Title. Author(s)Suzuki, Takuya; Hara, Hiroshi. CitationLife Sciences, 79(4): Issue Date Doc URL. Type. File Information.

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1 Title Difructose nhydride III nd sodium cprte ctivte cells Author(s)Suzuki, Tkuy; Hr, Hiroshi CittionLife Sciences, (): 01- Issue Dte Doc URL Type rticle (uthor version) File Informtion LFS-S-0-01.pdf Instructions for use Hokkido University Collection of Scholrly nd Ac

2 Elsevier Editoril System(tm) for Life Sciences Mnuscript Drft Mnuscript Numer: Title: Difructose nhydride III nd sodium cprte ctivte prcellulr trnsport vi different intrcellulr events in Cco- cells Article Type: Full Length Article Section/Ctegory: Keywords: Difructose nhydride III; Sodium cprte; Prcellulr trnsport; Tight junction; Cco- cells Corresponding Author: Dr Hiroshi Hr, PhD Corresponding Author's Institution: Hokkido University First Author: Tkuy Suzuki, PhD Order of Authors: Tkuy Suzuki, PhD; Hiroshi Hr, PhD Mnuscript Region of Origin: JAPAN Astrct: A nondigestile discchride, difructose nhydride (DFA) III, is known to ctivte clcium trnsport vi tight junctions (TJs); however, the chrcteristics of nd mechnisms for the increse in prcellulr trnsport induced y DFAIII hve not een clrified. We compred the effect of DFAIII with tht of sodium cprte (C), well-known enhncer of TJ permeility, on the chnges in TJ proteins, trnsport of prcellulr mrkers, nd effects of nine cellulr signling lockers using Cco- monolyers. The ddition of DFAIII (0-0 mmol/l) nd C (0- mmol/l) to the picl medium of the Cco- monolyers dose-dependently decresed trnsepithelil electricl resistnce (TER), which is n indictor of TJ permeility. The reduction with C ws much fster thn tht with DFAIII. Trnsport of the prcellulr mrkers of vrious moleculr weights (1-,00) ws elevted y the ddition of 0 mmol/l DFAIII nd mmol/l C. The trnsport rtes were much in the presence of C thn of DFAIII, while the reduction in

3 TER y two tretments ws similr (from 1,000 to 00 Ω*cm). Tretment with DFAIII nd C chnged the distriution of ctin filment nd cludin-1, ut not occludin, junctionl dhesion molecule-1, or zonul occludens-1; however, ltertions in the ptterns of the TJ proteins differed ccording to tretment. An inhiitor of myosin light chin kinse nd cheltor of intrcellulr clcium ion ([C+]i) ttenuted the TER reduction y C, ut not y DFAIII. These dt demonstrte tht the increse in TJ permeility induced y DFAIII results from the ltertions to ctin nd cludin-1 vi [C+]i-independent mechnisms.

4 Cover Letter 1 1) Title of the rticle: Difructose nhydride III nd sodium cprte ctivte prcellulr trnsport vi different intrcellulr events in Cco- cells ) Nmes of uthors: Tkuy Suzuki 1, Hiroshi Hr 1 ) Affilition of the uthors: 1 Division of Applied Bioscience, Grdute School of Agriculture, Hokkido University, Kit-, Nishi-, Kit-ku, Spporo, Jpn, 00- Jpn Society for the Promotion of Science,, 1-Bncho, Chiyod-ku, Tokyo, Jpn, -1 ) Corresponding uthor: 1 1 Nme: Miling ddress: Hiroshi Hr Kit-, Nishi-, Kit-ku, Spporo 00-, JAPAN 1 1 Telephone numer: Fx numer: e-mil ddress: hr@chem.gr.hokudi.c.jp

5 Mnuscript Astrct A nondigestile discchride, difructose nhydride (DFA) III, is known to ctivte clcium trnsport vi tight junctions (TJs); however, the chrcteristics of nd mechnisms for the increse in prcellulr trnsport induced y DFAIII hve not een clrified. We compred the effect of DFAIII with tht of sodium cprte (C), well-known enhncer of TJ permeility, on the chnges in TJ proteins, trnsport of prcellulr mrkers, nd effects of nine cellulr signling lockers using Cco- monolyers. The ddition of DFAIII (0-0 mmol/l) nd C (0- mmol/l) to the picl medium of the Cco- monolyers dose-dependently decresed trnsepithelil electricl resistnce (TER), which is n indictor of TJ permeility. The reduction with C ws much fster thn tht with DFAIII. Trnsport of the prcellulr mrkers of vrious moleculr weights (1-,00) ws elevted y the ddition of 0 mmol/l DFAIII nd mmol/l C. The trnsport rtes were much in the presence of C thn of DFAIII, while the reduction in TER y two tretments ws similr (from 1,000 to 00 Ω cm ). Tretment with DFAIII nd C chnged the distriution of ctin filment nd cludin-1, ut not occludin, junctionl dhesion molecule-1, or zonul occludens-1; however, ltertions in the ptterns of the TJ proteins differed ccording to tretment. An inhiitor of myosin light chin kinse nd cheltor of intrcellulr clcium ion ([C + ] i ) ttenuted the TER reduction y C, ut not y DFAIII. These dt demonstrte tht the increse in TJ permeility induced y DFAIII results from the ltertions to ctin nd cludin-1 vi [C + ] i -independent mechnisms. Keywords Difructose nhydride III, Sodium cprte, Prcellulr trnsport, Tight junction, Cco- cells Arevitions C, clcium; C, sodium cprte; DFA, difructose nhydride; FD, fluorescein isothiocynte-conjugted dextrn; FITC, fluorescein isothiocynte; JAM, junctionl dhesion molecule; LDH, lctte dehydrogense; LY, lucifer yellow CH dipotssium slt; MLCK, myosin light chin kinse; PLC, phospholipse C; TJ, tight junction; ZO-1, zonul occludens 1 1

