studies

672A AASLD ABSTRACTS HEPATOLOGY, October, 2015
C57BL6 wild-type or RIP3-deficient (RIP3 -/- ) mice were fed highfat
choline-deficient (HFCD) or methionine and choline-deficient
(MCD) diets, with subsequent histological and biochemical
analysis of hepatic damage. In primary murine hepatocytes,
necroptosis and oxidative stress were also assessed after necrostatin-1
treatment or RIP3 silencing. We show that in chronic
liver disease patients, RIP3 levels were significantly increased
and correlated with steatohepatitis histological severity (at least
p < 0.05). Circulating markers of necrosis and TNF-α, as well
as liver MLKL phosphorylation were increased in NAFLD (at
least p < 0.05). Likewise, RIP3 and MLKL protein levels and
TNF-α expression were increased in the liver of HFCD and
MCD diet-fed mice (at least p < 0.05). Moreover, RIP3 and
MLKL sequestration in the insoluble protein fraction of NASH
mice liver lysates, strongly suggesting necroptosis activation,
represented an early event during stetatohepatitis progression
(at least p < 0.05). Functional studies in primary murine hepatocytes
established the association between TNF-α-induced RIP3
expression, activation of necroptosis and oxidative stress (p <
0.05). Strikingly, RIP3 deficiency attenuated MCD diet-induced
liver injury, steatosis, inflammation, fibrosis and oxidative stress
(at least p < 0.05). In conclusion, necroptosis is increased in
the liver of NAFLD patients and in experimental models of
NASH. Further, TNF-α triggers RIP3-dependent oxidative stress
during hepatocyte necroptosis. As such, targeting necroptosis
appears to arrest or at least impair NAFLD progression.
Disclosures:
Helena Cortez-Pinto - Advisory Committees or Review Panels: Norgine, Lundbeck;
Speaking and Teaching: Janssen, Gilead Janssen
The following authors have nothing to disclose: Marta B. Afonso, Pedro M.
Rodrigues, Tânia Carvalho, Marta Caridade, Paula Borralho Nunes, Rui E. Castro,
Cecília M. Rodrigues
941
Over-expression of HNF4α attenuated lipotoxicity in
non-alcoholic fatty liver disease model
Jai Sun Lee 1 , Dae Won Jun 2 , Seung Min Lee 3 , Yong Kyun Cho 4 ,
Eun Chul Jang 5 , Sang Bong Ahn 6 ; 1 Department translational medicine,
Hanyang University Graduate School of Biomedical Science
and Engineering, Seoul, Korea (the Republic of); 2 Department
of Internal Medicine, Hanyang University College of Medicine,
Seoul, Korea (the Republic of); 3 Department of Food and Nutrition,
Sungshin Women’s University, Seoul, Korea (the Republic
of); 4 Department of Internal Medicine, Kangbuk Samsung Hospital,
Sungkyunkwan University, School of Medicine, Seoul, Korea
(the Republic of); 5 Department of Occupational and Environmental
Medicine, Soonchunhyang University Cheonan Hospital, Cheonan,
Korea (the Republic of); 6 Department of Internal Medicine, Eulji
University School of Medicine, Seoul, Korea (the Republic of)
Background: Hepatocyte nuclear factor 4α (HNF4α) is known
as a master regulator of liver-specific gene expression. The
effects of HNF4α on non-alcohol fatty liver disease (NAFLD)
at transcriptional level are largely unknown. In this study, we
evaluated the role of HNF4α in NAFLD model. Methods: High
fat (HF) diet was fed to rats for 20 weeks to induce NAFLD. To
make vitro NAFLD model, palmatic acid (PA) treated in HepG2
cells for 24 hours. Later, liver-specific gene expressions were
evaluated. Using microporator, a transcription factor, HNF4α
was over-expressed in HepG2 cells. After transfection, MTT
assay, Tunnel assay, FACS, Nile-red staining, and qPCR gene
evaluation were performed. Results: Hepatic gene expression
of Foxa2 and GATA4 were up regulated in HF diet fed rats
compared to control group. But HNF4α mRNA expression was
decreased in diet induced fatty disease model. In HepG2 cells,
palmatic acid treatment also decreased HNF4α mRNA expression;
however, interestingly, the oleic acid treatment did not
affect the HNF4α mRNA expression. HepG2 cells over-expressing
HNF4α through transfection showed a protective effect
against palmatic acid-induced apoptosis. HNF4α over-expression
was also associated with increased fatty acid oxidation
and VLDL secretion. The HepG2 cells over-expressing HNF4α
also exhibited increased the mRNA expression of enzymes
related to VLDL secretion (MTTP and ApoB) and fatty acid oxidation
(ACOX and CYP4A1). Moreover HNF4α over-expression
was also involved in decreased fatty acid uptake. The
HepG2 cells over-expressing HNF4α also exhibited decreased
the mRNA expression of fatty acid uptake mediator (CD36
and FATP2). Conclusions: HNF4α attenuated lipotoxicity by
increased VLDL secretion and fatty acid oxidation in NASH
model.
