studies

610A AASLD ABSTRACTS HEPATOLOGY, October, 2015
7 days. Oxysterols and bile acids in the liver and brain were
extracted, and analyzed by HPLC or LC/MS. Hepatic mRNA
were determined by RT-PCR. Primary hepatocytes were also
isolated from the same strains of mice. Cultures were incubated
with [ 14 C]-cholesterol and formed [ 14 C]-oxysterols/bile acids
were analyzed. Results: In wild type mice, hepatic CYP7B1
mRNA expression was suppressed (>80%Ñ) after StARD1 overexpression.
SHP mRNA expression was markedly increased
(300%É), while CYP7A1(40%Ñ) and CYP8B1(80%Ñ) mRNAs
were decreased. Interestingly, gallbladder bile acid concentration
in these mice was 30% higher as compared to control
mice. Primary hepatocyte culture experiments showed that the
rate of bile acid synthesis increased 3-fold after StARD1 overexpression.
These observations implied that an additional bile
acid synthetic pathway is present which compensates for the
CYP7A1 and the CYP27A1/CYP7B1 pathways. StARD1 overexpressed
liver accumulated 27-Hydroxycholesterol(HC) and
25HC. Also found was unexpected high level of 24HC(Ratio of
24, 25, 27HC, 6:1:6). Level of 24HC in the brain was identical
to control. When primary hepatocytes were incubated with
[ 14 C]-cholesterol following StARD1 overexpression, wild type
hepatocytes formed all three [ 14 C]-oxysterols, 24HC, 25HC
and 27HC(ratio, 3:1:10). However, hepatocytes isolated from
CYP27A1 -/- mice formed only [ 14 C]-25HC. These observations
show that 24HC originates from liver mitochondrial CYP27A1.
Furthermore, when StARD1 was overexpressed in CYP7B1 -/-
mouse liver in vivo and in vitro, 27HC and 25HC accumulated
but, not 24HC; suggesting 24HC is responsible for an alternative
metabolic pathway to bile acids. Conclusion: The results
indicate that hepatic CYP27A1 catalyzes not only 25- and
27-hydroxylations, but also 24-hydroxylation. Since it has been
reported that 24HC is metabolized by CYP7B1, CYP39A and
CYP7A1, this oxysterol may be an important intermediate in a
second alternative pathway to bile acids.
Disclosures:
William M. Pandak - Employment: Virginia Commonwealth University, Veterans
Affairs Medical Center
The following authors have nothing to disclose: Genta Kakiyama, Dalila M.
Marques, Kuniko Mitamura, Hajime Takei, Hiroshi Nittono, Daniel Rodriguez-Agudo,
Gregorio Gil, Huiping Zhou, Phillip B. Hylemon
805
Bile acid induced cholestatic liver injury involves TLR9
initiated inflammatory signals in mouse hepatocytes
Shi-Ying Cai, Xinshou Ouyang, Albert Mennone, Carol J. Soroka,
Wajahat Z. Mehal, James L. Boyer; Yale Univ, New Haven, CT
Background: The inflammatory response plays an important
role in cholestatic liver injury where bile acid (BA) induction
of proinflammatory cytokines in hepatocytes may initiate this
event. However, the signaling pathways involving BA stimulation
of cytokine production remain elusive, although BA injures
mitochondria in hepatocytes. Toll-like receptors (TLR) respond
to both endogenous sterile insults and pathogen recognitions
and play a critical role in tissue damage initiated inflammatory
responses. TLR9 has been characterized as an intracellular
DNA receptor that, upon activation, stimulates cytokine production
as part of the innate immune response. Aim: To determine
Tlr9’s role in BA induced liver injury. Methods: Hepatocytes
isolated from wild-type (WT) and Tlr9 knockout (KO) mice were
treated with BAs. Mitochondrial damage was assessed using a
JC-1 assay and Western blot. Q-PCR was used to detect inflammatory
chemokine gene expression. WT and Tlr9 KO mice
were subjected to bile duct ligation (BDL) or sham operation
for 7 days. Plasma biochemistry, liver gene expression, and
liver histology were examined. Results: At pathophysiological
concentration (≥25 mM), taurocholic acid (TCA) caused mitochondrial
membrane potential changes in mouse hepatocytes.
