studies

540A AASLD ABSTRACTS HEPATOLOGY, October, 2015
steatohepatitis. PCB-induced nuclear receptor activation only
partially explained these findings, although the other ‘off target’
mechanisms involved are unknown. These observations
taken together led us to investigate the hypothesis that PCB-mediated
EGFR inhibition alters downstream targets including the
mechanistic target of rapamycin (mTOR) – a master regulator
of hepatic energy metabolism in NASH. Methods: Mice
were fed either a control diet (CD) or 42% milk fat high fat
diet (HFD) and treated with either corn oil or Aroclor1260
(PCB mixture, 20 mg/kg by one time gavage) for 12 weeks.
Livers were removed and lysed for western blot analysis of
pEGFR Y1173, EGFR, pmTOR, mTOR, and β-actin. In vitro
cell culture assays were conducted with HepG2 and AML-12
cells. Cells were treated with either EGF and EGFR inhibitor,
or EGF alone, or in combination with Aroclor 1260; proteins
were extracted for western blot analysis. Results: CD fed mice
did not develop either steatosis or hepatic necro-inflammation.
While HFD treated mice developed steatosis, only those co-exposed
to HFD and Aroclor 1260 developed hepatic inflammation
and steatohepatitis consistent with NASH. Co-exposed
mice also had significant alterations in hepatic glucose and
lipid metabolism. Analysis of liver samples showed an impact
of diet on EGFR expression in which HFD decreased EGFR
expression relative to CD. Aroclor-exposed mice demonstrated
decreased phosphorylated EGFR at Y1173 independent of
diet. Mice exposed to PCBs also had decreased phosphorylated
mTOR and decreased total mTOR expression. In HepG2
or AML-12 cells, Aroclor 1260 exposure was found to diminish
EGFR phosphorylation at Y1173 relative to the positive control.
Conclusion: These data demonstrate that HFD can lead
to decreased hepatic EGFR expression and organopollutant
exposure can act as a ‘second hit’ further decreasing EGFR
signaling and altering mTOR activity. These may be the first
data on environmental chemicals altering tyrosine kinase signaling
in NASH. These findings require further study but may
represent a new mechanism by which environmental pollution
may contribute to NASH and the impaired liver regeneration
associated with steatosis. Because mTOR inhibition is generally
anti-inflammatory, additional mechanisms of PCB-induced
hepatic inflammation should be investigated.
Disclosures:
Matthew C. Cave - Advisory Committees or Review Panels: Intercept, Abbvie;
Consulting: Abbvie, Diapharma; Grant/Research Support: Merck, Gilead, Intercept,
Conatus, Lumena, Cepheid, Tobira, Galectin, Bayer; Speaking and Teaching:
BMS, Abbvie, Gilead, Janssen, Genentech
The following authors have nothing to disclose: Josiah Hardesty, Keith C. Falkner,
Heather B. Clair, Banrida Wahlang, Russell A. Prough
667
Skeletal muscle hyperammonemia causes mitochondrial
functional abnormalities in cirrhosis
Gangarao Davuluri 2 , Allawy Allawy 2,6 , Samjhana Thapaliya 2 ,
Dharmvir Singh 2 , Avinash Kumar 2 , Julie H. Rennison 2 , Rafaella
Nascimento e Silva 2 , Cynthia Tsien 3 , David R. Van Wagoner 4 ,
Sathyamangla V. Naga Prasad 4 , Hoppel Charles 5 , Takhar Kasumov
2 , Srinivasan Dasarathy 1 ; 1 Department Of Gastroenterology
and Hepatology, Cleveland Clinic, Cleveland, OH; 2 Pathobiology,
Cleveland Clinic, Cleveland, OH; 3 Gastroenterology, Toronto
General Hospital, Toronto, ON, Canada; 4 Molecular Cardiology,
Cleveland Clinic, Cleveland, OH; 5 Pharmacology and Medicine,
Case Western Reserve University, Cleveland, OH; 6 Department Of
Internal Medicine, Cleveland Clinic, Cleveland, OH
Background. Skeletal muscle is a recognized metabolic partner
to the liver for ammonia disposal and tissue specific adaptive
responses occur during hyperammonemia. However, it is
