1 (Fgfr1) and its ligands regulate insulin and glucose transporter 2 (Glut2) expression. Thus,GDNF influence on these factors could contribute to the increased β-cell mass and improvedglucose homeostasis seen in GDNF transgenic (GDNF-tg) mice. Aim: To; (1) examine thepotential role of cell proliferation on GDNF-stimulated β-cell mass increase and resistanceto streptozotocin (STZ)-induced hyperglycemia; and (2) investigate the involvement of Pdx1,Sox9, Fgfr1 and Hes1 in GDNF-stimulated β-cell mass increase and glucose homeostasis.Methods: GDNF-tg mice and their WT littermates (8-11 Weeks old) were used. Capan-1pancreatic ductal cells were used for In Vitro studies. Proliferation was assessed by immuno-staining for Ki67. Hyperglycemia was induced by two consecutive injections of 75 mg STZ/Kg body weight. Gene expression was assessed by both RT-PCR and immunostaining.Results: Untreated GDNF-tg mice had 4.9 fold higher β-cell proliferation rate than WTmice, (6.05 ± 0.6 % and 1.22 ± 0.3%, respectively; P<0.001), and this increased rate ofproliferation persisted 14 days following STZ treatment (5.47 ± 1.7% in GDNF-tg mice and0.97 ± 5.5% in WT mice, 5.64 fold difference, P<0.01). GDNF-tg mice also had significantlyhigher Pdx1 and Hes1 mRNA expression than WT mice (1.8 fold and 3.68 fold (P<0.001),respectively). Consistent with this, immunofluorescent staining revealed 1.5 fold (P<0.05)higher Pdx1 expression in β-cells in GDNF-tg mice pancreata than in WT mice pancreata.Pancreata from GDNF-tg mice also showed significantly higher expression of Sox9 and Fgfr1mRNA compared to WT mice. In Vitro, GDNF (100 ng /ml) treatment induced a 1.86 fold(P<0.05) increase in Sox9 mRNA expression in Capan-1 cells. Conclusions: These datasuggest that GDNF promotes proliferation of β-cells and also stimulates β-cell regenerationfollowing islet injury. GDNF-induced increase in proliferation is associated with increasedexpression of β-cell progenitors. Thus, GDNF may have potential as a therapeutic agent fortreating type 1 diabetes.

W1823

SRC Signaling Pathways Recruit Cell-Cell Adhesion and Microtubular-Microfilamentous Systems in Cholecystokinin-Stimulated Pancreatic AciniFumihiko Nozu, Michio Imawari

Background: We previously demonstrated that cholecystokinin (CCK) stimulates the Src-RhoA-Phosphoinositide 3-kinase (PI3K) pathways and RhoA and RhoA effector, ROCK-IIprevent basolateral pancreatic enzyme secretion and caerulein-induced acute pancreatitis.Although several lines of evidence have been accumulated that p120 catenin (p120) associateswith not only G12 and Rho but also microtubules in various cell types, the precise interactionbetween cell-cell adhesion and microtubular- microfilamentous systems has not been clarifiedin pancreatic acini. Aim: We attempted to evaluate the association of Src-RhoA-PI3K path-ways, ROCK-II, Rho guanine nucleotide exchange factor, Vav-2, p120, E-cadherin (EC) andmicrotubules in CCK-stimulated pancreatic acini. Methods: Isolated acini were obtainedfrom male Sprague-Dawley rats. Intact acini were incubated with or without CCK. Srcinhibitor, 4-amino-5- (4-chlorophenyl)-7-(tert-butyl) pyrazolo [3, 4-d] pyrimidine (PP2),Rho inhibitor, pravastatin, PI3K inhibitor, wortmannin and microtubules destabilizer, col-chicine were preincubated before CCK stimulation. Protein expressions of Src, RhoA, ROCK-II and PI3K, Vav-2, p120 and EC were analyzed by Western immunoblotting (WB) andtheir interactions were examined by immunoprecipitation (IP). Amylase secretion was alsomeasured. Results: In WB study, CCK (10 pM - 10 nM) enhanced the expressions of Src,RhoA, ROCK-II, PI3K, Vav-2, p120 and EC. The pretreatment of PP2 (5-100 µM) inhibitedthe expressions of Src, RhoA, ROCK-II, PI3K, Vav-2, p120 and EC in CCK-treated pancreaticacini. The pretreatment of pravastatin (1-100 µM) inhibited the protein expressions of RhoA,ROCK-II, PI3K, Vav-2, p120 and EC. The pretreatment of wortmannin (0.1-3 µM) inhibitedthe expressions of PI3K, Vav-2, p120 and EC. In IP study, p120 was co-immunoprecipitatedwith EC, Src, RhoA and PI3K in response to CCK stimulation. Vav-2 was co-immunoprecipit-ated with Src, RhoA and PI3K in response to CCK stimulation. CCK stimulates amylasesecretion in biphasic manner and the maximum secretion was seen with the concentrationof CCK 100 pM. The pretreatment of PP2, pravastatin and wortmannin inhibited amylasesecretion in CCK-treated pancreatic acini without altering basal secretion. On the otherhands, the pretreatment of colchicine (1-10 mM) and PP2 (40-100 µM) shifted the maximumresponse dose of CCK from 100 pM to 1 nM and potently diminished the expression ofSrc, RhoA, ROCK-II, PI3K, Vav-2, p120 and EC. Conclusion: We conclude that Src signalingpathways recruit not only cell-cell adhesion via Vav-2 and p120 but also microtubular-microfilamentous systems in CCK-stimulated pancreatic acini.

