studies

680A AASLD ABSTRACTS HEPATOLOGY, October, 2015
and a decrease in mRNA for markers of inflammation and stellate
cell activation CD-68 and α-SMA, respectively, in a DEN
model of cirrhosis. Furthermore, ND-654 markedly decreased,
in a dose dependent manner, DEN-induced protein expression
of inflammatory cytokines including MCP1, TNFα and LIF, and
fibrotic markers such as TIMP1 and FGF21 and other markers
including Serpin E1, Leptin and Resistin. Conclusions: These
data demonstrate that liver-directed inhibition of ACC can
impact multiple steps in the pathogenesis of fatty liver disease
to NASH and cirrhosis .
Disclosures:
Geraldine Harriman - Employment: Nimbus Discovery
Jeremy R. Greenwood - Employment: Schrodinger Inc.; Patent Held/Filed: Nimbus
Therapeutics; Stock Shareholder: Schrodinger Inc.
Kenneth K. Tanabe - Patent Held/Filed: EGF SNP and risk for HCC, EGFR inhibition
and HCC, gene signature for prognosis in cirrhosis
Bryan C. Fuchs - Consulting: Collagen Medical; Grant/Research Support: Nimbus
Therapeutics
Rosana Kapeller - Employment: Nimbus Therapeutics; Stock Shareholder: Aileron
Therapeutics
The following authors have nothing to disclose: Danielle K. DePeralta, Omeed
Moaven, Lan Wei, Sathesh P. Bhat, William Westlin, H. James Harwood
958
Metformin targets a phosphoSTAT3-miRNAs pathway
to inhibit lipid droplets accumulation and intracellular
inflammation in vitro and in vivo
Natalia Pediconi 1 , Silvia Di Cocco 3 , Silvia Piconese 3 , Federica
Mori 4 , Laura Belloni 2 , Abigail D. Nunn 2 , Tullio Scopigno 2 , Vincenzo
Barnaba 3 , Giovanni Blandino 4 , Sabrina Strano 4 , Massimo
Levrero 3,2 ; 1 Dept of Molecular Medicine, Sapienza University,
Rome, Italy; 2 Center for Life NanoScience (CNLS), IIT-Sapienza,
Rome, Italy; 3 Dept of Internal Medicine - DMISM, Sapienza University,
Rome, Italy; 4 Translational Oncogenomic Unit, Regina Elena
Cancer Institute, Rome, Italy
Background and aim: Accumulation of triglyceride-containing
lipid droplets (LDs) within hepatocytes in NAFLD patients is a
potentially reversible process, although sustained activation of
inflammatory signaling pathways leads to non-alcoholic steatohepatitis
(NASH) that can evolve into cirrhosis and HCC.
The prevalence of NAFLD is increased with aging, however
the molecular mechanism for aging-induced fatty liver remains
poorly understood. Here we investigated the role of a phosphoSTAT3-miRNAs
pathway in LD accumulation and the
activation of intracellular inflammatory pathways in vescicular
steatosis. Methods: DMSO-differentiated human non-transformed
HepaRG cells treated with oleic acid were used as a
cellular model of vescicolar steatosis. C57BL/6 mice treated or
not with metformin for 78 weeks were used as an in vivo model
of aging-induced fatty liver. Results: dHepaRG cells treated
with oleic acid show: a) an accumulation of intracellular lipids
with an increase of both total cellular lipid volume and lipid
droplets number, as evaluated by picosecond CARS (coherent
anti-stoke Raman spettroscopy) mycroscopy; b) the generation
of reactive oxygen species (ROS); c) deregulated lipid metabolism
and liver-specific genes, including PDK4, PLIN4, SLC2A1,
ALB and ALDOB; d) activation of an intracellular inflammatory
response, with the upregulation of NFkB and IFN-a regulated
genes and the activation of the phosphoSTAT3/IL6 pathway.
