studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 663A
920
Deletion of orphan nuclear receptor small heterodimer
partner prevents development of nonalcoholic steatohepatitis
Chunki Kim, Mikang Lee, Yoonkwang Lee; Integrative Medical
Sciences, Northeast Ohio Medical University, Rootstown, OH
An orphan nuclear hormone receptor Small Heterodimer
Partner (SHP) is involved in lipid and bile acid metabolism.
Our earlier studies have shown that deletion of SHP protects
the mice from diet-induced obesity and hepatic steatosis. In
order to explore a potential role of SHP in the development
of NASH, we quantified inflammatory gene expression in the
mice fed a westernized diet (WestD) for 6 month. The longterm
diet regimen induced mRNA levels of inflammatory genes
such as TNFa, IL-1b, CXCL-1, and iNOS in wild type (WT)
mice. However, the expression of these genes was significantly
down-regulated in SHPKO counterparts. In order to find out
whether the reduction of inflammatory responses is due to
SHP gene deletion or lower hepatic lipid level, we generated
ApoE/SHP double knockout mice (ApoE/SHPKO). ApoEKO
mouse model has been shown to be susceptible to diet-induced
inflammation but resistant to hepatic steatosis. One week-
WestD regimen significantly induced the expression of the
pro-inflammatory genes in ApoEKO mice but not in ApoE/
SHPKO mice without evidence of hepatic TG accumulation in
either genotype as expected, suggesting SHP, not hepatic TG
level, is responsible for the observed inflammatory responses.
To study NASH development in SHPKO mice further, the mice
were challenged with methionine and choline-deficient (MCD)
diet for 6 weeks. SHPKO mice were strongly protected from
NASH development evidenced by reduced pro-inflammatory
gene expression, reduced F4/80 positive macrophage infiltration,
decreased mRNA levels of fibrosis-related genes such
as collagen 1a1, TGF-b1, TIMP1, and a-SMA, which were all
significantly induced in WT mice. In addition, hepatocyte specific-SHPKO
(AlbCre/floxedSHP) mice challenged with MCD
diet also showed significant protection in NASH development,
as assessed with qPCR, immunofluorescence, Sirius red staining,
compared to floxed SHP counterparts. The current study
identifies the role of SHP as pro-inflammatory in the liver and
will provide new insights into development of NASH and its
possible therapeutic or preventative approaches.
Disclosures:
The following authors have nothing to disclose: Chunki Kim, Mikang Lee, Yoonkwang
Lee
921
Differential carbonylation of proteins in human fatty
and nonfatty NASH
Colin T. Shearn, David J. Orlicky, Dennis R. Petersen; Department
of Pathology, University of Colorado Anschutz Medical Campus,
Aurora, CO
Objective: In the liver, a contributing factor in the pathogenesis
of non-alcoholic fatty liver disease is oxidative stress leading
to the accumulation of highly reactive electrophilic α/β unsaturated
aldehydes. The objective of this study was to determine
if significant differences were evident when evaluating carbonylation
in human end-stage fatty nonalcoholic steatohepatitis
(fNASH) compared to end stage non-fatty NASH (nfNASH).
Methods: Using age-matched pooled hepatic tissue obtained
from healthy humans and patients diagnosed with end stage
nfNASH or fNASH, overall carbonylation was assessed by
immunohistochemistry (IHC) and LC-MS/MS of streptavidin
purified hepatic whole cell extracts treated with biotin hydrazide.
Identified carbonylated proteins were further evaluated
using bioinformatics analyses. Results: Picrosirius red staining
revealed extensive fibrosis in both fNASH and nfNASH which
corresponded with increased 4-HNE staining. Although significantly
elevated when compared to normal hepatic tissue,
no significant differences in overall carbonylation and fibrosis
were evident when comparing fNASH with nfNASH. Mass
spectrometric analysis revealed a total of 184 carbonylated
proteins. Of these, 51 were unique to nfNASH, 32 unique to
fNASH, 33 unique to normal hepatic tissue and 27 common to
all groups. DAVID and Gene Ontology bioinformatic pathway
analysis of hepatic carbonylated proteins revealed a propensity
for increased carbonylation of carboxylic acid catabolic
process, generation of precursor metabolites as well as energy
and proteolysis in nfNASH. Whereas carbonylation of proteins
implicated in protein complex biogenesis and regulation of
apoptosis occurred in fNASH. Using LC-MS/MS analysis, in
nfNASH a nonenaldehyde adduct was identified on Lys 235
on the cytoskeletal protein vimentin. Conclusions: These results
suggest that cellular factors regulating mechanisms of protein
carbonylation may be different depending on pathological
diagnosis of NASH. Furthermore these studies are the first to
use LC-MS/MS analysis of carbonylated proteins in human
NAFLD and begin exploring possible mechanistic links with
end stage cirrhosis due to fatty liver disease and oxidative
stress.
Disclosures:
The following authors have nothing to disclose: Colin T. Shearn, David J. Orlicky,
Dennis R. Petersen
922
Lysosomal cholesterol in Kupffer cells, particularly when
oxidized, contributes to murine steatohepatitis
Sofie Walenbergh 1 , Tom Houben 1 , Tim Hendrikx 1 , Patrick van
Gorp 1 , Mike Jeurissen 1 , Marie-Hélène Lenders 1 , Marion J. Gijbels
1 , Jogchum Plat 1 , Marten H. Hofker 2 , Christoph J. Binder 3 ,
Dieter Luetjohann 4 , Fons Verheyen 1 , Ger H. Koek 1 , Ronit Shiri-Sverdlov
1 ; 1 Maastricht University, Maastricht, Netherlands;
2 University Medical Center Groningen, Groningen, Netherlands;
3 Research Center for Molecular Medicine of the Austrian Academy
of Sciences, Vienna, Austria; 4 Institute of Clinical Chemistry and
Clinical Pharmacology, Bonn, Germany
Background and Aims: Recently, the importance of lysosomes
within the metabolic syndrome, including fatty liver disease,
is gaining increasing attention. It has been suggested that
macrophages during atherosclerosis as well as Kupffer cells
(KCs) during hepatic inflammation demonstrate properties of
an acquired lysosomal storage disorder. So far, it is unclear
whether there is a causal relationship between lysosomal cholesterol
accumulation (LCA) in KCs and hepatic inflammation.
Additionally, the specific contribution of the oxidized LDL
(oxLDL) fraction to LCA, its concomitant effect on lysosomal
function and on hepatic inflammation is unexplored. Methods:
Hematopoietic deficiency of the mutant Niemann-Pick type C1
(NPC1 mutant ) protein was used as a tool to induce LCA in KCs
of hyperlipidemic mice. To induce high levels of anti-oxLDL antibodies,
NPC1 mutant -transplanted mice were immunized every
two weeks with heat-inactivated pneumococci until the end
of the experiment. Results: Compared to wildtype, NPC1 mutant
-transplanted mice displayed severe hepatic inflammation
and fibrosis. Anti-oxLDL immunization of NPC1 mutant -transplanted
mice improved cholesterol metabolism, lysosomal dysfunction
and liver inflammation, in contrast to non-immunized
NPC1 mutant -transplanted mice. Conclusions: A direct causal link
exists between LCA in KCs and hepatic inflammation. Rather