W1824

Dietary Protein and Amino Acids Regulate the Synthesis of PancreaticDigestive EnzymesMaria Dolors Sans, Stephen J. Crozier, Nancy L. Vogel, John A. Williams

Background: Feeding stimulates protein synthesis and protein translation in mouse pancreaswithout changes in mRNA levels for digestive enzymes. Little is known about how specificacute dietary composition regulates digestive enzyme synthesis. We hypothesize that aminoacids from dietary protein are necessary as a signal as well as a substrate for pancreaticdigestive enzyme synthesis after a meal. Methods: male ICR mice were fasted for 16h(control group) and re-fed for 2h with the isocaloric diets: AIN-93G (93G) (18% protein),diet with protein replaced with amino acids (AA), diet without protein (No Prot) and dietwithout leucine (No Leu). Total pancreatic protein synthesis was analyzed by the floodingdose technique, and polysomal profiles of pancreas samples were obtained by U.V. monitoringof pancreas homogenates separated on a sucrose gradient. The activation of different transla-tion factors in pancreas was also analyzed. Results: Feeding a normal diet (93G) resultedin a significant increase in the incorporation of 3H-Phe into pancreatic protein (144±11%).This parameter was also increased in the AA group (128±10%), while the No Prot groupshowed a significant reduction (65±7%) compared to the fasted group. Analysis of polysomalprofiles from the different groups showed a correlation with total protein synthesis results;the 93G diet increased the polysomal fraction compared to the fasted group, and the Noprot diet reduced the same fraction, compared to the re-fed 93G diet group. The presence/absence of protein in the diets did not modify the stimulation of the Akt/mTOR pathwayafter re-feeding, as indicated by an increase on the phosphorylation status of Akt (to about300%) and ribosomal protein S6 (to about 500%) on all re-fed groups compared to the

T : 11501$$CH204-02-08 16:47:15 Page 723Layout: 11501B : o

A-723 AGA Abstracts

fasted group. Plasma concentration of amino acids was reduced to less than 5% in the NoProt group, and plasma insulin levels were increased to over 80 µU/ml in all three re-fedgroups, compared to the fasted group (5.8±0.9 µU/mL). To test whether the absence of anessential amino acid in the diet was having an effect on these parameters, mice were re-fedwith the No Leu diet and found that total pancreatic protein synthesis was strongly inhibited(to 41±3% of the fasted group) and eIF2α phosphorylation was increased to 250±20% ofcontrol. This group also showed an increase on the General Control nonderepressible 2(GCN2) kinase phosphorylation (160±16%), compared to the fasted and 93G diet groups,usually associated with inhibition of protein synthesis. Conclusions: Protein and aminoacids are necessary as a signal as well as a substrate for pancreatic digestive enzyme synthesisafter a meal.

W1825

Effect of Combination of Curcumin and Rosiglitazone On Experimental AcutePancreatitisPratibha Khosla, Promila Pandhi, Samir Malhotra, Ritambhara Nada, Surinder Rana,Deepak K. Bhasin

