April 2000

monitored . At kill, blood was collected for determination of glucose andinsulin levels and the gastrointestinal organs were weighed and sampled forhistological analysis . Epithelial proliferation in the crypts was assessed byimmunohistochemical staining for PCNA (proliferat ing cell nuclear anti­gen). Statistical analysis was by ANOVA. Results: Compared to vehicletreatment, body weight and food intake were dose-responsively decreasedby 14 percent and 16 percent respectively following 500 microgramslkg/day BTC (p< 0.01). BTC increased water intake by 36 percent and urineoutput by 76 percent (p< 0.(01). BTC decreased serum insulin levels (p<0.05) but serum glucose levels were not significantly affected. The wetweights of the duodenum and small intestine were increased by 16 percentand 19 percent, respectively (p< 0.05) and total gut weight expressed as afraction of body weight was increased by 25 percent (p< 0.001). AlthoughBTC significantly increased crypt depth in all regions of the small intestineand colon, the terminal ileum was most responsive, (increased by 47percent, p< 0.01), with a 37 percent increase in villus height and a 32percent increase in the PCNA labelling index (p< 0.01). Villus heights inthe other small intestinal regions were not significantly affected by beta­cellulin. Conclusions : Exogenously administered betacellulin manifested adiuresis, suggesting a possible role in fluid homeostasis. BTC also stimu­lated growth of the small and large intestine, the increased responsivenessof the terminal ileum suggesting therapeutic application in conditionsassociated with ileal insufficiency, such as the short bowel syndrome.

2931EVALUATION OF REGENERATED LIVER USING [l.13C]PHE.NYLALANINE BREATH TEST.Yukimoto Ishii, Satoshi Asai, Tadashi Kohno, Shigeru Suzuki, TakerohMazaki, Kimitoshi Katoh, Hideki Masuda, Koichi Ishikawa, ShigetomiIwai, Nihon Univ Sch of Medicine, Tokyo , Japan; Frontier TechnologyResearch Institute, Tokyo Gas, Yokohama , Japan.

(Objective) We examined the relationship between changes in the amountof hepatic tissue proteins (TP), amount of DNA, and phenylalanine hy­droxylase (PHA) activity and the results of [1-13C]phenylalanine (13Cphe)

breath test in hepatic regeneration in a rat model of 70% hepatectomy, toassess its usefulness for evaluating hepatic regeneration . (Material andMethods) Male Wistar rats (Z50-Z80 g) were subjected to 70% hepatec­tomy under anesthesia with Nembutal. One, 2, 3, 7, and 14 days postop­eratively, 30 mg/kg 13Cphe was intravenously injected into the femoralvein, and the increase in exhaled 13C0 2 (AJ3C02) was measured for 15minutes. Simple laparatomy was performed in control rats. Following thebreath test, the regenerated liver was removed and weighed. The amount ofhepatic tissue proteins, amount of DNA, and PHA activity were determinedusing the methods described by Lowry et al., Volkin et al., and Herber etal., respectively . (Results) The correlation coefficients (r) between LWIBW% and PAH activity, between DNA and PAH activity , and between TPand PAH activity were 0.723, 0.720, and 0.739, respectively . Breath testgraphs revealed that LW/BW%, DNA and TP showed the best correlationswith the peak value (PV) of A13C0

2 (LW/BW%: r=0.714, DNA: r=0.658,TP: r=O.64), and the correlation coefficient (r) between PAH activity andPV was 0.633. (Conclusion) These results suggest that measurement ofPAH activity in regenerated liver is an effective method for evaluatingregeneration after hepatectomy. Moreover , 13Cphebreath test is consideredto be highly useful for assessing regenerated liver, and the peak value of the13Cphe breath test graph seems to be an effective indicator for evaluatingregenerated liver.

2932HEPATOCYTE GROWTH FACTOR (HGF) ACTIVATES SOMECELLULAR EVENTS INVOLVED IN ENTEROCYTE-LIKE DIF·FERENTIATION OF COLON CANCER EPITHELIAL CELLS.Stephanie Kermorgant, Valerie Dessirier, Miguel Mjm Lewin, ThereseLehy, INSERM U 10, Paris, France.

Background & Aims. Hepatocyte growth factor (HGF) exerts morphogeniceffects during embryogenesis and cell transformation . We have reportedthat expression of HGF and its receptor c-Met as well as the funtionality ofc-Met appears very soon in human fetal digestive tissues, suggesting a rolefor HGF/c-Met in morphogenesis ofthese tissues. Here, the potential effectof HGF on some events involved in intestinal cell differentiation wasassessed on human colon cancer epithelial Caco-2 cells, which differentiatespontaneously into enterocytes after confluence. Methods . Cells were cul­tured without or with HGF (10 ng/ml) until 35 days post-plating . Alkalinephosphatase activity, expressions of c-Met, E-cadherin, villin, Grb-z-asso­ciated binding protein (Gab-I), protein kinase C-o (PKCo) were examinedin preconfluent and postconfluent cells by Northern blot and/or RT-PCRand western immunoblotting. Results. In Caco-Z cells cultured withoutHGF, c-Met expression augmented after confluence (r-day 5 post-plating),then decreased from day 15 post-plating . There was a 5-fold and 7-foldincrease for villin and E-cadherin expression, respectively, between days 3and 35 post-plating. HGF continuously added to culture medium shortenedthe growth phase up to confluence by -one day, significantly enhancedalkaline phosphatase activity, villin and Ecadherin protein expression atall time-points examined. The highest stimulation was observed on day 4post-plating (x 8 for villin and x 7 for E-cadherin) as compared with cellscultured without HGF. Gab-I was expressed in Caco-Z cells and HGFstimulated expression and tyrosine phosphorylation of c-Met and Gab-I . Itstimulated also PKCa expression . Since a potential role of PKCa in thedifferentiation of Caco-Z cells has been postulated, G06976, a PKC

AGAA559

inhibitor was added to the culture medium in presence or not of HGF. Itinhibited the HGF-induced villin increase before confluence and enhancedvillin expression in control cells after confluence. Conclusions : HGF ac­celerates Caco-Z cell differentiation and stimulates some molecules in­volved either in intestinal metabolism or in enterocyte phenotype acquisi­tion during the process of differentiation. These HGF-promoted eventsappear mediated partly by Gab-I and also by PKC, probably PKCa.