6 Introduction Difructose nhydride III (DFAIII; di-d-fructo-furnose 1, :, dinhydride) is nondigestile discchride produced y inulin fructotrnsferse, n enzyme of Arthrocter sp. H- (Sito nd Tomit, 000). Our recent studies demonstrted tht ingestion of DFAIII enhnced intestinl clcium (C) sorption in rts (Mineo et l., 00; Mitmur et l., 00; Suzuki et l., 1) nd humns (Shigemtsu et l., 00). One of the mjor mechnisms responsile for the increse in C sorption is the promotion of prcellulr C trnsport through the direct stimultion of DFAIII in the smll intestine (Mineo et l., 00; Mitmur et l., 00; Shigemtsu et l., 00; Suzuki et l., 1). Some cellulr events my e involved in the promotion of prcellulr C trnsport induced y DFAIII. We lso showed tht DFAIII enhnced prcellulr C trnsport in humn intestinl Cco- cells (Suzuki nd Hr, 00), which indictes tht the Cco- monolyer is useful model of the smll intestinl epithelium for exmining the mechnisms underlying the promotion of prcellulr C trnsport y DFAIII. C sorption in the smll intestine proceeds y two routes, n ctive trnscellulr trnsport nd pssive prcellulr trnsport (Bronner, 1; Bronner nd Pnsu, 1). Trnscellulr trnsport is sturle, regulted y 1, -dihydroxy vitmin D, nd minly confined to the duodenum (Bronner, 1; Bronner nd Pnsu, 1). Prcellulr trnsport is n unsturle process tht requires grdient of C concentrtions etween the luminl nd solterl sides, nd occurs throughout the intestines (Bronner, 1; Bronner nd Pnsu, 1). Prcellulr pssge of ions, wter, nd some wter-solule molecules is regulted y tight junctions (TJs) locted on the uppermost portion of the lterl plsm memrnes of djcent cells (Mitic et l., 000). The TJ is multiprotein complex composed of oth trnsmemrne nd cytosolic proteins, lthough their functions re not completely understood. Three integrl trnsmemrne proteins of TJs hve een identified: occludin (Furuse et l., 1), cludin (Furuse et l., 1), nd junctionl dhesion molecule (JAM) (Mrtin-Pdur et l., 1). Occludin nd cludin re tetr-spnning trnsmemrne proteins. Occludin plys n importnt role in the orgniztion of the TJ, ut does not pper to e essentil for its ssemly (Furuse et l.,

7 ; Sitou et l., 000). The cludin fmily hs een expnded to memers, nd they re known to ply crucil role in the gte-keeping function of TJs (Furuse et l., 00). JAM is single trnsmemrne protein tht hs een shown to prticipte in the seling of the TJs (Liu et l., 000). The croxyl terminl ends of these three trnsmemrne proteins interct with zonul occludens 1 (ZO-1), cytosolic plque protein (Bzzoni et l., 000; Furuse et l., 1; Itoh et l., 1). Severl TJ proteins, including occludin nd ZO-1, ssocite with the ctin cytoskeleton, which lso regultes the function of TJs (Itoh et l., 1; Wittchen et l., 1). Prcellulr trnsport vi TJs is physiologiclly regulted y vrious intrcellulr signlings (Krczewski nd Groot, 000). Extrcellulr stimuli, including food fctors, drugs, nd chemicls, re reported to ffect the prcellulr trnsport through signl trnsduction (Nusrt et l., 000). Sodium cprte (C), medium-chin ftty cid, hs een shown to increse prcellulr trnsport vi the contrction of ctin filments following the phosphoryltion of myosin light chins (MLCs) y C + /clmodulin-ctivted MLC kinse (MLCK) in Cco- cells (Lindmrk et l., 1). Lysophosphtidic cid increses TJ permeility cused y ctin contrction vi the Rho-kinse-dependent pthwy (Hirse et l., 001). Hydrogen peroxide lso induces ltertions in occludin nd ZO-1 vi the ctivtion of phosphtidylinositol -kinse nd protein tyrosine kinse, nd enhnces TJ permeility in Cco- cells (Sheth et l., 00). Protein tyrosine kinse prticiptes in numer of cellulr signl cscdes, nd the tyrosine phosphoryltion of occludin ffects its locliztion nd interction with ZO-1, -, nd - (Kle et l., 00). Stimultion of protein kinse A or C is lso known to regulte prcellulr permeility in epithelil cells (Kottr nd Vnk, 00; Stenson et l., 1). We reported tht DFAIII enhnced prcellulr trnsport with the induction of C + signling in Cco- cells (Suzuki nd Hr, 00). Therefore, we suggest tht the promotion of prcellulr trnsport y DFAIII results from ltertions in the cytoskeleton or TJ proteins vi n intrcellulr event fter DFAIII recognition y puttive sensory system in Cco- cells. Prcellulr pthwys re importnt for the trnsport of ions nd wter, while TJs form physicl rrier to the diffusion of llergens, toxins, nd pthogens. Uncontrolled increses in intestinl

8 permeility or disruption of TJ function llow the entry into the lood strem of these undesirle molecules. Thus, it is importnt to evlute the chrcteristics of the increse in prcellulr trnsport y DFAIII. The ims of the present study were to exmine the effects of DFAIII on the ltertions in TJ proteins nd trnsport of prcellulr mrkers of vrious moleculr weights, nd to exmine the effects of the following cellulr signling lockers on the ction of DFAIII: inhiitors of phospholipse C (PLC), MLCK, protein kinse C, lph-suunit of Gi protein, denylte cyclse, protein tyrosine kinse, phosphtidylinositol -kinse, nd Rho-ssocited protein kinse nd cheltor of intrcellulr C +. And we lso compred the ction of DFAIII with tht of C, s C is well-known enhncer of TJ permeility nd the mechnism underlying the ction of C hs een extensively investigted.

