studies

644A AASLD ABSTRACTS HEPATOLOGY, October, 2015
879
Ethanol-Mediated Lipocalin 2 Induction Selectively
Impairs Hepatic Chaperone-Mediated Autophagy and
Causes Alcoholic Steatosis in Mice
Xudong Hu 1,2 , Jiayou Wang 1,3 , Alvin Jogasuria 1 , Kwangwon Lee 1 ,
Jiashin Wu 1 , Min You 1 ; 1 Pharmaceutical Sciences, Northeast Ohio
Medical University (NEOMED), Rootstown, OH; 2 Department of
Biology, Shanghai University of Traditional Chinese Medicine,
Shanghai, China; 3 Department of Anatomy, Guangzhou University
of Chinese Medicine, Guangzhou, China
Lipocalin-2 (LCN2) [also named as SIP24/24 p
3 in mouse and
neutrophil gelatinase-associated lipocalin (NGAL) in human]
has been recently implicated as a critical player in alcohol-induced
fatty liver disease (AFLD). However, the cellular and
molecular mechanisms for its role in mediating detrimental
action of ethanol in the liver are poorly understood. We investigated
the in vivo function of LCN2 in the development of AFLD
by pair-feeding wild-type (WT) and LCN2 knockout (Lcn2KO)
mice using Lieber-DeCarli ethanol-containing diets for 4 weeks.
In WT mice, chronic ethanol administration significantly inhibited
the mRNA expression of hepatic chaperone-mediated autophagy
activity with decreased heat shock protein (Hsp) a8
(Hspa8), Hsp90aa1 and hsp70 compared to pair-fed controls.
Remarkably, mRNA levels of Hspa8, Hsp90aa1 or hsp70 in
the livers of chronically ethanol-fed Lcn2KO mice were completely
restored to the pair-fed control levels. Consistently, the
mRNA levels of lysosome-associated membrane protein type
2A (LAMP-2A) and co-chaperone BAG2, two molecules also
known to participate in chaperone-mediated autophagy, were
significantly inhibited in ethanol-fed WT mice but partially
and significantly restored in the livers of ethanol-fed Lcn2KO
mice. Those results clearly demonstrate that LCN2 deficiency
selectively stabilizes and activates hepatic chaperone-mediated
autophagy in chronically ethanol-fed Lcn2KO mice. The
autophagic flux status was further estimated by quantitating the
protein levels of p62, an autophagy-selective substrate. Compared
to WT control mice, chronic ethanol feeding substantially
inhibited p62 protein levels, and LCN2 deficiency suppressed
p62 to the similar extent. Interestingly, chronic ethanol administration
to Lcn2KO mice augmented inhibition of p62 protein
expression compared to all other groups. Consistent with these
in vivo findings, the mRNAs of Hspa8, Hsp90aa1 and hsp70
were significantly reduced by treatment of recombinant LCN2
in a dose-dependent manner in mouse AML-12 hepatocytes.
Collectively, our studies demonstrate that ethanol exposure
selectively inhibits hepatic chaperone-mediated autophagy
and disrupts autophagic flux via LCN2 induction in mice. Ethanol-mediated
disruption of hepatic chaperone-mediated autophagy
and autophagic flux via LCN2 contributes, at least in
part, to the development of alcoholic fatty liver in mice.
Disclosures:
The following authors have nothing to disclose: Xudong Hu, Jiayou Wang, Alvin
Jogasuria, Kwangwon Lee, Jiashin Wu, Min You
Disclosures:
The following authors have nothing to disclose: Kyota Fukazawa, Alexander A.
Vitin, Kenneth Martay, Jorge Reyes
880
Hepatocyte-specific depletion of UBXD8 accelerates
fibrosis in a mouse non-alcoholic steatohepatitis model
Norihiro Imai 1,2 , Yoji Ishizu 1 , Teiji Kuzuya 1 , Takashi Honda 1 ,
Kazuhiko Hayashi 1 , Masatoshi Ishigami 1 , Yoshiki Hirooka 1 , Toyoshi
Fujimoto 2 , Hidemi Goto 1 ; 1 Gastroenterology and Hepatology,
Nagoya University Graduate School of Medicine, Nagoya, Japan;
2 Anatomy and Molecular Cell Biology, Nagoya University Graduate
School of Medicine, Nagoya, Japan
[Background] We previously reported that UBXD8 in lipid
droplets is a key molecule for the degradation of lipidated
ApoB in hepatocytes. More recently, we found that hepatocyte-specific
UBXD8-deficient mice (U8-LKO) fed a high-fat
diet develop periportal macrovesicular steatosis accompanied
by a decrease in VLDL secretion (Imai et al, PLOS ONE 10,
e0127114, 2015). These results demonstrated that UBXD8 is
a crucial factor to regulate VLDL secretion from the liver and
suggested that dysfunction of UBXD8 may aggravate or even
cause NAFLD/NASH. To address this possibility, we studied
U8-LKO mice fed a NASH model diet. [Method] U8-LKO mice
and their age-matched littermates (control) were fed with three
different diets: (1) normal chow diet (CLEA Rodent Diet CE-2),
(2) high-fat diet (CLEA Rodent Diet Quick Fat; 14.4% fat), and
(3) choline-deficient high-fat diet (CDHF, Oriental Yeast; 60%
fat). Blood samples were analyzed biochemically. Paraffin sections
were stained with hematoxylin and eosin or Sirius red to
evaluate morphological changes. [Results] (1) With a normal
diet, liver sections did not exhibit any morphological difference
between U8-LKO and control mice. No sign of hepatic fibrosis
or steatosis was observed. (2) When mice were fed a high-fat
diet for 30 weeks, U8-LKO mice showed periportal steatosis,
as we reported previously. Even when steatosis was prominent,
no sign of fibrosis was observed in control or U8-LKO mice.
(3) When mice were fed a CDHF diet for two weeks, serum
AST and ALT levels were greater in U8-LKO mice than in control
mice (AST, 331 IU/l vs. 227 IU/l; ALT, 334 IU/l vs. 238
IU/l). Serum bilirubin and ALP levels were markedly higher
in U8-LKO mice than in control mice (bilirubin, 96.7 μg/dl
vs. 47.5 μg/dl, p = 0.036; ALP, 840 IU/l vs. 288 IU/L, p =
0.023), although food consumption and body weight changes
were similar in both groups. Under these conditions, the liver
of U8-LKO mice had a lumpy surface, and marked periportal
and perisinusoidal fibrosis was observed histologically. Control
mice showed no fibrosis of the liver. The Sirius red-positive
area was also significantly greater in U8-LKO mice than in
control mice (4.67% vs. 0.75%, p = 0.004). [Conclusion] With
only two weeks of CDHF diet feeding, U8-LKO mice exhibited
remarkable fibrosis; this was not observed with a normal or
simple high-fat diet. These results indicate that UBXD8 functionality
can be largely compensated for in the normal dietary
condition or even with mild fat loading, but that UBXD8 plays
a crucial role when the liver is exposed to intense dietary challenge.
It would be worthwhile to determine if UBXD8 functions
similarly in the human liver.
Disclosures:
Hidemi Goto - Grant/Research Support: MSD, Roche, Bayer, Bristol-Myers, Eisai,
Ajinomoto, Otsuka, Astra, Tanabe, Takeda
The following authors have nothing to disclose: Norihiro Imai, Yoji Ishizu,
Teiji Kuzuya, Takashi Honda, Kazuhiko Hayashi, Masatoshi Ishigami, Yoshiki
Hirooka, Toyoshi Fujimoto