studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 665A
925
A novel transcriptional repressor Hairy and Enhancer
Split 6 mediates hepatic lipid homeostasis through inhibition
of Pparg2 expression
Chunki Kim, Mikang Lee, James P. Hardwick, Yoonkwang Lee;
IMS, Northeastern Ohio Univ. College of Med, Rootstown, OH
Hepatic steatosis is a required prelude for development of
non-alcoholic fatty liver diseases. Pparg is a member of nuclear
hormone receptor superfamily and a master regulator for white
adipocyte differentiation and lipid storage. Uprising hepatic
Pparg2 level reprograms liver for lipid storage instead of for
VLDL secretion or fatty acid oxidation, which leads to hepatic
fat accumulation in pathophysiological conditions such as
obesity and diabetes. Our earlier study suggested that small
heterodimer partner and retinoic acid receptor coordinately
regulates Pparg2 gene expression via directly regulating a
novel transcriptional repressor Hairy and Enhancer Split 6
(Hes6) mRNA level. In the suggested regulatory paradigm,
Hes6 represses Pparg2 gene expression by inhibiting DNAbound
HNF4a transcriptional activity. To explore function of
Hes6 in hepatic lipid mobilization, we overexpressed Hes6
using adenovirus in the livers of mice fed a westernized diet
for 2 months. The targeted overexpression reduced Pparg2
mRNA level by 60% and hepatic triglyceride accumulation by
30% compared to the levels obtained from mice injected with
adenoviral empty vector. We also silenced hepatic Hes6 using
adenoviral shRNA and fed the mice western diet for additional
2 weeks. The adenoviral shHes6 effectively silenced Hes6
gene expression by approximately 90% and increased hepatic
fat accumulation and Pparg2 mRNA level by 50% and 6 fold,
respectively. In subsequent experiments, we utilized transient
transfection and gel mobility shift assays to identify a specific
HNF4a binding element in the Pparg2 promoter. Indeed, an
Hnf4a binding consensus sequence was identified at -903bp
from transcription start site. Deletion or point mutation of the
sequence in a luciferase reporter containing the Pparg2 promoter
abolished Hes6-mediated repression in HepG2 cells.
Chromatin immunoprecipitation and gel mobility shift assays
further confirmed direct recruitment and binding of Hnf4a to
the site. The data strongly prove that expression of Pparg2 is
maintained low by coordinate repression by Hes6 and Hnf4a
in normal liver and thus the Hes6-Hnf4a-Pparg2 transcriptional
axis is considered as a critical determinant for hepatic lipid
homeostasis.
Disclosures:
The following authors have nothing to disclose: Chunki Kim, Mikang Lee, James
P. Hardwick, Yoonkwang Lee
926
Effects of Dietary Different Doses of Copper and High
Fructose Feeding on Rat fecal Metabolome
Ming Song 1 , Xiaoli Wei 2 , Xinmin Yin 2 , Dale Schuschke 3 , Imhoi
Koo 2 , Craig J. McClain 1,4 , Xiang Zhang 2 ; 1 Department of Medicine/GI,
University of Louisville, Louisville, KY; 2 Chemistry, University
of Louisville, Louisville, KY; 3 Physiology, University of Louisville,
Louisville, KY; 4 Robley Rex VAMC, Louisville, KY
Background/Aims: The gut microbiota play a critical role in
the pathogenesis of nonalcoholic fatty liver disease (NAFLD)
through altering the gut metabolites and gut barrier function.
Increased fructose consumption and inadequate copper intake
are two critical risk factors in the development of NAFLD. The
aim of this study was to determine the effect of different dietary
doses of copper and high fructose feeding on fecal metabolites
in a rat model. Methods: To gain insights into the role of gut
microbiota, male weanling Sprague-Dawley rats were exposed
to different dietary levels of copper (adequate copper, 6ppm;
marginal copper, 1.5ppm; supplemental copper, 20ppm) with
and without high fructose (30% fructose, w/v, in the drinking
water was given ad lib) intake for 4 weeks. Fecal metabolites
of each rat were then analyzed by comprehensive two-dimensional
gas chromatography time-of-flight mass spectrometry
(GC×GC-TOF MS). In parallel, liver tissues were assessed by
histology and triglyceride assay. Results: Our data showed
that high fructose feeding led to obvious hepatic steatosis in
both marginal copper deficient rats and copper supplementation
rats. Among the 38 metabolites detected with significant
abundance alteration between groups, short chain fatty acids
(SCFAs) were markedly decreased with excessive fructose
intake, irrespective of copper levels. C15:0 and C17:0 long
chain fatty acids (LCFAs), produced only by bacteria, were
increased by either high copper level or high fructose intake.
In addition, increased fecal urea and malic acid paralleled
the increased hepatic fat accumulation. Conclusion: GC×GC-
TOF MS analysis of rat fecal samples revealed distinct fecal
metabolome profiles associated with the dietary high fructose
and copper level, with some metabolites possibly serving as
potential noninvasive biomarkers of fructose induced-NAFLD.
Disclosures:
Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco,
Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline;
Speaking and Teaching: Roche
The following authors have nothing to disclose: Ming Song, Xiaoli Wei, Xinmin
Yin, Dale Schuschke, Imhoi Koo, Xiang Zhang
927
Liver sinusoid endothelial cell derived bone morphogenetic
protein binding endothelial regulator (BMPER)
induces iron overload of high-fat diet induced non-alcoholic
fatty liver mice
Takumu Hasebe 1 , Koji Sawada 1 , Shunsuke Nakajima 1 , Hiroki
Tanaka 2 , Takaaki Ohtake 3 , Mikihiro Fujiya 1 , Yutaka Kohgo 3 ;
1 Medicine, Asahikawa Medical University, Asahikawa, Japan;
2 Gastrointestinal Immunology and Regenerative Medicine, Asahikawa
Medical University, Asahikawa, Japan; 3 Gastroenterology,
International University of Health and Welfare Hospital,
Otawara, Japan
[Background] Excessive irons frequently coexist with non-alcoholic
fatty liver (NAFL), which induces hepatic inflammation
and fibrosis. Down regulation of iron regulatory protein
hepcidin and its inducer bone morphogenetic protein (BMP)
signals can be the central cause of iron overload, however
the mechanism in NAFL is still controversial. To investigate
the mechanism of iron overload in NAFL, we performed transcriptome
analysis using high throughput sequencer. [Methods]
Male C57BL/6 mice were fed on regular or high-fat diet for 16
weeks. Internal iron was evaluated by plasma iron, ferritin or
hepatic iron content. Whole RNA sequencing was performed
as transcriptome analysis using Ion Proton (Life Technologies).
Altered expressions of genes comparing between regular and
high-fat diet mice were as follows; fold change > 1.5, p value
< 0.05 (Student’s t test). Altered gene expressions in mice liver
were confirmed by RT-PCR. Plasma hepcidin concentration was
measured by LC-MS/MS. Localization of protein expression
in mice liver was analyzed by immunofluorescence. Hepatocytes
and liver sinusoid endothelial cells were isolated from
regular mice by collagenase perfusion to assess expressions
of iron regulating molecule by RT-PCR. [Results] High-fat diet
mice showed significant obesity and fatty liver, however neither
hepatic inflammation nor fibrosis. Plasma iron and ferritin were
increased in high-fat diet mice, whereas hepatic iron content