9 Mterils nd methods Chemicls Difructose nhydride III (DFAIII; di-d-fructo-furnose 1, :, dinhydride) ws kindly provided y Nippon Beet Sugr MFG., Ltd. (Oihiro, Jpn). Rhodmine phlloidin ws purchsed from Moleculr Proe Inc. (Eugene, OR). Mouse nti-occludin-fitc conjugte, rit nti-cludin-1, rit nti-zo-1, rit nti-jam-1, nd got nti-rit IgG-FITC conjugte were purchsed from Zymed lortories Inc. (Sn Frncisco, CA). U1 (n inhiitor of PLC), BAPTA-AM ( cell-permele cheltor of C + ), ML- (n inhiitor of MLCK), H- (n inhiitor of protein kinse C), nd pertussis toxin (n inhiitor of lph-suunit of Gi protein) were purchsed from Sigm Chemicl (St. Louis, MO). SQ (n inhiitor of denylte cyclse), genistein (n inhiitor of protein tyrosin kinse), LY00 (n inhiitor of phosphtidylinositol -kinse), nd Y- (n inhiitor of Rho-ssocited protein kinse) were purchsed from Cliochem (Sn Diego, CA). [ H]-mnnitol (specific ctivity, 0 TBq/mol) ws purchsed from Americn Rdioleled Inc. (St. Louis, MO). All other chemicls were otined from Wko Pure Chemicl Industries, Ltd. (Osk, Jpn). Cell culture Cco- cells (HTB-; Americn Type Culture Collection, Rockville, MD) were propgted nd mintined in high-glucose (. g D-glucose/L) DMEM supplemented with 0 ml/l decomplemented fetl ovine serum, mmol/l sodium icronte, 1 mmol/l sodium pyruvte, 0,000 U/L penicillin, nd 0 mg/l streptomycin, nd djusted to ph. (Suzuki nd Hr, 00). The cells were seeded into permele polyester memrne filter supports (Trnswell, 1 mm dimeter, 0. µm pore size, 1.0 cm growth re; Corning Costr Co., Cmridge, MA) t density of,000 cells/cm. Prior to ll experiments, cultures were mintined for t lest d fter seeding. The medium ws refreshed every d. Cultures were used for experiments etween pssge nd 0. Mesurement of tight junction permeility Trnsepithelil electricl resistnce (TER) cross the cell monolyers ws mesured using commercil pprtus (Millicell-ERS; Millipore Co., Billeric, MA). The Cco- cells grown on the

10 permele memrne filters were rinsed with pre-wrmed HBSS [1 NCl,. NHCO, 0. N HPO,. KCl, 0. KH PO, 1. CCl, 0. MgCl, 0.1 MgSO,. D-glucose,.0 L-glutmine, nd HEPES (mmol/l), ph.] t C. The solterl nd picl chmers of the cells were thed in 1.0 nd 0. ml pre-wrmed HBSS t C, respectively. After 0-min equilirtion period, the experiments were initited y dding DFAIII (0,, 0,, or 0 mmol/l) or C (0,,., or mmol/l) to the picl chmer. TER ws mesured efore (0 min) nd t severl points fter the strt of incution (1-10 min). The experiments using C were crried out using stndrd HBSS in the solterl chmer nd C + /Mg + -free HBSS in the picl chmer to void precipittion of the ftty cid. These conditions hd no effect on TER cross the monolyers. To exmine the osmotic effect of DFAIII on TJ permeility, 0 mmol/l ethylene glycol or DFAIII ws dded to the picl or solterl chmers in the following comintions: picl chmer/solterl chmer; none/none; DFAIII/none; none/dfaiii; ethylene glycol/none; none/ethylene glycol; DFAIII/ethylene glycol. TER cross the Cco- monolyers ws mesured efore nd fter incution for h with ech of the six tretments. Trnsport of prcellulr mrkers for 1 h ws ssessed from the picl to the solterl chmer in the monolyers fter -h exposure to 0 mmol/l DFAIII or 0.-h exposure to mmol/l C. Monolyers incuted for h without DFAIII or C were used s the control group. Mnnitol (contining 0 MBq/L), lucifer yellow CH dipotssium slt (LY), nd FITC-conjugted dextrn (FD-; verge moleculr weight,00), (FD-;,00), 0 (FD-0; 1,00), nd 0 (FD-0;,00) were used s prcellulr mrkers, nd were dded into the picl chmer t finl concentrtion of 0 µmol/l. The concentrtions of LY nd FDs in the solterl solution were determined y mesuring fluorescence (CAF-1; JASCO Interntionl Co., Ltd., Tokyo, Jpn). The rdioctivity of [ H]-mnnitol in the solterl solution ws mesured y mens of liquid scintilltion counting system (LSC-0; Alok, Tokyo, Jpn) (Suzuki nd Hr, 00). To clrify the cellulr signling pthwy involved in the promotive effect of DFAIII on TJ permeility, nine signling lockers were used s follows: U1, µmol/l; BAPTA-AM, 0

11 µmol/l; ML-, 0 µmol/l; H-, 0 µmol/l; Y-, µmol/l; SQ, 00 µmol/l; genistein, 00 µmol/l; LY00, µmol/l; pertussis toxin, 00 µg/l. Signling lockers except for pertussis toxin were dded to the picl side of the monolyers during the equilirtion (1 h) nd experimentl ( h) periods. Pertussis toxin ws dded to the Cco- cell medium h prior to the experiment. The TER cross the monolyers ws mesured efore nd fter incution for h with or without 0 mmol/l DFAIII. The effect of four signling lockers, BAPTA-AM, ML-, H-, nd Y-, on the C-induced enhncement of TJ permeility ws exmined in the sme mnner. The TER cross the monolyers ws mesured efore nd fter 0.-h incution with or without mmol/l C. Immunofluorescent locliztion of TJ proteins nd F-ctin Cco- cell monolyers grown on the permele memrne filters were used fter -h exposure to 0 mmol/l DFAIII or 0.-h exposure to mmol/l C. The monolyers incuted for h without DFAIII nd C were used s the control group. For F-ctin stining, the cell monolyers were fixed in.% formldehyde for 0 min, permeilized in ice-cold 0.% Triton X-0, nd incuted with,000 U/L rhodmine phlloidin for 0 min t room temperture. For immunostining of TJ proteins, the cell monolyers were fixed nd permeilized with methnol for min t -0 C nd were incuted with % norml got serum for 0 min t room temperture. For cludin-1 nd ZO-1, the cell monolyers were incuted with cludin-1 or ZO-1 ntiody (1:0 dilution) for 1 h t room temperture. For JAM-1, the monolyers were incuted with JAM-1 ntiody (1:0 dilution) for h t C. The cell monolyers were incuted with FITC-conjugted nti-rit IgG (1:0 dilution) for 1 h t room temperture. For occludin, the cell monolyers were incuted with occludin ntiody-conjugted FITC (1:0 dilution) for h t C fter incution with % norml mouse serum. For doule stining of F-ctin nd cludin-1, the monolyers fixed with methnol were used. All monolyers were mounted in 1% n-propyl gllte in 0% glycerol nd nlyzed y confocl microscopy (Zeiss LSM ; Zeiss, Thornwood, NY). Mesurement of lctte dehydrogense ctivity Cco- cell viility ws ssessed y mesuring the relese of lctte dehydrogense (LDH)

