studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 307A
194
Enteral Obeticholic Acid Prevents Hepatic Cholestasis in
Total Parenteral Nutrition-Fed Neonatal Pigs
Douglas Burrin 1 , Yanjun Jiang 1 , Zhengfeng Fang 2 , Barbara Stoll 1 ,
Gregory J. Guthrie 1 , Hongtao Wang 3 , Ignacio R. Ipharraguerre 4,5 ,
Jose J. Pastor 4 ; 1 USDA Children’s Nutrition Research Center,
Department Pediatrics, Baylor College of Medicine, Houston,
TX; 2 Animal Nutrition Institute, Sichuan Agricultural University,
Chengdu, China; 3 Pediatric Gastroenterology, Hepatology, Nutrition,
Department Pediatrics, Baylor College of Medicine, Houston,
TX; 4 Lucta SA, Barcelona, Spain; 5 Institute of Human Nutrition and
Food Science, University of Kiel, Kiel, Germany
Total parenteral nutrition (TPN) is a vital support for neonatal
infants with congenital or acquired gastrointestinal (GI) disorders
and requiring small bowel resection. An adverse outcome
associated with prolonged TPN use is parenteral nutrition
associated cholestasis (PNAC). We previously showed that
enteral chenodeoxycholic acid (CDCA) treatment reduced
PNAC. We hypothesized that the protective effects of CDCA
were mediated by modulation of FXR-target genes involved
in bile acid homeostasis. The aim of the current study was to
compare the physiological effects of a selective FXR agonist,
obeticholic acid (OCA) vs CDCA on hepatic bile acid homeostasis
in TPN-fed piglets. Term, newborn pigs were assigned
to receive complete TPN (PN), TPN + enteral CDCA (30 mg/
kg), or TPN+enteral OCA (0.5, 5, 15 mg/kg) daily for 19 d.
The daily parenteral lipid was Intralipid given at 10 g/kg. Endpoints
of PNALD and bile acid homeostasis were measured.
We found that, compared to PN pigs, treatment with high dose
of OCA (OCA5 and OCA15), but not CDCA and low dose
of OCA (OCA0.5), reduced serum PNAC markers including
bilirubin, gamma-glutamyl transferase (GGT), total bile acid, triglyceride,
and very-low-density lipoprotein. Compared to PN,
OCA 5 and 15, but not OCA 0.5 or CDCA, reduced the total
plasma bile acid concentration by increasing the proportional
transfer of hepatic bile acid into the gallbladder, suggesting
increased bile flow. TPN-induced ductopenia as measured by
percentage of intact bile ducts per portal tract was prevented
by OCA suggesting preservation of bile ducts. The major bile
acids in plasma were glyco-conjugated forms of CDCA, hyocholic
acid and hyodeoxycholic acid. OCA5 and OCA15, but
not CDCA, suppressed hepatic expression of CYP7A1, while
CYP27A1 and CYP8B1 mRNA remained unchanged. The bile
acid detoxification enzyme CYP3A29 mRNA was inhibited by
both CDCA and OCA treatments. OCA, but not CDCA upregulated
hepatic mRNA involved in hepatobiliary bile acid and bilirubin
transport into bile including bile salt export pump (BSEP),
multidrug resistance protein 1(MDR1), and multidrug resistance
protein 4 (MRP4). OCA5 and OCA15 induced hepatic and
ileal FGF19 expression more than CDCA in pigs. We further
found that OCA5 and OCA15, but not CDCA, inhibited the
hepatic expression of inflammatory marker interleukin-8. Contrary
to our previous study, we found that CDCA did not prevent
PNAC. We suspect that this was due to the higher lipid
load infused. We conclude that enteral OCA is more effective
than CDCA in prevention of PNAC via upregulation of FXR-target
genes involved in preservation of hepatobiliary transporters
and bile duct function.
Disclosures:
The following authors have nothing to disclose: Douglas Burrin, Yanjun Jiang,
Zhengfeng Fang, Barbara Stoll, Gregory J. Guthrie, Hongtao Wang, Ignacio R.
