studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 541A
plasma concentration of glutamine accompany cirrhosis. Here
we show that hyperammonemia activates a specific molecular
program in the skeletal muscle increasing the cellular demand
for leucine that is transported into the mitochondria to potentially
serve as a metabolic substrate for glutamine synthesis.
Methods. Studies were performed in the skeletal muscle from
patients with cirrhosis, hyperammonemic portacaval anastomosis
rat, differentiated murine C2C12 myotubes during hyperammonemia
appropriate controls. Myotubes were exposed
to either ammonium acetate (10mM) or leucine (5mM) or a
combination of leucine and ammonium acetate. Expression of
mTOR, its target signaling proteins, global repressor of protein
synthesis eIF2α and intracellular amino acid deficiency sensor,
GCN2 expression were quantified by immunoblots. System L
leucine transporter (LAT1) and glultamine transporter SLC38A2
were quantified by real time PCR. Rate of protein synthesis was
quantified using 3 H phenylalanine incorporation. Cellular and
mitochondrial leucine uptake in myotubes was quantified using
3 H leucine uptake and amino acids quantified using HPLC.
Results. Skeletal muscle from cirrhotic patients and the PCA
rat showed increased phosphorylation of eIF2α and reduced
mTOR activation compared to that from respective controls.
Murine myotubes showed activation of general control derepressed-2
(GCN2) and its target, eIF2α, a global repressor of
protein synthesis during hyperammonemia. This was accompanied
by impaired mTOR signaling and reduced protein synthesis.
Both the molecular perturbations and impaired protein
synthesis were reversed by L-leucine supplementation in the
medium. Lack of induction of GADD34 expression, an eIF2α
phosphatase during hyperammonemia resulted in prolonged
phosphorylation and activation of eIF2α. Increased expression
of leucine exchanger, LAT1 but not glutamine transporter,
SLC38A2 in both human muscle and murine myotubes were
observed during hyperammonemia. Conclusions. We demonstrate
a novel amino acid starvation response in the skeletal
muscle during hyperammonemia that responds specifically to
leucine supplementation despite increased leucine transport
and intracellular concentrations. These observations combined
with mitochondrial transport of leucine suggest that increased
demand due to cataplerosis results in increased mitochondrial
transport and activation of a specific program of translational
repression mediated by persistent activation of eIF2α and
impaired mTOR1 signaling.
Disclosures:
The following authors have nothing to disclose: Gangarao Davuluri, Dawid Krokowski,
Bo-Jhih Guan, Dharmvir Singh, Allawy Allawy, Avinash Kumar, Rafaella
Nascimento e Silva, Ashok Runkana, Samjhana Thapaliya, Chenyang Zhao,
Thomas Hamilton, Sathyamangla V. Naga Prasad, Maria Hatzoglou, Srinivasan
Dasarathy
669
Orchestration of BH3-only proteins Bim, Bid and Puma
controls hepatocyte apoptosis in Bcl-xL/Mcl-1 knockout
mice
Yoshinobu Saito, Hayato Hikita, Takahiro Kodama, Yuto Shiode,
Yasutoshi Nozaki, Yugo Kai, Yuki Makino, Tasuku Nakabori,
Satoshi Tanaka, Satoshi Aono, Ryotaro Sakamori, Naoki Hiramatsu,
Tomohide Tatsumi, Tetsuo Takehara; Gastroenterology
and Hepatology, Osaka University Graduate School of Medicine,
Suita, Japan
Background and Aim: Liver humanized mouse models using
Alb-uPA SCID mice and Alb-HSVtk NOG mice have been
reported to provide a lot of information on human liver physiology
and pathology. However, these mouse models are not
appropriate for investigating chronic inflammation and liver
cancer because these mice are immune-deficient and could not
survive more than one year. Immune tolerance is considered
to be established during embryonic or neonatal stage. In this
study, we investigated the engraftment efficacy of allogeneic or
xenogeneic hepatocytes into the fetal liver by in utero transplantation
in immune-competent mice. Method: As immune-competent
recipient mice, we used hepatocyte-specific Mcl-1 knockout
(KO) mice, which cause spontaneous hepatocyte apoptosis
in life (Liver Injury mice), and hepatocyte-specific Mcl-1 and
Bcl-xL double KO (DKO) mice, which show a decreased number
of hepatocytes on embryonic (E) 18.5 day and die within
one day after birth due to hepatic failure (Liver Impairment
mice). Primary hepatocytes isolated from ubiquitously green fluorescent
protein (GFP) transgenic (Tg) mice or primary human
hepatocytes were administered into these recipient mice on E
16.5 day via vitelline vein. These embryos were given birth by
cesarean section and these livers were investigated at birth or
at the age of 6 weeks. Results: Hepatocyte-transplanted wildtype
mice and Liver Injury mice showed scattered embolization
areas at birth by HE staining of the liver sections. The embolization
was considered to be caused by transplanted hepatocytes.
In contrast, Liver Impairment mouse livers escaped from
embolization. In livers of wild-type mice and Liver Injury mice
transplanted with GFP Tg hepatocytes, GFP-positive cells were
observed forming a lot of clusters. While repopulation rate was
not different between livers of wild-type mice and those of Liver
Injury mice at birth (35.8% (3.6-48.3) and 29.8% (20.4-39.1),
respectively), that of Liver Impairment was 7.8% (3.3-16.7).
Meanwhile, repopulation rate at the age of 6 weeks was 0% in
wild-type mice and 4.2% (2.1-8.0) in Liver Injury mice. In livers
of wild-type and Liver Injury mice transplanted with primary
human hepatocytes, embolization was similarly observed at
birth by HE staining, suggesting that transplanted hepatocytes
might be engrafted. Conclusion: Our results suggests that transplantation
of primary hepatocytes into the fetal liver of Liver
Injury mice, but not Liver Impairment mice, have the possibility
of generating humanized liver mice without immunodeficiency.
Disclosures:
Hayato Hikita - Grant/Research Support: Bristol-Myers Squibb
Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD
K.K., Bristol-Meyer Squibb, Mitsubishi Tanabe Pharma Corparation, Toray Industories
Inc. ; Speaking and Teaching: MSD K.K., Bristol-Meyer Squibb, Janssen
Pharmaceutical Companies
The following authors have nothing to disclose: Yoshinobu Saito, Takahiro
Kodama, Yuto Shiode, Yasutoshi Nozaki, Yugo Kai, Yuki Makino, Tasuku Nakabori,
Satoshi Tanaka, Satoshi Aono, Ryotaro Sakamori, Naoki Hiramatsu,
Tomohide Tatsumi
670
Sustained Inhibition of Liver Regeneration in Mice following
Combined Elimination of MET & EGFR Signaling
Shirish Paranjpe, William C. Bowen, Meagan Haynes, Anne Orr,
Wendy M. Mars, Jianhua Luo, George K. Michalopoulos; Pathology,
University of Pittsburgh, Pittsburgh, PA
Acute loss of liver mass following toxic or surgical means elicits
hepatic regeneration and restoration of liver tissue to its original
mass, indicating a tightly regulated growth process. PHx
in mice and rats results in rapid induction of more than 100
genes, (that are not expressed in resting liver) & activation
of multiple signaling pathways. EGFR & MET, the two major
mitogenic receptor tyrosine kinases, are activated within 15 -
30 minutes following a PHx. The MET-EGFR signaling pathway
plays a significant role in is activated within 15-30 minute
following a partial hepatectomy (PHx) in mice and rats. The
role played by these two pathways during liver regeneration
was investigated by utilizing mice that had c-met deleted in the
liver using a tamoxiphen inducible Cre-loxP system. Mice were