studies

612A AASLD ABSTRACTS HEPATOLOGY, October, 2015
life and stronger biliary enrichment (approx. 30x) of Z-BOX A
compared to Z-BOX B. In parallel, Z-BOX A and Z-BOX B triggered
an instantaneous and significant reduction of bile flow
(1 min vs. 3.5 min after bolus injection; n = 5; p = 0.0002).
Regardless their reported vasoconstrictive properties in cerebral
arterioles, Z-BOX A and Z-BOX B did not affect hepatic
hemodynamics. However, Z-BOX A and Z-BOX B showed a
dose-dependent decrease in cellular metabolic rate (IC 50
=
383 mmolL -1 and 141 mmolL -1 , respectively), while only Z-BOX
A induced a cytoskeletal remodeling of cultured hepatocytes.
Conclusions: Higher order metabolites of heme are elevated
under conditions of hyperbilirubinemia and are able to impair
bile production and cellular integrity.
Disclosures:
The following authors have nothing to disclose: Raphael A. Seidel, Franziska
Schleser, Christoph Sponholz, Frans Cuperus, Sandor Nietzsche, Georg Pohnert,
Michael Bauer
809
NF-kB is critically important for the up-regulation of
organic solute transporter OSTα/OSTβ in cholestasis
Natarajan Balasubramaniyan, Frederick J. Suchy; Pediatrics, University
of Colorado Denver, Aurora, CO
The up-regulation of the organic solute transporter OSTα/
OSTβ in cholestasis provides an alterative pathway for excretion
of bile acids and other cholephiles across the hepatocyte
basolateral membrane.However, the mechanisms underlying
this adaptation have been enigmatic. The mouse and human
Ostα/β(OSTα/β) promoters contain functional FXR and LRH
elements, which mediate, respectively, positive and negative
feedback regulation by bile acids in normal liver, but these
binding sites don’t explain how this transport system is up-regulated
in cholestasis when FXR-dependent pathways are compromised.We
identified previously unknown,well-conserved NF-kB
binding sites in the Ostα(OSTα) and Ostβ(OSTβ)promoters.
On chromatin immunoprecipitation(ChIP) analysis of Huh-7
cells,over-expressed NF-kB was recruited to these sites. In vivo
ChIP assays done with liver nuclei obtained from mice after 3
days of CBDL or 6 hours post LPS injection, showed markedly
increased recruitment of NF-kB p65 to the NF-kB binding sites
of the Ostα and Ostβ promoters and a diminution of FXR at its
binding sites (FXRE). Next Huh-7 cells were transiently transfected
with plasmid vectors containing the OSTα and OSTβ
promoter sequences that included the NFkB response element
linked to luciferase as reporter. In contrast to the inhibitory
effect of NFkB p65 on activity of the BSEP promoter, expression
of NFkB p65 and p50 subunits together synergistically
activated the OSTα and OSTβ promoters in a dose-dependent
fashion. The inhibitors IkBα and IkBSR blocked induction by
p65 and p50. Previous studies have shown that NF-kB, acting
as a transcriptional activator, recruits a coactivator complex
that has striking similarities to that recruited by nuclear receptors
which usually includes the cyclic AMP response element
binding protein (CREB)-binding protein (CBP) and p300.CBP/
p300 is particularly important in possessing intrinsic histone
acetyltransferase activity and providing a platform for recruitment
of a variety of coactivator proteins required for NF-kB-dependent
gene expression. In vivo ChIP assays using liver nuclei
obtained from both cholestatic mouse models and an antibody
that recognizes the homologous CBP and p300 proteins
showed a markedly increased recruitment of CBP/p300 to the
NF-kB sites in the Ostα and Ostβ promoters compared with
controls. In contrast, there was significant depletion of CBP/
p300 at the FXRE of Ostα and Ostβ in these models compared
with controls. These studies reveal a novel, NF-kB-dependent
mechanism for the up-regulation OSTα/OSTβ in cholestasis,
providing an alternative export pathway to prevent accumulation
of hydrophobic bile salts and other toxic products.
Disclosures:
The following authors have nothing to disclose: Natarajan Balasubramaniyan,
Frederick J. Suchy
810
The Ileal Bile Acid Transporter Inhibitor A4250 Modulates
Bile Acid Synthesis and Decreases Serum Bile
Acids.
Hanns-Ulrich Marschall 1 , Per-Göran Gillberg 2 , Hans Graffner 2 , Leif
Rikner 2 ; 1 Department of Molecular and Clinical Medicine, Sahlgrenska
Academy, Institute of Medicine, University of Gothenburg,
Gothenburg, Sweden; 2 Albireo, Gothenburg, Sweden
Background and Aim: Reabsorption of BAs from the intestine
by ileal bile acid transporter (IBAT) is pivotal for the enterohepatic
circulation of BAs and sterol homeostasis. We aimed to
study BA metabolism in a phase 1 trial with the selective IBAT
inhibitor A4250. Methods: In a double-blind, multiple ascending
dose design, A4250 capsules or matching placebo was
administered for 7 days; 1 mg once daily; 3 mg once daily
and 1.5 mg twice daily, respectively. Each group consisted of
8 healthy volunteers whereof 6 received active compound and
2 placebo. BAs were measured by HPLC-MS in plasma and
feces, FGF19 and C4 in plasma. Results: No serious adverse
events occurred and all participants finished the trial per protocol.
Plasma total BAs, and FGF19 decreased, whereas plasma
C4 and fecal BAs increased. The majority of fecal BAs in the
A4250 groups were the primary BAs cholic and chenodeoxycholic
acids. Conclusion: A4250 is a highly efficient and
well-tolerated compound that by blocking the IBAT in the terminal
ileum interrupts the enterohepatic circulation of BAs, which
should be of benefit in patients with cholestatic liver diseases.
Disclosures:
Hanns-Ulrich Marschall - Consulting: Albireo
Per-Göran Gillberg - Employment: Albireo
Hans Graffner - Employment: Albireo
Leif Rikner - Consulting: Albireo AB
811
Inhibition of Adenylyl Cyclase 5 reduces cyst growth in
mice with polycystic liver disease (ADPKD) caused by
defective Polycystin-2
Carlo Spirli 1 , Ambra Villani 1 , Valeria Mariotti 1,2 , Luca Fabris 3 ,
Romina Fiorotto 1 , Mario Strazzabosco 1,2 ; 1 Yale University, Section
of Digestive Diseases, New Haven, CT; 2 Department of Surgery
and Interdisciplinary Medicine, University of Milano-Bicocca,
Milan, Italy; 3 Department of Molecular Medicine, University of
Padova, Padua, Italy
Genetic defects in Polycystin-1 (PC1) or Polycystin-2 (PC2)
cause polycystic liver disease associated with ADPKD (PLD). In
PC-2 defective cystic cholangiocytes, the interaction of STIM1
(the molecular sensor that mediates Ca 2+ entry following
ER-Ca 2+ decrease) with ORAI channels is uncoupled accounting
for a reduced Store Operated Calcium Entry (SOCE) and
low intracellular and Endoplasmic Reticulum (ER) Ca 2+ levels.