studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 855A
also offer potential for novel biomarkers and therapeutic targets.
Disclosures:
Puneet Puri - Advisory Committees or Review Panels: Health Diagnostic Laboratory
Inc.; Consulting: NPS Pharmaceuticals Inc.
Andrew R. Joyce - Independent Contractor: Venebio Group, LLC, algorithmRx,
LLC; Management Position: Venebio Group, LLC, algorithmRx, LLC
Richard K. Sterling - Advisory Committees or Review Panels: Merck, Vertex, Salix,
Bayer, BMS, Abbott, Gilead, Baxter, Jansen; Grant/Research Support: Merck,
Roche/Genentech, Pfizer, Gilead, Boehringer Ingelheim, Bayer, BMS, Abbott
Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,
Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,
Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet
Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,
GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier
The following authors have nothing to disclose: Amon Asgharpour, Mohammad
S. Siddiqui, R. Todd Stravitz, Velimir A. Luketic, Scott C. Matherly, Kalyani Daita,
Susan A. Walker, Stephanie Taylor, Christian E. Ammons, Faridoddin Mirshahi
1310
Ethanol Exposure Inhibits Hepatocellular Lipophagy by
Inactivating the Small Regulatory GTPase Rab7
Ryan J. Schulze 1 , Shaun Weller 1 , Micah B. Schott 1 , Karuna
Rasineni 2 , Barbara Schroeder 1 , Carol A. Casey 2 , Mark A.
McNiven 1 ; 1 Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN; 2 University of Nebraska Medical Center, Omaha,
NE
Alcohol consumption is a well-established risk factor for the
onset and progression of fatty liver disease, in which an estimated
90% of heavy drinkers are thought to develop significant
liver steatosis. For these reasons, an increased understanding
of the molecular basis for alcohol-induced hepatic steatosis
is urgently required. Recently, it has become clear that autophagy,
a catabolic process of intracellular degradation and
recycling, plays a key role in hepatic lipid metabolism. We
have previously shown that the small GTPase Rab7, known
to regulate membrane trafficking and fusion events in the late
endocytic pathway, also acts as a central orchestrator of hepatocellular
lipophagy, a selective form of autophagy in which
lipid droplets (LDs) are specifically targeted for turnover by
the autophagic machinery (Schroeder et al., 2015 and article
highlight by Carmona-Gutierrez et al., 2015). Nutrient starvation
resulted in Rab7 activation on the surface of the LD as
well as on degradative compartments such as the lysosome.
This activation resulted in the mobilization of triglycerides
stored within the LDs for energy production. The GOAL of
this study was to test whether the steatotic effects of alcohol
exposure are a result of perturbations in the Rab7-mediated
lipophagic pathway. Rats fed a 6-week Lieber-DeCarli ethanol-containing
diet accumulated a significantly higher amount
of fat in their hepatocytes. Interestingly, primary hepatocytes
isolated from either these ethanol-fed rats or cultured VA-13
hepatocytes (HepG2 cells expressing alcohol dehydrogenase)
contained enlarged and juxtanuclear-localized multivesicular
bodies (MVBs) and lysosomes that exhibited impaired motility
and fewer productive interactions with LDs. Importantly, similar
morphological changes in these organelles are observed
in hepatocytes subjected to an siRNA-mediated knockdown
of Rab7. Consistent with these defects in the MVB and lysosomal
compartments, we observed a marked 80% reduction
in Rab7 activity in cultured hepatocytes as well as a complete
block in starvation-induced Rab7 activation in primary hepatocytes
isolated from chronic ethanol-fed animals. In summary,
these findings support a direct mechanism whereby acute or
chronic ethanol exposure inhibits Rab7 activity, resulting in
the impaired transport, targeting, and fusion of the autophagic
machinery with LDs, leading to an accumulation of hepatocellular
lipids and hepatic steatosis. This study was generously supported
by funding from the NIDDK [5R37DK044650 (MAM)],
the NIAAA [5R01AA020735 (MAM and CAC)], the Mayo
Clinic Center for Cell Signaling in Gastroenterology, and the
Robert and Arlene Kogod Center on Aging.
Disclosures:
The following authors have nothing to disclose: Ryan J. Schulze, Shaun Weller,
Micah B. Schott, Karuna Rasineni, Barbara Schroeder, Carol A. Casey, Mark
A. McNiven
1311
Alcohol modifies the composition of the intrahepatic
macrophage pool through FOXO3-dependent and cell
type specific apoptosis
Zhuan Li, Jie Zhao, Steven A. Weinman; Department of Internal
Medicine, University of Kansas Medical Center, Kansas City, KS
Hepatic macrophages are a heterogeneous population consisting
of both resident and infiltrating macrophages that carry
out diverse pro- and anti-inflammatory functions necessary
for homeostasis, disease progression and injury repair. The
mechanisms that regulate macrophage subsets in alcoholic
liver disease are not yet understand. We recently showed that
FOXO3 protects the liver from alcohol-induced inflammation.
It also induces S574 phosphorylation of FOXO3 which selectively
enhances expression of pro-apoptotic genes and induces
apoptosis. FOXO3-dependent apoptosis was induced in macrophages
by LPS. The AIMS of this study were to determine
whether FOXO3-dependent macrophage apoptosis protects
the liver from alcohol through changes in intrahepatic macrophage
populations. METHODS: ChIP assays assessed promoter
binding. qPCR was used to measure gene expression. Mouse
peritoneal macrophages were treated with LPS for 24 hours or
differentiated with INF-γ or IL-4 before LPS treatment. Mice were
gavaged once with alcohol or fed a Lieber-DiCarli alcohol diet
for 3 wks. RESULTS: LPS treatment of THP-1 monocytes induced
FOXO3 S574 phosphorylation, enhanced expression of TRAIL,
suppressed Bcl-2 and induced apoptosis. A similar result was
obtained with peritoneal macrophages from wild-type (wt)
mice but none of these effects occurred in FOXO3 -/- macrophages.
To determine if apoptosis was selective for macrophage
type, we separately measured LPS-induced apoptosis in
INF-γ differentiated macrophages (M1) and IL-4 differentiated
macrophages (M2). Only M1 macrophages underwent apoptosis
and this was FOXO3 dependent. Acute alcohol gavage
resulted in nearly a 50% decrease of hepatic macrophage
number by 9 hours in wt but not FOXO3 -/- mice and this loss
of macrophages was selective for cells with M1 markers.
Unlike acute gavage, chronic ethanol feeding had no effect
on hepatic macrophage number or M1/M2 ratio in wt mice
but increased the M1/M2 ratio by 50% in FOXO3 -/- mice.
In the FOXO3 -/- mice there was a strong positive correlation
between M1 pro-inframammary macrophage number and ALT
(P