japan; Keio Univ Sch ofMedicine, Tokyo, Japan; Hokkaido Univ, Sch of Veternary Medicine,Sapporo, Japan.

Background and Aims: Germinated barley foodstuff (GBF) contains pro­tein and insoluble dietary fiber. We have previously shown in ulcerativecolitis patients and colitis model that GBF feeding attenuates mucosaldamage by increasing luminal butyrate levels (Aliment Pharmacol Ther12:1225, 1998). However, the detailed mechanism remains unclear becauseof its heterogeneous nature. The present study was carried out to (1)evaluate the active ingredient in GBF, (2) to examine its effect on the repairprocess in colonic inflammation, and (3) to estimate its effect on luminalmicroflora by using a dextran sulfate sodium (DSS) colitis model. Meth­ods: Colitis was induced by feeding a 0.5 % to 3.5 % DSS containing dietto male SD rats. (1) Active ingredient: GBF was fractionated enzymaticallyinto fiber- and protein-rich fractions. Each fraction was administered toDSS-colitis rats. Clinical signs, cecal short chain fatty acid (SCFA) con­centrations and serum aI-acid glycoprotein (AAG) were determined. (2)Effect on mucosal repair: GBF with or without sulfasalazine (SASP), orSASP alone was administered to rats after the onset of colitis. Seven daysafter initial treatment, the number of epithelial cells in H&E sections wereevaluated morphologically in a blind fashion and serum AAG was deter­mined. (3) Effect on microflora: Bacterial microflora and SCFA measure­ments in the luminal content was performed in colitis rats treated with orwithout GBF. Results: (1) GBF and GBF-fiber significantly attenuatedclinical signs of colitis (p<0.05) and decreased serum AAG (35.7 in GBF,43.6 in GBF-fiber vs 262.3 in control; p.g/ml p<O.OOl), with a significantincrease in cecal butyrate production (20.1 in GBF, 23.4 in GBF-fiber, 2.1in control p. moll g, p<0.05), while GBF-protein did not. (2)Treatment ofGBF alone and GBF with SASP significantly accelerated colonic epithelialrepair (76% in GBF, 70% in GBF with SASP vs 41% in control, p<0.05)and improved clinical signs (p<0.05). These effects were more potent thanwith SASP alone. (3) GBF feeding led to a significant increase in Eubac­terium (8.2 in GBF vs 6.8 in control; log Ig, p<0.05) and a decrease inBacteroidaceae (p<0.05) were observed. Furthermore, Bifidobacteriumwas also increased along with a significant increase on butyrate production

AGAA73

(p<0.05). Conclusions: These findings suggest that the fiber fraction ofGBF may effectively enhance luminal butyrate production by Eubacteriumand Bifidobacterium, and thereby accelerate colonic epithelial repair incolitis.

564

A NON-TOXIC HEAT SHOCK PROTEIN 70 INDUCER, GERA­NYLGERANYLACETONE, EFFECTIVELY RESTORES THEHEAT SHOCK RESPONSE IN GASTRIC MUCOSA OF PROTEIN­MALNOURISHED RATS.Kazuhito Rokutan, Tomoko Kawai, Shigetada Teshima, Tsukasa Kawa­hara, Takeshi Nikawa, Kyoichi Kishi, Sch of Medicine, Univ of To­kushima, Tokushima, Japan.