Mitochondrial specific proteins cytochrome C, AIF and ER
specific protein Grp78 were detected in the cytosolic fraction
by Western blot analysis, indicating mitochondria damage
and ER stress, whereas no ROS were detected. Cxcl2 induction
was significantly less in Tlr9 KO hepatocytes than in WT
hepatocytes (KO: 3.7-fold v/s WT: 14-fold) after treatment with
100mM TCA for 24 hr. The synthetic Tlr9 ligand CpG ODNs
and BAs demonstrated synergistic effects in stimulating Cxcl2
expression in mouse hepatocytes, consistent with Tlr9’s involvement
in BA induction of Cxcl2 expression. After BDL, plasma
ALT, ALP and BA levels and liver BA levels were also significantly
lower in Tlr9 KO mice compared to WT BDL mice, confirming
a role for Tlr9 in mediating cholestatic liver injury. Bile
duct proliferation, assessed by liver H&E histology and CK19
labeling, and hepatic levels of Ccl2 and Cxcl2 mRNA were
also significantly less in Tlr9 KO than in WT mice after BDL.
Conclusion: BA injures mitochondria resulting in DNA release
and triggers an innate immune response by activating Tlr9.
Preventing mitochondrial damage or blocking Tlr9 activation
may be new strategies for treating cholestasis.
Disclosures:
James L. Boyer - Advisory Committees or Review Panels: Pfizer; Consulting:
abbvie
The following authors have nothing to disclose: Shi-Ying Cai, Xinshou Ouyang,
Albert Mennone, Carol J. Soroka, Wajahat Z. Mehal
806
Proteomics analysis of rat canalicular membrane
reveals the expression of several P4-ATPases in liver
Pururawa M. Chaubey, Bruno Stieger; Department of Clinical
Pharmacology and Toxicology, University Hospital Zurich, Zurich,
Switzerland
Background: Transport processes in the canalicular membrane
are key elements in bile formation and are the driving force
for the enterohepatic circulation of bile salts. The canalicular
membrane is constantly exposed to a hostile environment due
to the detergent action of the high conentration of canalicular
bile salts. Aims: To obtain more insights into the molecular entities
rendering the canalicular membrane resistant to the detergent
action of bile salts, the proteome of highly purified rat
canalicular membrane vesicles was determined. Methods: Isolated
canalicular membrane vesicles (JCB 98:991,1983) from
Sprague Dawley rats were stripped from adherent proteins
(JCB 93:97,1982), deglycosylated, protease digested and subjected
to shot gun proteomic analysis. Expression of P4-ATPases
was confirmed by RT-PCR and Western blotting. Localization of
P4-ATPases was carried out with immunofluorescence methodology
on liver sections. Results: We identified 1745 unique
proteins in canalicular vesicles. A comparative analysis with a
renal brush border membrane proteome and liver proteomes
revealed a large set of membrane and membrane associated
proteins (1287) specifically identified in our study, possibly
because we enriched for membrane proteins. Assignment to
functional categories of the newly identified proteins revealed
an over-representation of transporter specific categories. We
identified several transporters out of which 19 members are
from SLC families and 11 members are from ABC families.
Additionally, we also identified P4-ATPases (ATP8A1, ATP8B1,
ATP9A, ATP11A, & ATP11C) and a P5-ATPase (ATP13A1).
Four of 5 identified P4-ATPases are not known to be expressed
in liver. Furthermore, expression analysis of P4-ATPases using
RT-PCR and Western blotting could verify the expression of
identified P4-ATPase with a rather high expression of ATP11C.
Finally, ongoing immunofluorescence analysis demonstrates