2933

EFFECT OF GLUCAGON-LIKE PEPTIDE 2 ON FUNCTIONALAND TRANSCRIPTION FACTOR GENE EXPRESSION IN THESMALL INTESTINE.Paul Anthony Kitchen, Natalie F. Barley, Anthony F. FitzGerald, NikkiMandie, Mohammed Ghatei, Steve Bloom, Robert A. Goodlad, AlastairForbes, Julian Rf Walters, St Mark ' s Hosp, Middlesex, United Kingdom;Imperial Coli Sch of Medicine, London, United Kingdom; HistopathologyUnit, ICRF, London, United Kingdom; St Mark's Hosp, London, UnitedKingdom.

Background: Glucagon-like peptide Z (GLP-Z) is a trophic factor that hasbeen shown to stimulate proliferation in the small intestine of rats fed bytotal parenteral nutrition (TPN). Two genes involved with absorption ,sucrase-isomaltase (SI) and ileal bile acid binding protein (IBABP), arepredominately expressed in the jejunum and ileum respectively. Further­more intestinal gene expression may be regulated by transcription factors,caudal-related homeobox protein (Cdx-Z) and hepatocyte nuclear factor-4(HNF-4). Aims: To investigate the effects in the small intestine of GLP-Zon cell proliferation, representati ve functional gene expression, and toelucidate whether transcription factor gene expression is a regulatory step.Methods: Rats were established on TPN for 7 days. The treatment groupreceived 40 mcg/day ofGLP-Z intravenously with the TPN diet. In additionto the TPN controls , animals fed chow ad libitum were also included. Atautopsy the small intestine was weighed. Tissue was obtained from thejejunum and terminal ileum. Northern blots were probed in parallel withpreviously characterized eDNA probes: intestinal transcription factors,Cdx-Z and HNF-4; functional genes, 51 and IBABP; 18S ribosomal RNAto standardize for RNA loading. Tissue was also fixed in Camoy' s fluidfrom these regions and later microdissected to determine the changes inmetaphase arrest scores/crypt , as a marker of cell proliferation (CP).Results: The mean small intestinal weights of GLP-Z treated or oral fed ratswere two fold greater than in the TPN controls (p<O.ool). GLP-Z doubledthe mean CP in both the jejunum and ileum compared to TPN fed rats(p<O.ool). Mean expression of sucrase-isomaltase was increased byGLP-Z compared to TPN controls in both the jejunum (x1.7, p=O.08) andileum (xI.6, p<0.05). Mean expression was increased xl.8 by GLP-Z forIBABP (p<O.OI) and HNF-4 (p<0.05). However mean expression ofCdx-Z for the TPN and GLP-Z groups were as follows: 100% and IZO%respectively in jejunum and ZOO% and 180% in ileum (all expressed as %of TPN jejunum). There were no significant differences between thesegroups. Conclusion: GLP-Z reversed atrophy of the small intestine in TPNfed rats and significantly increased cell proliferation and differentiation inthe jejunum and ileum. Furthermore GLP-Z may up-regulate HNF-4 geneexpression.

2934

GASTRIN·RELEASING PEPTIDE RECEPTOR (GRP·R) FUNC·TIONAL EXPRESSION VARIES WITH THE DEGREE OF CEL·LULAR DIFFERENTIATION IN COLON CANCER.Sean W. Lee, Waddah A. Alrefai, Richard V. Benya, Robert E. Carroll,Univ of Illinois at Chicago, Chicago, IL.

GRP is a mitogen that acts upon a specific receptor. GRP/GRP-R are notnormally expressed by epithelial cells lining the colon, but are aberrantlyexpressed by most colon cancers and cancer cell lines . We recently showedin a correlative study of previously resected human colon cancers thatGRP/GRP-R expression was linked to tumor cell differentiation (Am JPhysiol 1999; Z76: G655). To directly evaluate the role of tumor celldifferentiation on GRP-R expression, we studied pre and post-confluentCaCo-Z cells. CaCo-Z cells at 50% confluence (5 days post-plating) lackcell-cell adhesions , show little nuclear polarity, and biochemically do notexpress markers of differentiation . The relatively undifferentiated status ofthese cells was confirmed by failing to find evidence of dra (down regu­lated in ;!denoma) expression by RT-PCR. However, pre-eonfluent cellsstrongly expressed GRP-R and proliferated in response to GRP (EC50 10nm). eH]thymidine incorporation in CaCo-Z cells exposed to 100 nm GRPincreased > 4-fold. The GRP-R specific antagonist [D-Phe6

] bombesinmethyl ester attenuated this response, indicating that functional GRP-Rcapable of causing cell proliferation were present. In contrast, just-conflu ­ent CaCo-Z cells (10 days post-plating) were columnar and possessedcell-cell adhesions characteristic of a mature epithelium, as well as ex­pressed dra by RT-PCR. Confluent CaCo-Z cells did not express GRP-Rand did not show evidence of eH]thymidine incorporation in response toagonist exposure. Conclusion: GRP-R expression is only found in prolif­erating CaCo-Z cells and is temporally linked to their stage of differenti­ation.