Background: Protein malnutrition is inevitable for chronically ill patientswith wasting diseases and is linked to cachexia, immune dysfunction, andpoor prognosis. Protein-malnourished patients occasionally suffer fromsevere gastric mucosal damage when confronted with additionally stressfulsituations. However, it is usually difficult to effectively improve the nutri­tional state due to the underlying diseases. We report here that proteinmalnutrition inhibits heat shock protein (HSP) 70 induction in rat gastricmucosa and impairs the mucosal defense against stress ulcer. We alsoexamined whether a non-toxic HSP70 inducer, geranylgeranylacetone(GGA), effectively improved the mucosal integrity by stimulating HSP70induction under protein malnutrition. Methods: The present experimentswere approved by the Animal Care Committe, University of Tokushima.Male Wistar rats fed a 5% or a 20% casein diet for 3 weeks were exposedto restraint and water-immersion stress, and the ulcer index was measured.The activation of heat shock factor 1 (HSFl) was examined by gel mobilityshift assay with the heat shock element oligonucleotide. The level s ofHSP70 mRNA and protein were measured by Northern blotting andWestern blotting, respectively. Results and Conclusion: Protein malnutri­tion did not change the HSFllevel in the gastric mucosa, while the proteindeficiency attenuated the HSFI activation and inhibited the HSP70 mRNAexpression and HSP70 induction after exposure to the stress, leading toaggravate mucosal damage. A single or short-term administration of GGA(200 mglkg twice a day) to the protein-malnourished rats for up to oneweek failed to stimulate the heat shock response, while administration ofGGA for longer than 2 weeks restored the HSP70 induction, and 3 weekslater, the HSP70-inducing capacity of gastric mucosa of the protein­malnourished rats recovered to the level of normally nourished rats. Theteatment of protein deficient rats with GGA for 3 weeks induced higherresistance against stress ulcer than that in control rats, suggesting othermechanism besides HSP70-inducing action may be involved in the en­hanced mucosal integrity by GGA. GOA has been widely used in Japan asan antiulcer drug for more than 15 years without any serious adverseeffects. Our results suggest that GOA may have a potential benefit for theprevention of stress ulcer in chronically and/or critically ill patients withprotein malnutrition.

565

FERMENTATION PRODUCTS OF ALCOHOLIC BEVERAGESMALEIC AND SUCCINIC ACID STIMULATE CCK RELEASE.Karlheinz Kiehne, Jan H. Egberts, Cornelia Wilgus, Stephan Teyssen,Ulrich R. Folsch, Karl-Heinz Herzig, I. Dept of Internal Medicine, Chris­tian-Albrechts-Univ, Kiel, Germany; Univ of Heidelberg, Mannheim, Ger­many.

Alcoholic beverages with low ethanol content are powerful stimulants ofgastrin release and gastric acid output in humans (Singer et al. 1991).Subsequent studies revealed that fermentation but not distillation producedthe stimulators (Teyssen et al. 1997). Recently, maleic and succinic acidhave been identified as stimulants of gastric acid secretion from fermentedalcoholic beverages (Teyssen et al. 1999). Furthermore, wine and beersignificantly increased meal-stimulated pancreatic secretion in humans(Hajnal et al. 1990). Therefore, in this study we investigated the effect ofmaleic and succinic acid on CCK release using isolated perfused mucosalcells and the neuroendocrine cell line STC-l. Methods: Isolated cells wereobtained by incubation of rat prox. small bowel in Ca2+ -free buffer. Aftercentrifugation, washing, filtration the cells were mixed with Sephadex G50and transferred into a perfusion apparatus and continously perfused. Theneuroendocrine cell line STC-l was seeded (l05 cells/ml) into 6-well platesreaching more than 90% confluence after 3-4 days. CCK determination wasmeasured using a pancreatic bioassay system. Maleic and succinic acid didnot affect amylase secretion from isolated acini. Results: In isolated duo­denal cells, maleic and succinic acid dose-dependently stimulated CCKrelease from 2,7:t0,3 to B,I:tl,7 and 27,1:t3,4pM at lmM, an amount,which is found in finished beer (Piendl 1980). At lOOnM succinic but notmaleic acid still stimulated CCK release to 8±0,9pM. In addition, in theneuroendocrine cell line STC-I maleic and succinic acid dose-dependentlystimulated CCK release from 3,1:to,3 to 8,9:t 1,5 and Il,7:t2,3pM at100nM, respectively. Pretreatment of the cells with the voltage-gatedL-type calcium channel blocker diltiazem significantly inhibited maleicand succinic acid stimulated CCK release. Conclusion: These data identifymaleic and succinic acid as potent direct stimuli of CCK release viaincreases in intracellular calcium mediated by voltage-gated L-type cal­cium channels.