12 1 1 in the picl nd solterl solutions of the monolyers incuted with DFAIII (0,, 0,, or 0 mmol/l) for h or C (0,,., or mmol/l) for h. LDH ctivity ws determined using commercilly ville kit (LDH CII-Test Wko; Wko Pure Chemicl Industries). Sttisticl nlysis All vlues re expressed s mens ± SEM. The TER is expressed s Ω cm of surfce re of the monolyer. The trnsport of prcellulr mrkers cross the monolyers re expressed s nmol of ech mrker trnsferred/cm of surfce re or percentge increse ginst the vlues of the control group, nd the two vlues were logrithmiclly trnsformed efore sttisticl nlyses, s heteroscedsticity ws present within ech dt. Sttisticl nlyses were performed y repeted mesure -wy ANOVA or 1-wy ANOVA followed y Duncun multiple rnge test (Duncn, 1). A difference with P < 0.0 ws considered significnt. These sttisticl nlyses were done y the generl liner models procedure of the Sttisticl Anlysis Systems progrm (version.0; SAS Institute Inc., Cry, NC). 1 1

13 Results Effects of DFAIII nd C on TER cross the cell monolyers nd LDH relese Incution time, DFAIII concentrtion, nd the interction etween them ll hd significnt effects on TER (P < 0.001, -wy ANOVA, Fig. 1A). DFAIII dose-dependently nd grdully decresed TER cross the monolyers from 0 min to min fter the strt of incution. There were no decreses with mmol/l DFAIII nd no differences in the TER t ny dose of DFAIII t 0 min. At 0, 0,, nd 10 min, vlues of TER with 0,, nd 0 mmol/l DFAIII were lower thn those without DFAIII or with mmol/l DFAIII. Incution time, C concentrtion, nd the interction etween them lso hd significnt effects on TER (P < 0.001, -wy ANOVA, Fig. 1B). C dose-dependently nd rpidly decresed TER fter the strt of incution. For ll concentrtions tested, TER reched its lowest vlue t 1 min. The TER vlues for,., nd mmol/l C were lower thn the vlues without C nd the vlues for. nd mmol/l C were lower thn tht for mmol/l C t ll time points except efore incution. There were no differences in LDH ctivity, n indictor of cell memrne dmge, in the picl nd solterl solutions fter -h incution with 0,, 0,, nd 0 mmol/l DFAIII (Tle 1). The ddition of C dose-dependently incresed LDH relese to the picl nd solterl solutions fter -h incution. The LDH ctivity in the picl solution ws higher in the monolyers incuted with,., nd mmol/l C thn in those incuted without C. The specificity of promotive effect of DFAIII on TJ permeility Apicl pplictions of DFAIII [DFAIII/none (picl chmer/ solterl chmer) nd DFAIII/ethylene glycol] clerly decresed TER vlues cross the monolyers for h (Tle ); however, the solterl ppliction of DFAIII hd smller effect on the TER thn did the picl ppliction, nd the vlue in the none/ DFAIII tretment ws higher thn those in the two picl DFAIII tretments. The solterl ppliction of ethylene glycol hd no effect on TER. There were no differences in TER cross the Cco- monolyers efore the -h incution mong the six tretments.

14 Effects of DFAIII nd C on trnsport of prcellulr mrkers The trnsport rtes of LY nd the four FDs with vrious moleculr weights were higher in the monolyers incuted with 0 mmol/l DFAIII thn in those incuted without DFAIII (Fig. A). The % increses ginst the control group were lso significnt for ll mrkers except mnnitol (Fig. B). In the monolyers incuted with mmol/l C, the trnsport rtes nd the % increses of ll mrkers were much higher thn those in the control nd the DFAIII groups. The trnsport rtes of FDs in ll groups decresed with increses in the moleculr weight of the FDs. The rtes of FD-0 in the control group nd the vlues of FD-0 nd -0 with DFAIII tretment were lower thn those of FD- with the sme tretments. With increses in the moleculr weight of the FDs, the % increse in FD trnsport tended to e lrger with C tretment, ut to e lower with DFAIII tretment. The % increses of FD-0 nd -0 were significntly lower thn those of FD- nd - in the DFAIII group. The trnsport rtes of mnnitol were higher thn those of other mrkers for ech tretment, nd the vlue ws incresed y C, ut not y DFAIII. The rte of LY ws lower thn tht of FD- in the control group. In this experiment, we lso checked TER to confirm similr reductions in the TER etween the monolyers fter -h exposure to 0 mmol/l DFAIII nd 0.-h exposure to mmol/l C. The TER with DFAIII nd C were lower thn the control vlues for ll mrkers, nd the reductions in TER induced y DFAIII nd C were very similr t round 00 Ω cm (Tle ). There were no differences in the TER efore incution with ech mrker (dt not shown). Altertions in F-ctin nd TJ proteins induced y DFAIII nd C Stronger stining of the F-ctin ws oserved with C tretment thn with the control nd DFAIII tretments on the djcent orders of Cco- cells in the perijunctionl imges (Fig. ). In the monolyers treted with DFAIII, F-ctin ws strongly stined only t the points where or cells were in contct. The four TJ proteins, occludin, cludin-1, JAM-1, nd ZO-1, were oserved s continuous pericellulr nds in the perijunctionl imges of the control monolyers (Fig. ). In the monolyers treted with DFAIII, strong fluorescent intensity of immunostined cludin-1 ws oserved t the

15 points where or cells were in contct, which ws similr to F-ctin stining nd ws distinguishle from the control monolyers. In the monolyers treted with C, dispersed nd frgmented stining pttern of cludin-1 ws oserved. The distriutions of occludin, JAM-1, nd ZO-1 in the monolyers were not ffected y tretment with DFAIII or C. The perijunctionl imges of doule immunostined F-ctin nd cludin-1 showed their co-locliztion in the control monolyers (Fig. A-C). In the monolyers treted with DFAIII, the co-loclized redistriutions of F-ctin nd cludin-1 were oserved t the cell corners (Fig. D-F). Strong stining of the F-ctin ws oserved on the orders of the cells treted with C; however, the stining pttern of F-ctin ws not identicl to the dispersed loction of cludin-1, which showed s red (Fig. G-I). Effect of signling lockers on DFAIII- nd C-induced reductions in TER In ll sets of experiments without inhiitors, TER cross the monolyers treted with DFAIII ws much lower thn tht in those without DFAIII (Fig. A-I). The DFAIII-induced reduction in TER ws not ffected y tretment with ny of the signling lockers tested except for n inhiitor of denylte cyclse (SQ), y which TER in the presence of the inhiitor ws slightly, ut significntly, higher thn TER without the inhiitor in the DFAIII-treted monolyers. In control monolyers, no lockers hd ny effects on the TER cross the monolyers. TER with the C tretment ws reduced to less thn hlf of the vlue of the control monolyers, nd the reduced TER ws prtly reversed y the inhiitors MLCK (ML-) nd cell-permele C + cheltor (BAPTA-AM) (Fig. ). The inhiitors of Rho-ssocited protein kinse (Y-) nd protein kinse C (H-) hd no effect on the C-induced reduction in TER.

