studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 885A
1377
Branched-chain amino acids prevent hepatic fibrosis
and the development of liver tumors by suppressing
TGF-β signaling
Kai Takegoshi 1 , Masao Honda 1 , Hikari Okada 1 , Riuta Takabatake
1 , Takayoshi Shirasaki 1 , Naoto Matsuzawa 1 , Toshinari
Takamura 1 , Takuji Tanaka 2 , Shuichi Kaneko 1 ; 1 Kanazawa University,
Kanazawashi, Japan; 2 Tohkai Cytopathology Institute, Gifu,
Japan
BACKGROUND & AIMS Oral supplementation with branchedchain
amino acids (BCAAs) in patients with liver cirrhosis
potentially suppresses the incidence of hepatocellular carcinoma
and improves event-free survival. However, the detailed
mechanisms by which BCAAs act on hepatic fibrosis have not
been elucidated fully. METHODS BCAAs were administered to
atherogenic and high-fat (Ath&HF) diet-induced nonalcoholic
steatohepatitis (NASH) model mice and platelet-derived growth
factor C transgenic (Pdgf-c Tg) mice. Liver histology, tumor
incidence, and GeneChip expression profiles were evaluated.
To investigate the mechanisms of the effect of BCAAs, in vitro
studies were performed using a human stellate cell line (Lx-2),
rat liver progenitor cell-like cells (WB-F344), mouse primary
hepatic stellate cells (MHSC), and mouse primary hepatocytes
(MPH). RESULTS Mice fed the Ath&HF diet for 30 weeks developed
liver fibrosis, whereas mice fed the Ath&HF diet containing
BCAAs showed markedly reduced fibrosis. After 60 weeks,
73.5% of mice fed the Ath&HF diet developed liver tumors,
while BCAAs reduced tumor incidence to 30.8%. BCAAs also
prevented liver fibrosis of Pdgf-c Tg mice and reduced tumor
incidence. Gene expression analysis demonstrated the significant
resolution of pro-fibrotic genes, pro-oncogenic genes, cancer
stem cell markers (EpCAM and CD90), and impaired lipid
metabolism gene expression by BCAA supplementation. In
vitro, in stellate cells, BCAAs restored the transforming growth
factor (TGF)-β1-stimulated expression of pro-fibrotic genes and
inhibited the trans-differentiation of MHSC to myofibroblast-like
cells. In hepatocytes, BCAAs prevented TGF-β1 and palmitate
co-stimulated apoptosis signaling and restored impaired fatty
acid β oxidation gene expression in MPH. Moreover, in progenitor
cells, BCAAs reduced the TGF-β1-stimulated spheroid
formation of WB-F344 cells. We found BCAAs inhibited TGF-β
signaling by preventing the nuclear localization of the histone
acetylating transcription factor NF-Y. Knockdown of NF-YA
by small interfering RNA attenuated the TGF-β1-stimulated
expression of p-AKT and p-Smad2/3. CONCLUSIONS BCAA
supplementation prevents hepatic fibrosis and the development
of liver tumors in the NASH mouse model and Pdgf-c Tg mice,
possibly by suppressing TGF-β signaling through an interaction
with NF-Y. These results highlight a new mechanism of the
anti-fibrotic and anti-tumor effect of BCAAs in the liver and
could be utilized for the treatment of chronic liver disease.
Disclosures:
Hikari Okada - Employment: Kanazawa University
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Kai Takegoshi, Masao Honda,
Riuta Takabatake, Takayoshi Shirasaki, Naoto Matsuzawa, Toshinari Takamura,
Takuji Tanaka
1378
Membrane type I-matrix metalloprotease-cleaved collagens
derived from hepatic stellate cells play critical role
in liver regeneration after partial hepatectomy in rats
Akihiro Yoneda 1,2 , Miyuki Nishimura 2 , Naoko Urushihara 2,3 ,
Tomoko M. Sudo 2,3 , Kenjiro Minomi 2,3 , Yasuaki Tamura 1 , Yoshiro
Niitsu 2 ; 1 Department of Molecular Therapeutics, Center for Food &
Medical Innovation, Institute for the Promotion of Business-Regional
Collaboration, Hokkaido University, Sapporo, Japan; 2 Department
of Molecular Target Exploration, School of Medicine, Sapporo
Medical University, Sapporo, Japan; 3 Hokkaido Laboratory, Nitto
Denko Corporation, Sapporo, Japan
Background and Aims: The liver is well known to exhibit a
remarkable ability to regenerate after partial heptatectomy
(PHx). Liver regeneration is regulated by various events such as
the remodeling of extracellular matrices (ECMs) and proliferation
of hepatic cells stimulated by growth factors and cytokines.
However, the precise molecular mechanism remains controversial.
Since hepatic stellate cells (HSCs) are main producer of
ECMs and also hepatic growth factor (HGF) in the damaged
liver, HSCs has been postulated to play an important role in
liver regeneration. We have recently demonstrated that membrane
type I-matrix metalloprotease (MT1-MMP) of activated
HSCs cleaved collagens secreted from themselves to expose
RGD motif which interacted with integrins, thereby transduced
survival signal and sustained their proliferation in an autocrine
manner (Birukawa et al. J. Biol. Chem. 2014). The purpose of
this study was to explore the possibility that MT1-MMP-cleaved
collagens stimulate liver regeneration after PHx in a paracrine
manner. Methods: Proliferations of hepatic cells after 70% PHx
in rats were examined by immunohistochemistry and flow cytometric
analysis. Hepatocytes isolated from EGFP-transgenic
rats were co-cultured with activated HSCs. In vivo gene silencing
system was performed using vitamin A-coupled liposome
reported previously (Sato et al. Nature Biotechnol. 2008).
Results: Proliferation of hepatocytes was coincide with activation
of HSCs during the early phase of liver regeneration
after 70% PHx. The proliferative hepatocytes localized in the
vicinity of activated HSCs. Co-culture system demonstrated
that activated HSCs stimulated the proliferation of hepatocytes
while gene silencing of MT1-MMP or Hsp47, collagen-specific
chaperone, in activated HSCs completely inhibited their
proliferation. In vivo silencing of MT1-MMP or Hsp47 genes
also resulted in remarkable suppression of liver regeneration
after 70% PHx in rats. MT1-MMP-cleaved collagen I and HGF
induced hepatocyte proliferation via the engagement of integrin
b1 with c-Met tyrosine kinase receptor followed by the phosphorylation
of c-Met and activation of focal adhesion kinase.
We further found that proliferation of Sox9+ hepatic progenitor
cells (HPCs) in close proximity to activated HSCs was transiently
detected during the late phase of liver regeneration after
70% PHx. Co-culture system demonstrated that activated HSCs
induced the proliferation of HPCs while gene silencing of MT1-
MMP or Hsp47 in activated HSCs completely inhibited their
proliferation. Conclusion: MT1-MMP-cleaved collagens from
HSCs play a pivotal role in liver regeneration after PHx.
Disclosures:
Miyuki Nishimura - Grant/Research Support: Nitto Denko
The following authors have nothing to disclose: Akihiro Yoneda, Naoko Urushihara,
Tomoko M. Sudo, Kenjiro Minomi, Yasuaki Tamura, Yoshiro Niitsu