studies

646A AASLD ABSTRACTS HEPATOLOGY, October, 2015
883
Lipid-induced Endoplasmic Reticulum Stress But Not
Hepatic Steatosis Contributes to Blockage of Autophagosome-Lysosome
Fusion
Koichiro Miyagawa, Shinji Oe, Yuichi Honma, Masaru Harada;
University of Occupational and Environmental Health, Kitakyushu,
Japan
Purpose: Autophagy is crucial for intracellular quality control of
proteins and intracellular organelles in various tissues. Several
studies showed that blockage of hepatic autophagic degradation
system occurred in obese, and that had an important role
in the development of nonalcoholic fatty liver disease (NAFLD).
However, the mechanism of this blockage has not been fully
elucidated. In this study, we evaluated how lipid overload is
linked to impairment of autophagy in hepatocytes. Methods:
We investigated the effect of lipid overload on hepatic autophagy
in cultured hepatocytes-derived cells treated with monounsaturated
fatty acids (MUFAs) or saturated fatty acids (SFAs).
Autophagic flux was analyzed by immunoblotting and fluorescence
microscopy. We also evaluated the relationship between
hepatic steatosis, endoplasmic reticulum (ER) stress, and autophagy
in this model. Results: SFAs but not MUFAs induced ER
stress and accumulation of autophagosomes despite of lower
accumulation of lipid droplets compared with MUFAs. Microtubule-associated
protein 1 light chain 3 (LC3) turnover assay
demonstrated that SFAs induced the reduction of the rate of
lysosomal degradation of autophagosomes without change
in autophagosome synthesis. Moreover, we monitored autophagic
flux in cells transfected with mRFP-GFP tandem fluorescence-tagged
LC3 (tf-LC3). GFP fluorescence is quenched in
the lysosome but mRFP is not, indicating that GFP and mRFP
are co-localized before the fusion with lysosomes. SFAs but not
MUFAs induced GFP and mRFP overlapping, indicating that
SFAs induced accumulation of autophagosomes. SFAs also
suppressed co-localization of mRFP and lysosome-associated
membrane protein 1 (Lamp1), suggesting that SFAs-induced
impairment of autophagic flux was due to blockage of autophagosome-lysosome
fusion. Furthermore, we assessed lysosomal
acidification. Cells treated with SFA were labeled for
Lysotracker Red and then stained with Lamp1. We could not
identify Lysotracker-negative Lamp1 in SFAs-treated cells as
well as vehicle, suggesting that lysosomal acidification was not
impaired by SFAs treatment. We also revealed that SFAs did
not affect cathepsin activity. These results suggested that SFAs
impaired autophagosome-lysosome fusion without change in
lysosomal function. Moreover, we found that this impairment
occurred in an ER stress-dependent manner. Conclusions: The
disturbance of autophagic flux in NAFLD is due to the blockage
of autophagosome-lysosome fusion, and that involves in the
degree of ER stress but not the intracellular accumulation of
lipid droplets.
Disclosures:
The following authors have nothing to disclose: Koichiro Miyagawa, Shinji Oe,
Yuichi Honma, Masaru Harada
884
CHOP and the Unfolded Protein Response Regulate
NF-κB Activity through IRAK2 in the Pathogenesis of
Non-Alcoholic Steatohepatitis
Jeffrey A. Willy 1 , James L. Stevens 1 , Howard C. Masuoka 2 , Ronald
C. Wek 1 ; 1 Biochemistry and Molecular Biology, Indiana University
School of Medicine, Indianapolis, IN; 2 Department of Medicine,
Indiana University School of Medicine, Indianapolis, IN
Free fatty acid (FFA) induction of cell death is an important feature
of NASH and has been associated with disruption of the
endoplasmic reticulum and activation of the Unfolded Protein
Response (UPR). The mechanisms by which this stress pathway
results in deleterious effects at the organ and cellular levels
are not well understood. In this study, we examined the role of
the UPR in NASH. The UPR features transcriptional and translational
control of gene expression involved in stress remediation,
including the transcription factor CHOP, which can trigger
death processes during chronic endoplasmic reticulum stress.
Primary hepatocytes and HepG2 cells were exposed to physiologic
levels of either saturated or unsaturated FFA. We found
that the saturated FFA palmitate, but not the unsaturated FFA
oleate, induced hepatocyte cell death as a result of inhibition
of autophagic flux in a CHOP-dependent manner. Additionally,
saturated FFA exposure resulted in activation of NF-κB through
expression of IRAK2, resulting in secretion of many cytokines,
including TNFα and IL-8, which contribute to both hepatic cell
death and inflammation. Depletion of CHOP or the RelA subunit
of NF-κB in hepatocytes by shRNA alleviated both autophagy
and cytokine secretion resulting in enhanced cell viability
and lowered inflammatory responses during exposure to saturated
FFA. We next investigated the relevance of these findings
in human liver disease. We showed that the CHOP-IRAK2-
NF-κB pathway is upregulated in liver biopsies from patients
with NASH, in combination with inhibition of autophagic flux,
as measured by accumulation of LC3b and P62. Secretion of
CHOP-dependent cytokines such as TNFα and IL-8 are also
increased in the sera of NASH patients. Interestingly, while
UPR activation was conserved in primary rodent hepatocytes,
there was no induction of IRAK2 and NF-κB in primary rodent
hepatocytes following saturated FFA exposure, providing a
potential explanation for the shortcomings in regards to inflammation
and fibrosis in current rodent models of NASH relative
to the human disease. In conclusion, we have identified a novel
pathway that is upregulated in NASH patients that plays an
active role in both the hepatotoxicity and inflammation at the
level of the hepatocyte following excess saturated FFA. A better
understanding of the mechanism by which the UPR contributes
to hepatocyte toxicity during FFA exposure may provide new
approaches for the diagnosis and treatment of liver disease.
Disclosures:
The following authors have nothing to disclose: Jeffrey A. Willy, James L. Stevens,
Howard C. Masuoka, Ronald C. Wek
885
Deletion of G-protein-coupled bile acid receptor (Gpbar-
1, Tgr5) alleviates fasting induced hepatic steatosis by
altering hepatic lipid metabolism
Ajay C. Donepudi, Shannon M. Boehme, John Chiang; Northeast
Ohio Medical University, Rootstown, OH
Introduction: Bile acids and its receptors such as farnesoid X
receptor (Fxr) and G-protein-coupled bile acid receptor (Gpbar-
1, Tgr5) play a major role in nutrient homeostasis. Tgr5 has
been shown to regulate glucose homeostasis by regulating insulin
secretion. Tgr5 is also involved in regulating basal metabolic