16 Discussion DFAIII nd C dose-dependently decresed TER cross Cco- cell monolyers (Fig. 1), which is consistent with results of previous reports (Lindmrk et l., 1; Suzuki nd Hr, 00). However, the ptterns of the decreses in TER were distinct from ech other. Specificlly, the tretment with DFAIII lowered the TER much more slowly thn did C. This finding implies tht DFAIII nd C enhnce prcellulr trnsport vi different mechnisms. Tretment with C, ut not DFAIII, incresed LDH relese, n indictor of memrne dmge, in oth the picl nd solterl solutions (Tle 1). DFAIII ctivtes prcellulr routes without cell memrne dmge nd is not hrmful to intestinl cells even t high concentrtions. Prcellulr pssge of solutes is regulted y TJs, showing tht the TJs function not only s rrier to lrge molecules ut lso s trnsport pthwy for ions nd wter (Tsukit nd Furuse, 000; Tsukit et l., 001). The enhnced trnsport rtes of prcellulr mrkers were much higher in the C-treted monolyer thn in the DFAIII-treted one (Fig. A), lthough the electrolyte trnsport indicted y TER is identicl in the two tretments (Tle. ). The % increses in FD trnsport were enhnced y C, ut were reduced y DFAIII with increses in the moleculr weight of FDs (Fig. B). The enhncement of prcellulr trnsport is known to result from incresing oth the rdius nd numer of the pthwys constituted y TJs (Wtson et l., 001). These results indicte tht pore size of the prcellulr pthwys ctivted y DFAIII is smller thn tht of the pthwys ctivted y C. It hs een reported tht C tretment incresed the pore rdius in the TJ pthwy from to 0 ngstrom in the rt colon (Hyshi et l., 1). The excess increse in the pore size of TJ pthwys my llow trnsport of undesirle molecules; e.g., toxic compounds or llergens. Conversely, DFAIII ppers to e sfe s food dditive for the promotion of sorption vi TJs. The trnsport rtes of mnnitol nd FDs, which re electriclly neutrl, depended on their moleculr weight in ll the monolyers (Fig. A). However, the rtes of LY, hving negtive chrge, were lower thn the vlues of FD- for ech tretment though the moleculr weight of LY ( D) ws much smller thn tht of FD- (,00 D). The trnsport of solutes vi TJs depends on the chrge 1

17 s well s the moleculr size (Knipp et l., 1). Our result grees with tht of previous report in which the TJ permeility of Cco- monolyers ws higher for positive nd neutrl molecules thn for negtive ones (Knipp et l., 1). The ctivtion of prcellulr pthwys y oth DFAIII nd C ppers to result from ltertions to F-ctin nd cludin-1, not other TJ proteins (occludin, JAM-1, nd ZO-1) (Fig. nd ). Immunohistochemicl nlysis demonstrtes tht the ltertion of the ptterns of F-ctin nd cludin-1 were different etween DFAIII nd C groups. Tretment with DFAIII cused increses in the fluorescent signls of F-ctin nd cludin-1 t the cell corners, nd the oth signls were co-loclized (Fig. ). In contrst, the contrction of F-ctin fter tretment with C ws much stronger thn tht with DFAIII, nd the cludin-1 stining fter the C tretment showed discontinuous feture on the djcent order of the cells (Fig. ). Cludins re known to e essentil for the seling of TJs (Furuse et l., 00) nd the ctin cytoskeleton lso regultes TJ functions (Itoh et l., 1; Wittchen et l., 1). Therefore, the different chnges in the two TJ proteins my reflect different increses in the prcellulr trnsport of mrkers etween the two tretments. TJ proteins, especilly cludins, need to e continuously distriuted on the djcent orders of the cells, s shown in the control nd DFAIII groups, to regulte prcellulr trnsport. The discontinuous feture of cludin-1 induced y C my result in the formtion of the lrger pores nd llowing the permetion of lrger mrkers. Further investigtions my define the reltionship etween ltertions to TJ proteins nd chrcteristics of prcellulr trnsport. The contrction of ctin filments is reported to e cused y phosphoryltion of MLCs vi C + /clmodulin-ctivted MLCK nd Rho-kinse pthwys in the epithelil cells (Hirse et l., 001; M et l., 000). The C-induced reduction in TER ws prtilly, ut clerly, ttenuted y cell-permele cheltor of C + (BAPTA-AM) nd n inhiitor of MLCK (ML-) (Fig. ). This result shows tht one of the mjor mechnisms underlying the ction of C is ctin contrction following the ctivtion of MLCK, which is consistent with the results of previous report (Lindmrk et l., 1). Our recent study suggests tht C + signling my e involved in the promotive effect of DFAIII on 1