Ipharraguerre, Jose J. Pastor
195
Biliatresone causes GSH reduction in cholangiocytes,
leading to cholangiocyte damage, increased monolayer
permeability, and periductular myofibroblasts: insights
into the mechanism of biliary atresia
Orith Waisbourd-Zinman 1 , Hong Koh 2,3 , Pierre-Marrie Lavrut 2,4 ,
Shannon Tsai 2 , John R. Porter 5 , Michael Pack 2 , Rebecca G.
Wells 2 ; 1 Gastroenterology, Hepatology and Nutrition, Children’s
Hospital of Philadelpahia, Philadelpahia, PA; 2 Gastroenterology,
Perelman School of Medicine, University of Pennsylvania, Philadelphia,
PA; 3 Department of Pediatrcis, Division of Gastroenterology,
Hepatology and Nutrition, Yonsei University College of Medicine,
Severance Children’s Hospital, Seoul, Korea (the Republic of);
4 Pathology, Academic Hospital of Lyon, Lyon, France; 5 Biological
Sciences, University of the Sciences, Philadelphia, PA
Background: Biliary atresia (BA) is an acquired fibro-obliterative
disease of unknown etiology affecting the extrahepatic
bile ducts of the newborn. We previously reported that the
novel toxin biliatresone causes selective atresia of the extrahepatic
biliary tree in zebrafish and microtubule instability and
lumen obstruction in a cholangiocyte 3D culture model. The
toxin strongly and spontaneously binds to glutathione (GSH).
We hypothesized that GSH reduction is responsible for the
effects of the toxin. Methods: We used several mouse cholangiocyte
culture systems: 2D culture, 3D spheroid culture,
and ex vivo culture of neonatal extra-hepatic bile ducts. We
treated these with biliatresone and compounds that regulate
GSH, then stained and imaged the cells. We measured
GSH levels in cholangiocytes treated with the compounds at
different time points. In order to assess lumen permeability,
we used a rhodamine efflux assay with live cell imaging of
cholangiocytes in 3D cultures. Results: Cholangiocytes in 2D
culture demonstrate a 70% decrease in GSH 1h following
toxin treatment, with recovery to baseline by 6h. In 3D culture,
lowering GSH with buthionine sulfoximine (BSO) mimicked
the effects of biliatresone, with microtubule instability, loss of
polarity, and lumen obstruction. N-Acetyl-L-cysteine (L-NAC),
which increases GSH, protected 3D cultures from the effects
of biliatresone. Neonatal bile ducts cultured ex vivo showed
patchy disruption of the cholangiocyte monolayer and lumen
obstruction after treatment with biliatresone or BSO, with
protection by L-NAC but not the stereoisomer D-NAC. A time
course study of cholangiocytes in 3D culture treated with biliatresone
showed that microtubule injury occurred first, within 3
hours, followed by abnormalities in F-actin, polarity, and tight
junctions, and finally lumen obstruction. Rhodamine remained
in the lumens of vehicle-treated cholangiocyte spheroids for up
to 12 h, but effluxed within 3 h after biliatresone treatment,
suggesting increased epithelial permeability. In the ex vivo bile
duct model, staining for a-smooth muscle actin was increased
in the stroma surrounding the cholangiocyte layer after treatment
with biliatresone, BSO, or D-NAC plus biliatresone, but
remained at baseline levels after treatment with L-NAC plus biliatresone.
Conclusions: Biliatresone rapidly decreases GSH in
mouse cholangiocyte and bile duct cultures, initially disrupting
microtubules then compromising cholangiocyte polarity and
increasing lumen permeability. In vivo, this is a potential cause
of obstruction and periductular fibrosis and may be important
to the pathophysiology of BA.
Disclosures:
The following authors have nothing to disclose: Orith Waisbourd-Zinman, Hong
Koh, Pierre-Marrie Lavrut, Shannon Tsai, John R. Porter, Michael Pack, Rebecca
G. Wells