Background: Acute Pancreatitis is an inflammatory condition associated with significantmorbidity and mortality. Treatment is largely supportive with specific therapy lacking. Thereis thus a need to explore novel drug treatment strategies. Objective: To investigate the effectof combination of curcumin & rosiglitazone in acute experimental pancreatitis. Methods:Acute pancreatitis was induced in Wistar rats (150-250 g) by four intraperitoneal injectionsof caerulein (50 mg/kg) at one hour intervals. Drug/vehicle (dimethylsulfoxide) was adminis-tered thirty minutes before caerulein/saline administration. Curcumin was administered ina dose of 200 mg/kg and rosiglitazone in a dose of 0.5 mg/kg. The doses used were basedon earlier work conducted in our laboratory. The study was approved by the InstitutionalAnimal Ethics Committee. Animals were sacrificed one hour after last caerulein injection.Serum amylase and lipase levels were measured. Pancreas was rapidly removed and fixedin formaldehyde. Grading of interstitial edema, inflammation, hemorrhage and acinar cellnecrosis was performed on a scale of 0-4 on H&E stained slides. Apoptotic index wasevaluated using TUNEL (terminal transferase-mediated dUTP-biotin nick end labeling) andHoechst stained slides. The results were analyzed using one-way analysis of variance followedby Scheffe' test or Mann Whitney U test. P <0.05 was considered significant. Results:Caerulein administration led to significant pancreatic edema and inflammation (p<0.01)with marked rise in serum amylase and lipase levels (p<0.01) as compared to saline control.Combination (curcumin & rosiglitazone) caused significant reduction in amylase (11555 vs3664 SU/100 mL; p<0.01) and lipase levels (19.3 vs 5.7 U/mL; p<0.01) as compared tocaerulein alone. The combination also reduced edema and inflammation associated withcaerulein (histological score 4.6 vs 1.5; p<0.01). There was a trend towards lower apoptosisand necrosis scores in the combination group (p>0.05). Conclusion: Combined treatmentwith curcumin and rosiglitazone ameliorated histopathological and biochemical changesassociated with caerulein-induced experimental pancreatitis.

W1826

The Effect of Rosiglitasone, a Specific PPAR -Gamma Ligand, On theDevelopment of Experimental Sodium Taurocholate Induced AcutePancreatitis and the Role of Immunohistochemical ReactionKrzysztof Celinski, Beata Prozorow-Krol, Agnieszka Korolczuk, Grazyna Czechowska,Maria Slomka, Elzbieta Korobowicz, Halina Cichoz-Lach, Agnieszka Madro

INTRODUCTION: Acute pancreatitis is a disease with a multifactorial but not explainedmechanism. The results of recent experimental studies indicate the participation of PPAR-gamma agonists in the regulation of inflammatory processes. The aim: of the experimentwas to determine the effect of Rosiglitasone (a PPAR-gamma agonist) on the developmentof the sodium taurocholate induced acute pancreatitis. METHODS: The experiment wascarried out on male Wistar rats weighing 200-250 g. Acute pancreatitis was induced accordingto Aho and Henckel method by injecting 5% sodium taurocholate into the biliary-pancreaticduct (0.08 ml / 100 gm.c.). The animals were divided into 6 experimental groups consistingof 8 rats each. *Group 1-control *Group 2 were injected 0.9% NaCl into the biliary-pancreatic duct (0.08 ml / 100 gm.c.); experimental acute pancreatitis was induced in*Group 3A and B where the tissue was collected after 24h and 48h from the onset ofinflammation respectively. *Group 4A and 4B were administered Rosiglitasone(Avandia-GlaxoSmith Kline) at a dose of 50 mg/kgm. c. per os and the experimental material wascollected 24h or 48h from the onset of inflammation. *Group 5A and 5B were administeredboth sodium taurocholate and Rosiglitasone and the pancreas was collected after 24h and48h. The animals in *Group 6 were used to determine the survival time from the onsetof induced inflammation. The collected experimental material was used for histologicalexaminations and biochemical determinations (activity of lipase, amylase, bilirubin, amino-transferase ). The results of biochemical examinations were statistically analysed accordingto the U Mann-Whitney test. The immunohistochemical reaction for ICAM and nitrotyrosinehave been obtained. RESULTS: On the basis of these experiments it was found that theadministration of Rosiglitasone to the animals in Group 5A and 5B markedly decreased theintensity of the inflammatory response and reduced the incidence of necrosis in the pancreasof studied animals. Intraparenchymatous oedema was clearly reduced and the observedvacuolisation was limited and focal in character. The statistically significant decrease in theactivity of amylase, lipase, bilirubin and aminotransferase was observed in Group 5A and5B. The obtained immunohistochemical reaction for ICAM and nitrotyrosine show thedecrease of intensity of t5he inflammatory reaction in the experimental groups that weretreated with rosiglitasone. CONCLUSION: The administration of Rosiglitasone, the PPAR-gamma agonist, decreased the intensity of the inflammatory process in the course of sodiumtaurocholate induced acute pancreatitis.

AG

AA

bst

ract

s