18 prcellulr trnsport (Suzuki nd Hr, 00). However, BAPTA-AM nd ML- did not ttenute the reduction in TER induced y DFAIII (Fig. ). This finding revels tht the C + signling induced y DFAIII is not involved in the mechnisms underlying the ction of DFAIII. Also, n inhiitor of Rho-ssocited protein kinse (Y-) did not ffect the ction of DFAIII (Fig. A, C, nd E). The cellulr mechnisms leding to the ltertions to TJ proteins including the cludin fmily re not completely understood. The inhiitor of denylte cyclse (SQ) slightly, ut significntly, ttenuted the effect of DFAIII (Fig. G). However, tretment with SQ similrly incresed TER in the control group (ut not significntly), suggesting tht the inhiition of denylte cyclse incresed TER irrespective of DFAIII ction. The other signling lockers used did not ffect the ction of DFAIII. Therefore, other signling mechnisms my possily e involved in the ltertion to F-ctin, cludin-1, nd TER y DFAIII, nd we should consider lso mechnisms not involving intrcellulr signl trnsduction. The promotive effect of DFAIII on prcellulr trnsport is not cused merely y incresing solutes, which ws demonstrted y the result tht the picl ppliction of 0 mmol/l ethylene glycol hd no effect on TER (Tle ). Our recent study showed tht some nondigestile scchrides similrly decresed TER (Suzuki nd Hr, 00). In ddition, the ction of DFAIII ws not influenced y the solterl ppliction of ethylene glycol, which countercted the osmotic grdient etween the picl nd solterl sides, nd the solterl ppliction of DFAIII hd no sustntil effect on TER. These findings indicte tht specific common structures in the scchrides re recognized y puttive sensory system in the rush order memrne of Cco- cells nd ctivte prcellulr routes. However, the moleculr weight of ethylene glycol used s negtive control is smller thn those of the scchrides nd its physiochemicl properties my e different from the scchrides. We re conducting further studies to clrify the specificity of the sensory system for the nondigestile scchrides. In summry, DFAIII nd C reduced TER nd enhnced the trnsport of prcellulr mrkers with ltertions to cludin-1 nd F-ctin in Cco- cell monolyers; however, the ltertions 1

19 induced y DFAIII were distinguishle from those y C, nd the pore size of the prcellulr routes ctivted y DFAIII my e smller thn tht of those ctivted y C. Further, C prtilly enhnced prcellulr routes y ctivtion of MLCK, while the DFAIII-induced reduction in TER ws not ttenuted y ny signling lockers. The results of the present study demonstrte tht the cellulr mechnisms underlying the enhncement of prcellulr trnsport y DFAIII re distinct from those of C. Further informtion on the mechnisms is necessry to fully estlish the sfety of DFAIII for humn consumption. Acknowledgements We used the Rdioisotope Lortory of the Grdute School of Agriculture, Hokkido University. 1 1

20 References Bzzoni G., Mrtinez-Estrd O.M., Orsenigo F., Cordenonsi M., Citi S., Dejn E., 000. Interction of junctionl dhesion molecule with the tight junction components ZO-1, cingulin, nd occludin. Journl of Biologicl Chemistry (), Bronner F., 1. Clcium sorption-- prdigm for minerl sorption. Journl of Nutrition 1 (), 1-0. Bronner F., Pnsu D., 1. Nutritionl spects of clcium sorption. Journl of Nutrition 1 (1), -1. Duncn D., 1. Multiple rnge nd multiple F-test. Biometrics, 1-. Furuse M., Fujit K., Hiirgi T., Fujimoto K., Tsukit S., 1. Cludin-1 nd -: novel integrl memrne proteins loclizing t tight junctions with no sequence similrity to occludin. Journl of Cell Biology (), Furuse M., Ht M., Furuse K., Yoshid Y., Hrtke A., Sugitni Y., Nod T., Kuo A., Tsukit S., 00. Cludin-sed tight junctions re crucil for the mmmlin epiderml rrier: lesson from cludin-1-deficient mice. Journl of Cell Biology 1 (), -. Furuse M., Hirse T., Itoh M., Ngfuchi A., Yonemur S., Tsukit S., Tsukit S., 1. Occludin: novel integrl memrne protein loclizing t tight junctions. Journl of Cell Biology 1 ( Pt ), 1-1. Furuse M., Itoh M., Hirse T., Ngfuchi A., Yonemur S., Tsukit S., Tsukit S., 1. Direct ssocition of occludin with ZO-1 nd its possile involvement in the locliztion of occludin t tight junctions. Journl of Cell Biology 1 ( Pt 1), -1. Furuse M., Sski H., Fujimoto K., Tsukit S., 1. A single gene product, cludin-1 or -, reconstitutes tight junction strnds nd recruits occludin in firolsts. Journl Cell Biology 1 (), Hyshi M., Tomit M., Awzu S., 1. Trnscellulr nd prcellulr contriution to trnsport processes in the colorectl route. Advnced Drug Delivery Review, -0. 1

21 Hirse T., Kwshim S., Wong E.Y., Ueym T., Rikitke Y., Tsukit S., Yokoym M., Stddon J.M., 001. Regultion of tight junction permeility nd occludin phosphoryltion y Rho-p10ROCK-dependent nd -independent mechnisms. Journl of Biologicl Chemistry (1), -1. Itoh M., Furuse M., Morit K., Kuot K., Sitou M., Tsukit S., 1. Direct inding of three tight junction-ssocited MAGUKs, ZO-1, ZO-, nd ZO-, with the COOH termini of cludins. Journl of Cell Biology 1 (), -1. Itoh M., Ngfuchi A., Moroi S., Tsukit S., 1. Involvement of ZO-1 in cdherin-sed cell dhesion through its direct inding to lph ctenin nd ctin filments. Journl of Cell Biology 1 (1), -1. Kle G., Nren A.P., Sheth P., Ro R.K., 00. Tyrosine phosphoryltion of occludin ttenutes its interctions with ZO-1, ZO-, nd ZO-. Biochemicl nd Biophysicl Reserch Communictions 0 (), -. Krczewski J., Groot J., 000. Moleculr physiology nd pthophysiology of tight junctions III. Tight junction regultion y intrcellulr messengers: differences in response within nd etween epitheli. Americn Journl of Physiology Gstrointestinl nd Liver Physiology (), G0-G. Knipp G.T., Ho N.F., Brsuhn C.L., Borchrdt R.T., 1. Prcellulr diffusion in Cco- cell monolyers: effect of perturtion on the trnsport of hydrophilic compounds tht vry in chrge nd size. Journl of Phrmceuticl Sciences (), 1-. Kottr G., Vnk C., 00. The forskolin-induced opening of tight junctions in Xenopus gllldder epithelium is medited y protein kinse C. Cellulr nd Moleculr Biology (Noisy-le-grnd) (1), -. Lindmrk T., Kimur Y., Artursson P., 1. Asorption enhncement through intrcellulr regultion of tight junction permeility y medium chin ftty cids in Cco- cells. Journl of Phrmcology nd Experimentl Therpeutics (1), -. Liu Y., Nusrt A., Schnell F.J., Reves T.A., Wlsh S., Pochet M., Prkos C.A., 000. Humn 1