Treatment of oleate-exposed HepaRG with the S3I STAT3
inhibitor reduces bothe tryglicerides and ROS accumulation
and inhibits phosphoSTAT3 accumulation. We also found that
several STAT3/IL6 responsive miRNAs, including miR21 and
miR24, are upregulated after lipid overload, paralleling STAT3
activation. Chromatin immuno-precipitation (ChIP) experiments
showed that the oleate-dependent transcriptional deregulation
of these miRNAs correlates with phosphoSTAT3 bound to their
promoters. Treatment with S3I inhibits both oleate-dependent
miR21 and miR24 upregulation and phosphoSTAT3 binding to
their promoter. Treatment with metformin, an AMPK activator
widely used as anti-diabetic drug and known to reduce liver
steatosis in vivo, reduces phosphoSTAT3 accumulation, inhibits
miRNAs upregulation and reduces trygliceride accumulation in
fatty HepaRG. Importantly, a chronic 78 weeks treatment with
metformin prevents aging-induced steatosis in C57BL/6 mice
and reduces both liver and serum miR21 levels. Conclusions:
We show that a phosphoSTAT3-miRNAs intracellular inflammatory
pathway amenable to therapeutic targeting by metformin
is activated in response to lipid accumulation in differentiated
hepatocytes in vitro and in vivo.
Disclosures:
Massimo Levrero - Advisory Committees or Review Panels: BMS, Jansen, Gilead,
Tekmira, Galapagos, Medimmune; Speaking and Teaching: MSD, Roche
The following authors have nothing to disclose: Natalia Pediconi, Silvia Di
Cocco, Silvia Piconese, Federica Mori, Laura Belloni, Abigail D. Nunn, Tullio
Scopigno, Vincenzo Barnaba, Giovanni Blandino, Sabrina Strano
959
Exacerbation of non-alcoholic fatty liver disease
(NAFLD) in mice with myeloid cell-specific deletion of
FXR
Rachel McMahan 1 , Cara Porsche 1 , Moshe Levi 2 , Hugo R. Rosen 1 ;
1 Gastroenterology, University of Colorado, Aurora, CO; 2 Renal
Diseases & Hypertension, University of Colorado, Aurora, CO
Background: Nonalcoholic fatty liver disease (NAFLD) is estimated
to affect a third of the U.S. population. The progression
from benign steatosis to steatohepatitis in NAFLD has been
linked to increased recruitment of pro-inflammatory monocytes
and activation of liver macrophages. We have previously
shown that activation of the nuclear bile acid receptor FXR can
improve disease in murine models of NALFD and that in vitro
activation of FXR in macrophages inhibits pro-inflammatory
cytokine production. However, FXR is also expressed in other
cell types including hepatocytes, and the protective effects of
FXR may be through non-macrophage FXR signaling. Therefore,
we aimed to determine the specific role of macrophage
FXR expression in NALFD. Methods: To evaluate to role of
macrophage expression of FXR in NAFLD, we used the Cre-
Lox system to create myeloid-cell specific targeted mutants of
the FXR gene (FXR fl/fl LyzMCre). FXR fl/fl LyzMCre or littermate
controls were fed a western or control diet for 3 months and
NAFLD severity was determined by histological analysis,
hepatic gene expression and flow cytometric analysis of intrahepatic
immune cells isolated by collagenase and mechanical
digestion. Results: Loss of FXR expression in myeloid cells led to
a more severe histological phenotype with increased macrovesicular
steatosis and inflammatory foci compared to littermate
controls. In addition, western diet feeding increased hepatic
expression of pro-inflammatory genes (CCL2, TNF-alpha) and
pro-fibrotic genes (TIMP-1) in FXR fl/fl LyzMCre mice. Myeloid
cell deletion of FXR also led to increased recruitment of intrahepatic
pro-inflammatory monocytes and an increase in IL-17
producing T cells. Conclusions: Myeloid cell-specific deletion of
FXR leads to increased liver inflammation in a murine model of
NAFLD. Targeting FXR in macrophages may be important for
inhibiting hepatic inflammation and NAFLD development.
Disclosures:
Moshe Levi - Grant/Research Support: Intercept, Genzyme-Sanofi, Daiichi Sankyo,
Merck, Novartis
The following authors have nothing to disclose: Rachel McMahan, Cara Porsche,
Hugo R. Rosen