not known if increased metabolic and cellular stress induced
by elevated ammonia uptake by the skeletal muscle results in
defects in mitochondrial bioenergetic function. Methods. Skeletal
muscle from human cirrhosis and controls, hyperammonemic
portacaval anastomosis (PCA) rat and sham operated controls
and myotubes exposed to hyperammonemia were used. Flow
cytometry, using the fluorophores DCFDA and MitoSox, was
used to measure reactive oxygen species (ROS) in C2C12
myotubes during hyperammonemia. Oxidative modification of
proteins by immublots for carbonylated proteins, lipid peroxidation
by measuring thiobarbituric acid reactive substances
(TBARS) and ATP content by fluorometric assays. The site of
electron leak that generated the ROS was determined my quantifying
H 2
O 2
production by oxidation of fluorogenic indicator
amplexred in the presence of specific inhibitors (rotenone,
malonate, antimycin A,oligomycin) of the electron transport
chain. Cellular NAD+ and NADH were quantified by a fluorometric
assay with and without inhibitors of the ETC to determine
the mechanism of impaired electron flow. Results. Hyperammonemia
increased mitochondrial ROS in C2C12 myotubes
that was reversed by MitoTEMPO, a specific mitochondrial
ROS scavenger. ATP content was lower, and carbonylated
proteins and TBARS were significantly higher in the skeletal
muscle from patients with cirrhosis and the PCA rat and in
myotubes during hyperammonemia compared to the respective
controls. Lowering cellular ammonia concentration reversed
the low ATP content observed during hyperammonemia. Using
sequential blockers of the various electron transport chain complexes,
we demonstrate that Complex III was the major source
of electron leak and ROS production production in the skeletal
muscle during hyperammonemia. Finally, NAD+/NADH ratio
was decreased with lower NAD+ and increased NADH due to
impaired Complex I of the ETC NADH oxidase during hyperammonemia.
Conclusions. Our studies in a comprehensive
array of models show that skeletal muscle ammonia disposal
activates a sequence of metabolic responses that culminate in
disordered mitochondrial function. Notably, the reduction in
ATP content is accompanied by electron leak at complex III
and consequent generation of ROS during hyperammonemia.
Reduction in ammonia can potentially reverse the mitochondrial
bioenergetics dysfunction. These data provide evidence of
a novel mechanism of hyperammonemia-induced perturbations
in skeletal muscle cellular bioenergetics.
Disclosures:
The following authors have nothing to disclose: Gangarao Davuluri, Allawy
Allawy, Samjhana Thapaliya, Dharmvir Singh, Avinash Kumar, Julie H. Rennison,
Rafaella Nascimento e Silva, Cynthia Tsien, David R. Van Wagoner,
Sathyamangla V. Naga Prasad, Hoppel Charles, Takhar Kasumov, Srinivasan
Dasarathy
668
Hyperammonemia activates a leucine responsive amino
acid starvation stress response in the skeletal muscle
Gangarao Davuluri 2 , Dawid Krokowski 3 , Bo-Jhih Guan 3 , Dharmvir
Singh 2 , Allawy Allawy 2,6 , Avinash Kumar 2 , Rafaella Nascimento e
Silva 2 , Ashok Runkana 2 , Samjhana Thapaliya 2 , Chenyang Zhao 4 ,
Thomas Hamilton 4 , Sathyamangla V. Naga Prasad 5 , Maria Hatzoglou
3 , Srinivasan Dasarathy 1 ; 1 Department Of Gastroenterology
and Hepatology, Cleveland Clinic, Cleveland, OH; 2 Pathobiology,
Cleveland Clinic, Cleveland, OH; 3 Department of Pharmacology,
Case Western Reserve University, Cleveland, OH; 4 Immunology,
Cleveland Clinic, Cleveland, OH; 5 Molecular Cardiology, Cleveland
Clinic, Cleveland, OH; 6 Department Of Internal Medicine,
Cleveland Clinic, Cleveland, OH
Backgound. Skeletal muscle ammonia concentrations are
increased in cirrhosis and results in sarcopenia. Decreased
plasma and muscle concentrations of leucine and increased