22 junction dhesion molecule regultes tight junction reseling in epitheli. Journl of Cell Science (Pt 1), -. M T.Y., Ho N.T., Trn D.D., Bui V., Pedrm A., Mills S., Merryfield M., 000. Cytochlsin B modultion of Cco- tight junction rrier: role of myosin light chin kinse. Americn Journl of Physiology Gstrointestinl nd Liver Physiology (), G-G. Mrtin-Pdur I., Lostglio S., Schneemnn M., Willims L., Romno M., Fruscell P., Pnzeri C., Stoppcciro A., Ruco L., Vill A., Simmons D., Dejn E., 1. Junctionl dhesion molecule, novel memer of the immunogloulin superfmily tht distriutes t intercellulr junctions nd modultes monocyte trnsmigrtion. Journl of Cell Biology 1 (1), -1. Mineo H., Amno M., Chiji H., Shigemtsu N., Tomit F., Hr H., 00. Asorptive ctivity of clcium in the isolted cecl epithelium dptively incresed y week's feeding of difructose nhydride III in rts. Bioscience, Biotechnology, nd Biochemistry (), 1-. Mitmur R., Hr H., Aoym Y., Chiji H., 00. Supplementl feeding of difructose nhydride III restores clcium sorption impired y ovriectomy in rts. Journl of Nutrition 1 (), -. Mitic L.L., Vn Itllie C.M., Anderson J.M., 000. Moleculr physiology nd pthophysiology of tight junctions I. Tight junction structure nd function: lessons from mutnt nimls nd proteins. Americn Journl of Physiology Gstrointestinl nd Liver Physiology (), G0-G. Nusrt A., Turner J.R., Mdr J.L., 000. Moleculr physiology nd pthophysiology of tight junctions. IV. Regultion of tight junctions y extrcellulr stimuli: nutrients, cytokines, nd immune cells. Americn Journl of Physiology Gstrointestinl nd Liver Physiology (), G1-G. Sito K., Tomit F., 000. Difructose nhydrides: their mss-production nd physiologicl functions. Bioscience, Biotechnology, nd Biochemistry (), -1. Sitou M., Furuse M., Sski H., Schulzke J.D., Fromm M., Tkno H., Nod T., Tsukit S., 000. Complex phenotype of mice lcking occludin, component of tight junction strnds. Moleculr Biology of the Cell (1), -1. 1

23 Sheth P., Bsuroy S., Li C., Nren A.P., Ro R.K., 00. Role of phosphtidylinositol -kinse in oxidtive stress-induced disruption of tight junctions. Journl of Biologicl Chemistry (), -. Shigemtsu N., Okuhr Y., Shiomi T., Tomit F., Hr H., 00. Effect of difructose nhydride III on clcium sorption in humns. Bioscience, Biotechnology, nd Biochemistry (), -1. Stenson W.F., Esom R.A., Riehl T.E., Turk J., 1. Regultion of prcellulr permeility in Cco- cell monolyers y protein kinse C. Americn Journl of Physiology ( Pt 1), G-G. Suzuki T., Hr H., 00. Vrious nondigestile scchrides open prcellulr clcium trnsport pthwy with the induction of intrcellulr clcium signling in humn intestinl Cco- cells. Journl of Nutrition 1 (), 1-. Suzuki T., Hr H., Ksi T., Tomit F., 1. Effects of difructose nhydride III on clcium sorption in smll nd lrge intestines of rts. Bioscience, Biotechnology, nd Biochemistry (), -1. Tsukit S., Furuse M., 000. Pores in the wll: cludins constitute tight junction strnds contining queous pores. Journl of Cell Biology 1 (1), 1-. Tsukit S., Furuse M., Itoh M., 001. Multifunctionl strnds in tight junctions. Nture Reviews Moleculr Cell Biology (), -. Wtson C.J., Rowlnd M., Wrhurst G., 001. Functionl modeling of tight junctions in intestinl cell monolyers using polyethylene glycol oligomers. Americn Journl of Physiology Cell Physiology 1 (), C-C. Wittchen E.S., Hskins J., Stevenson B.R., 1. Protein interctions t the tight junction. Actin hs multiple inding prtners, nd ZO-1 forms independent complexes with ZO- nd ZO-. Journl of Biologicl Chemistry (),

24 Figure legends Fig Trnsepithelil electricl resistnce (TER) cross the Cco- cell monolyers incuted in the presence of up to 0 mmol/l difructose nhydride (DFA) III (A) or mmol/l sodium cprte (B) in the picl chmers. Vlues re mens ± SEM of monolyers. Mens not shring common letter re significntly different t ech time point (P < 0.0). Fig. Trnsport rtes of prcellulr mrkers cross the Cco- cell monolyers incuted for 1 h fter -h exposure to 0 mmol/l DFAIII or 0.-h exposure to mmol/l sodium cprte (A) nd the % increses ginst the control vlue (B). Cco- monolyers incuted for h without DFAIII or sodium cprte were used s the control group. Vlues re mens ± SEM of - monolyers. Mens not shring common letter in the sme tretment differ (P < 0.0). *Significntly different from the vlue of the control group for the sme mrker (P < 0.0). #Significntly different from the vlue of the DFAIII group for the sme mrker (P < 0.0). Fig. Confocl imges of ctin filments stined with rhodmine phlloidin (A, F, nd K) nd immunofluorescent stining of occludin (B, G, nd L), cludin-1 (C, H, nd M), JAM-1 (D, I, nd N), nd ZO-1 (E, J, nd O) in Cco- cell monolyers. A-E, Cco- monolyers incuted for h without DFIII or sodium cprte; F-J, Cco- monolyers incuted for h with 0 mmol/l DFAIII; E-O, Cco- monolyers incuted for 0. h with mmol/l sodium cprte, respectively. The rrows in pnel F indicte res of high intensity of fluorescence-leled F-ctin in the monolyers incuted with DFAIII. The rrowheds indicte res of high intensity of fluorescence-leled cludin-1 in the monolyers incuted with DFAIII. The imges re representtives of t lest monolyers, respectively. The scle r represents µm. Fig. Confocl imges of doule immunostined F-ctin (A, D, nd G) nd cludin-1 (B, E, nd H) 0

