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Bile Acid Feeding Induces Cholangiocyte Proliferation and Secretion: Evidence for Bile Acid–Regulated Ductal Secretion

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Bile Acid Feeding Induces Cholangiocyte Proliferation and Secretion: Evidence for Bile Acid–Regulated Ductal Secretion GASTROENTEROLOGY 1999;116:179–186 Bile Acid Feeding Induces Cholangiocyte Proliferation and Secretion: Evidence for Bile Acid–Regulated Ductal Secretion GIANFRANCO ALPINI,*,‡,§ SHANNON S. GLASER,* YOSHIYUKI UENO,¶ REBECCA RODGERS,* JO LYNNE PHINIZY,* HEATHER FRANCIS,* LEONARDO BAIOCCHI,* LEIGH A. HOLCOMB,§,࿣ ALESSANDRA CALIGIURI,* and GENE D. LESAGE* Departments of *Internal Medicine, ‡Medical Physiology, and ࿣Psychiatry and Behavioral Science, Scott & White Hospital and Texas A&M University Health Science Center College of Medicine, and §Central Texas Veterans Health Care System, Temple, Texas; and ¶Third Department of Internal Medicine, Tohoku University School of Medicine, Aobaku, Sendai, Japan Background & Aims: We have shown that taurocholate patic ductal mass.1,3 Cholangiocyte proliferation is closely (TC) and taurolithocholate (TLC) interact in vitro with coupled with increased DNA synthesis, SR gene expres- normal cholangiocytes, increasing DNA synthesis, se- sion, secretin-induced cAMP synthesis, and secretin- cretin receptor (SR) gene expression, and adenosine stimulated bicarbonate-rich choleresis.1–3 3Ј,5Ј-cyclic monophosphate (cAMP) synthesis. To fur- Bile acids (BAs) interact with cholangiocytes and alter ther extend these in vitro studies, we tested the cholangiocyte secretion and growth.4–8 To explain a hypothesis that bile acids (BAs) directly stimulate bicarbonate-rich hypercholeresis after administration of cholangiocyte proliferation and secretion in vivo. Methods: After feeding with TC or TLC (1% for 1–4 certain BA, a ‘‘cholehepatic shunt pathway’’ has been weeks), we assessed the following in vivo: (1) ductal proposed, suggesting that protonated BAs in the lumen 7 proliferation by both morphometry and immunohisto- of bile ducts are reabsorbed by cholangiocytes. Toxic chemistry for proliferating cell nuclear antigen (PCNA) hydrophobic BAs accumulate in both the blood circula- and measurement of [3H]thymidine incorporation; and tion and liver in patients with chronic liver disease. (2) the effect of secretin on bile secretion and bicarbon- Previous studies have shown toxicity of hydrophobic BAs 9,10 11 ate secretion in vivo. Genetic expression of H3-histone to hepatocytes and bile ducts. We have shown that, and SR and intracellular cAMP levels were measured in in vitro, both taurocholate (TC) and taurolithocholate isolated cholangiocytes. Results: After BA feeding, (TLC) increase DNA synthesis, SR gene expression, and there was an increased number of PCNA-positive chol- secretin-stimulated cAMP levels.4 Furthermore, we and angiocytes and an increased number of ducts com- others5,12 have shown functional and genetic expression of 3 pared with control rats. [ H]Thymidine incorporation, the sodium-dependent apical bile acid transporter (ABAT) absent in control cholangiocytes, was increased in in the cholangiocyte apical membranes. This Na1- cholangiocytes from BA-fed rats. In BA-fed rats, there was increased SR gene expression (approximately dependent ABAT probably plays an important role in the 2.5-fold) and secretin-induced cAMP levels (approxi- BA effects on cholangiocyte proliferation and secretion mately 3.0-fold) in cholangiocytes, which was associ- because BAs alter cholangiocyte function in vitro only in ated with de novo secretin-stimulated bile flow and the presence of sodium, and ABAT in cholangiocytes has bicarbonate secretion. Conclusions: These data indi- a Michaelis constant (Km) for TC similar to the BA cate that elevated BA levels stimulate ductal secretion concentration at which the maximum effects of BA on and cholangiocyte proliferation. cholangiocyte secretory and proliferative functions are observed in vitro.4,12 Thus, we propose that uptake of BAs from bile by ABAT initiates signals that modulate holangiocytes that line the intrahepatic biliary tree cholangiocyte proliferation and secretion.4,12 secrete water and electrolytes in response to hor- C In this study, we wanted to determine whether in- mones.1–3 Secretin stimulates ductal bile secretion by interacting with secretin receptor (SR) present only on cholangiocytes,2 resulting in increases in intracellular Abbreviations used in this paper: ABAT, apical bile acid trans- porter; BA, bile acid; BDL, bile duct ligation; CK-19, cytokeratin 19; 3,4 adenosine 3Ј,5Ј-cyclic monophosphate (cAMP) synthesis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GGT, g-glu- Cholangiocytes are normally in a quiescent state.3,4 In tamyl transferase; g-GT, g-glutamyltranspeptidase; KRH, Krebs– experimental models of ductal hyperplasia, such as bile Henseleit bicarbonate solution; SR, secretin