Disclosures:
The following authors have nothing to disclose: Jai Sun Lee, Dae Won Jun, Seung
Min Lee, Yong Kyun Cho, Eun Chul Jang, Sang Bong Ahn
942
Thioesterase Superfamily Member 2 (Them2) Reduces
Membrane Fluidity and Promotes Endoplasmic Reticulum
(ER) Calcium Loss in Response to Saturated Free
Fatty Acids: Pathogenic Role in Non-Alcoholic Fatty Liver
Disease (NAFLD)
Baran A. Ersoy, Yingxia Li, David E. Cohen; Medicine, Brigham
and Women’s Hospital and Harvard Medical School, Boston, MA
Background: NAFLD is associated with maladaptive increases
in hepatic glucose production in the setting of excessive nutrition
that are due in part to insulin resistance. Saturated free
fatty acids (FFA) contribute by reducing ER membrane fluidity.
This promotes insulin resistance, which is attributable in part to
depletion of ER calcium and ER stress. In addition, increased
cytosolic calcium levels promote hepatic glucose production
by activating calcium/calmodulin-dependent protein kinase 2
(CaMKII) and amp-activated protein kinase (AMPK). Genetic
ablation of Them2, an acyl-CoA thioesterase that preferentially
converts saturated fatty acyl-CoAs to FFA, protects against
depletion of ER calcium and ER stress in cell culture systems and
reduces hepatic glucose production in high fat fed mice. Aim:
This study was designed to determine the molecular mechanism
by which Them2 expression regulates ER calcium homeostasis
and hepatic glucose production. Methods: HEK 293E cells
were exposed for 6 h to 0.5 mM palmitic acid after treatment
with Them2 or scrambled (control) siRNA. ER calcium release
and reuptake were measured using Fluo-4 following treatment
of cells with the re-uptake inhibitor thapsigargin (2 mM). Total
ER calcium stores were measured following treatment of cells
with 5 mM ionomycin, an ionophore that creates calcium-permeable
membrane pores. Membrane fluidity was measured by
polarization anisotropy of diphenylhexatriene, as well as by
formation of pyrenedecanoic acid eximers using fluorescence
spectrometry. Activation of calcium-sensitive phosphoproteins
that promote hepatic glucose production was assessed by
immunoblot analysis. Results: In the absence of palmitic acid
treatment, Them2 knockdown reduced thapsigargin-mediated
loss of ER calcium by 40%. This was not attributable to reduced
ER calcium stores because knockdown of Them2 expression
did not alter ionomycin-mediated calcium release. Palmitic acid
treatment reduced ER membrane fluidity by 58%, reduced ER
calcium uptake by 11% and increased cytosolic calcium by
2-fold. These effects were abrogated by knockdown of Them2.
Moreover, reductions in cytosolic calcium following Them2
knockdown correlated with decreases in activation of both
CaMKII and AMPK. Conclusions: Them2 mediates the effects of
saturated FFA on ER membrane fluidity and calcium depletion,