25 in Cco- cell monolyers (C, F, nd I, overly imges). A-C, Cco- monolyers incuted without DFIII or sodium cprte for h; D-F, Cco- monolyers incuted with 0 mmol/l DFAIII for h; G-I, Cco- monolyers incuted with mmol/l sodium cprte for 0. h, respectively. The rrowheds indicte res of high intensity of fluorescence-leled cludin-1 in the monolyers incuted with DFAIII. The imges re representtives of t lest monolyers, respectively. The scle r represents µm. Fig. Trnsepithelil electricl resistnce (TER) cross the Cco- monolyers incuted for h with or without 0 mmol/l DFAIII in the presence or sence of ech signling locker. A, n inhiitor of phospholipse C (U1); B, cell-permele chelor of clcium ion (BAPTA-AM); C, n inhiitor of myosin light chin kinse (ML-); D, n inhiitor of protein kinse C; E, n inhiitor of Rho-ssocited protein kinse (Y-); F, n inhiitor of phosphtidylinositol -kinse (LY00); G, n inhiitor of denylte cyclse (SQ); H, n inhiitor of tyrosine kinse (genistein); I, n inhiitor of lph-suunit of Gi protein (pertussis toxin). Vlues re mens ± SEM of - monolyers. Mens not shring common letter in ech pnel differ (P < 0.0). Fig. Trnsepithelil electricl resistnce (TER) cross the Cco- monolyers incuted for 0. h with or without mmol/l C in the presence or sence of inhiitors of myosin light chin kinse (ML-), protein kinse C, nd Rho-ssocited protein kinse (Y-) nd cell-permele chelor of clcium ion (BAPTA-AM). Vlues re mens ± SEM of monolyers. Mens not shring common letter differ (P < 0.0). 1

26 Referee List 1 List of suggested referees 1) Dr. Gregory T. Knipp Assistnt Professor of Phrmceutics, Ernest Mrio School of Phrmcy, The Stte University of New Jersey, USA E-mill: gknipp@rci.rutgers.edu ) Dr Kevin Cshmn Professor of Deprtment of Food nd Nutritionl Sciences, University College, Western Rod, Cork, Irelnd E-mil: k.cshmn@ucc.ie ) Dr. Artursson Per Professor of Deprtment of Phrmcy, Uppsl University, PO Box 0, SE-1 Uppsl, Sweden. E-mil: per.rtursson@glenik.uu.se ) Dr. Atsutne Oht Professor of Fculty of Phrmceuticl Sciences, Josi Interntionl University, Chi, Jpn E-mil: oht@ttglol.net

27 Figure1 A TER cross the monolyers (Ω cm ) DFAIII concentrtion (mmol/l) c c c c c c c c Incution time (min) B TER cross the monolyers (Ω cm ) Sodium cprte concentrtion (mmol/l) c c 00 c c c c c c c c c c Incution time (min) Fig. 1 T. Suzuki et l.

28 Figure Trnsport rte of prcellulr mrker (nmol/h/cm ) A α A *# Mnnitol γ *# c * B LY β *# * B FD- Control 0 mmol/l DFAIII mmol/l Sodium cprte γ *# *# cd cd *# d * C γ D * * E FD- FD-0 δ FD-0 % increse ginst the control vlue (%) B *# C Mnnitol *# * A LY *# * A FD- * A FD- *# * B FD-0 *# * B FD-0 *# Fig. T. Suzuki et l.

29 Figure A B C D E F G H I J K L M N O Fig. T. Suzuki et l.

30 Figure A B C D E F G H I Fig. T. Suzuki et l.

31 Figure TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) No inhiitor No inhiitor No inhiitor U1 H- Control SQ TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) No inhiitor BAPTA-AM No inhiitor No inhiitor 0 mmol/l DFAIII A B C Y- Genistein TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) TER cross the monolyer (Ω cm ) D E F G H I c No inhiitor No inhiitor No inhiitor ML- LY00 Pertussis toxin Fig. T. Suzuki et l.

32 Figure TER cross the monolyer (Ω cm ) No inhiitor c No inhiitor BAPTA-AM ML- c Y- c H- Control mmol/l Sodium cprte Fig. T. Suzuki et l.

33 Tle1 1 Tle 1. Lctte dehydrogense ctivity in the picl nd solterl medium of Cco- monolyers incuted in the presence of up to 0 mmol/l DFAIII or mmol/l sodium cprte in the picl chmers Apicl side (mu/0.ml) Bsolterl side (mu/1.0ml) DFAIII 000 mmol/l ± 1 1. ±.1 0 mmol/l 1 ± 1. ± mmol/l ± 1. ± 1. 0 mmol/l ± 1. ± 1. 0 mmol/l ± 1. ± 1. Sodium cprte 0 mmol/l 1 ± 1 c.0 ± 0. mmol/l ± 1. ± 0.. mmol/l ± 0. ±. mmol/l ± 1. ±. Vlues re mens ± SEM, n =. Mens in column not shring common letter re significntly different (P < 0.0). Apicl nd solterl chmers of the cells were thed in 1.0 nd 0. ml HBSS, respectively. Arevitions used: DFA, difructose nhydride; FD, Fluorescein isothiocynte-conjugted dextrn; TER, trnsepithelil electricl resistnce.

34 Tle Tle. Trnsepithelil electricl resistnce (TER) cross the Cco- monolyers efore nd fter -h incution in the sence or presence of DFAIII or ethylene glycol in the picl or solterl chmers. Administrtion site TER cross the monolyers (Ω cm ) Apicl site Bsolterl site 0 h h None None 1 ± ± 1 DFAIII None 10 ± 0 ± c None DFAIII 1 ± 1 ± 0 Ethylene glycol None 1 ± 1 1 ± None Ethylene glycol 1 ± 10 ± DFAIII Ethylene glycol ± 01 ± c Vlues re mens ± SEM, n = -. Vlues in column not shring common letter re significntly different (P < 0.0). Arevitions used: DFA, difructose nhydride; TER, trnsepithelil electricl resistnce.

35 Tle Tle Trnsepithelil electricl resistnce (TER) of Cco- monolyer used in the trnsport experiment fter incution MW TER fter incution (Ω cm ) Control DFAIII Sodium cprte Mnnitol 00,1 ± 0 1 ± 1 ± Lucifer Yellow 00, 0 ± ± 1 ± FD- 0,00 ± 1 1 ± 1 ± FD-,00 1 ± 0 ± ± FD-0 1,00 1 ± 0 0 ± 1 ± FD-0,00 ± 0 1 ± 1 ± 1 Vlues re mens ± SEM, n = -. Vlues in row not shring common letter re significntly different (P < 0.0). Arevitions used: DFA, difructose nhydride; FD, Fluorescein isothiocynte-conjugted dextrn; MW, moleculr weight; TER, trnsepithelil electricl resistnce. 1