receptor; TC, taurocho- late; TLC, taurolithocholate. duct ligation (BDL) or 70% hepatectomy, cholangiocytes ௠ 1999 by the American Gastroenterological Association proliferate markedly, leading to enlargement of intrahe- 0016-5085/99/$10.00 180 ALPINI ET AL. GASTROENTEROLOGY Vol. 116, No. 1 creased biliary BA concentrations could directly stimu- were obtained as previously described.2,3,16,17 Briefly, after late in vivo cholangiocyte proliferation and ductal secre- standard collagenase perfusion,17 a cholangiocyte-enriched frac- tion in the absence of cholestasis or hepatic injury. We tion (40%–55% pure by g-GT) was obtained from intact 17 experimentally increased biliary BA concentration by portal tracts, as described by Ishii et al., and subsequently 3,17 feeding rats TC (a physiologically relevant BA) or TLC purified by immunoaffinity separation. (which has been shown to induce ductal hyperplasia in 8 13 Assessment of Intrahepatic Ductal Mass rodents ) for 1–4 weeks. There is enrichment and a in Normal and BA-Fed Rats 2–3-fold increase in the BA pool14 with the adminis- trated BA in BA-fed rats. In rats fed TC or TLC, we found Intrahepatic duct mass was calculated by point count 3,18 that BA concentrations were increased in bile but not in analysis by determination of the fraction of total matrix of 19 serum and that there was no biochemical or histological points overlapping bile ducts, stained for g-GT, in coded frozen liver sections (6 µm) from normal and BA-fed rats (n 5 evidence of hepatic damage or cholestasis, yet there was 12). In each liver section, the entire area was examined by cholangiocyte proliferation and increased ductal secretion. multiple photographs taken in a mosaic fashion. From the total number of points over hepatic tissue and the number of points Materials and Methods over g-GT–positive ducts, we calculated the volume percent of Animal Model and Materials liver occupied by ducts.3,18 Indirect immunohistochemistry was performed to detect Male Fischer 344 rats (125–150 g) from Charles River proliferating cellular nuclear antigen (PCNA) in proliferating (Wilmington, MA) were used in the present studies. The bile ducts. Briefly, formalin-fixed liver sections were deparaf- animals were kept in a temperature-controlled environment fined, followed by microwave treatment (5 minutes in water at (20–22°C) with alternating 12-hour light-dark cycles. The 500 W). After adequate blocking of endogenous peroxidase studies were conducted in rats fed 1% TC or 1% TLC, activity by methanol-peroxidase solution, nonspecific binding representing approximate doses of 275 and 260 µmol/day, was blocked by incubation with normal goat serum for 30 respectively, for 1–4 weeks. Rat chow containing 1% TC, 1% minutes at room temperature. Sections were incubated with TLC, or AIN 76 (control diet) was prepared by Dyets Inc. anti-PCNA antibody (clone PC-10; Oncogene Research Prod- (Bethlehem, PA). Rats were fasted overnight before experi- ucts, Cambridge, MA) at 4°C overnight. After several washes ments. Before each protocol, rats were anesthetized with sodium with cold phosphate-buffered saline, biotin-labeled secondary pentobarbital (50 mg/kg intraperitoneally). Reagents were pur- antibody at a dilution of 1:100 (Dako, Kyoto, Japan) was added chased from Sigma Chemical Co. (St. Louis, MO) unless otherwise for 1 hour at room temperature. For detection of this reaction, a indicated. N(g-L-glutamyl)-4-methoxy-2-naphthylamide, the peroxidase-labeled avidin-biotin complex with diaminobenzi- substrate for g-glutamyltranspeptidase (g-GT), was purchased dine (Dojin Chemical Co, Kumamoto, Japan) was used as a from Polysciences (Warrington, PA). Both TC and TLC were substrate. Sections were counterstained with hematoxylin and purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). examined with a microscope (Olympus Optical Co., BX 40, Japan); 300–400 cholangiocytes were analyzed for each inter- Determination of Liver Inflammation val. and Hepatic Damage 3 After TC or TLC feeding, the degree of liver inflamma- In Vivo Measurement of [ H]Thymidine tion was assessed by the grade system described by Davis and Incorporation in Cholangiocytes Madri.15 H&E-stained liver sections were graded for the degree We measured DNA synthesis of pure cholangiocytes of inflammation in a coded fashion as follows: 0, normal; 11, from control and BA-fed rats by measuring of [3H]thymidine triad-based; 12, triad-based with focal parenchymal inflamma- incorporation as described previously.3 tion without necrosis; 14, triad-based with lobular inflamma- tion with unicellular necrosis; and 15, confluent necrosis. To Measurement of H3-Histone and SR determine whether BA feeding induces liver damage, the Gene Expression serum levels of selected enzymes associated with hepatic damage (serum glutamic pyruvic transaminase [SGPT], serum Steady-state levels of H3-histone and SR messenger glutamic oxalacetic
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