studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 653A
(KO) mice were backcrossed into ApoE -/- mice, which are susceptible
to diet-induced NASH, to generate ApoE-LCN2 double
knockout (DKO) mice and ApoE -/- /LCN2-wild type (AKO)
control mice. LCN2-chimeric mice were generated through
a combined strategy of lethal irradiation and bone marrow
transplantation. After confirmation of successful reconstitution
of bone marrow cells, chimeric mice (n= 5 - 6 per group) were
fed with high fat high cholesterol (HFHC) diet for 12 weeks to
induce NASH. Results: HFHC diet feeding resulted in a marked
elevation in hepatic expression and circulating levels of LCN2
in AKO mice. Transplantation with LCN2-null bone marrow
cells dramatically abolished HFHC diet-evoked LCN2 elevation
in both liver and blood in AKO mice, whereas reconstitution of
wild-type bone marrow cells in DKO mice restored the hepatic
expression and circulating levels of LCN2. Functionally, transplantation
of LCN2-expressing bone marrow cells from AKO
mice into DKO mice was sufficient to restore the susceptibility
of the recipient mice to HFHC-induced liver inflammation
and damage, as evidenced by deteriorated liver histology
and elevated serum activities of alanine aminotransferase and
aspartate aminotransferase. By contrast, liver lesions triggered
by HFHC diet were dramatically inhibited in chimeric mice
bearing LCN2-deficient bone marrow cells. Likewise, HFHC
diet-elicited hepatic infiltration of inflammatory cells (including
macrophages and neutrophils) and expression of pro-inflammatory
cytokines (TNF-α and IL-1β) and chemokines (MCP-1
and CXCL-2) were significantly augmented in DKO mice by
transplantation of LCN2-expressing bone marrow cells from
AKO mice, but were markedly suppressed by reconstitution of
LCN2-null bone marrow cells from DKO mice into AKO mice
(P 0.05). Conclusions: Bone marrow-derived cells are the major
contributor to elevated LCN2 in both circulation and the liver in
murine model of NASH. LCN2 chimerism in bone marrow-derived
cells is sufficient to exert significant influence on HFHC
diet-induced NASH, suggesting that bone marrow-derived
inflammatory cells and neutrophil-derived LCN2 may serve as
promising therapeutic target for NASH.
Disclosures:
The following authors have nothing to disclose: Dewei Ye, Yu Wang, Karen S.
L. Lam, Aimin Xu
899
Key role of ASMase in the susceptibility of MAT1A deficient
mice to choline deficient diet-induced steatohepatitis
Cristina Alarcón-Vila 1 , Vicent Ribas 1 , Susana Nuñez 1 , Carmen
Garcia-Ruiz 1,2 , Jose Fernandez-Checa 1,2 ; 1 Instituto Investigaciones
Biomedicas Barcelona, CSIC, Barcelona, Spain; 2 Liver Unit, Hospital
Clinic-IDIBAPS, Barcelona, Spain
Non-alcoholic steatohepatitis (NASH) is an advanced stage of
fatty liver disease characterized by steatosis, oxidative stress,
fibrosis, inflammation and hepatocellular death, which can
progress to cirrhosis. Available treatments are ineffective due
to our poor understanding of the underlined mechanisms. Disturbed
methionine metabolism (e.g. increased homocysteine
and decreased S-adenosyl-L-methionine, SAMe) is known to
contribute to NASH. The first step in methionine metabolism
is its conversion to SAMe by the enzyme methionine adenosyltransferase,
MAT1A, which is expressed mainly in adult
liver. MAT1A deletion markedly depletes hepatic SAMe levels,
induces NASH and increases liver proliferation. Acid sphingomyelinase
(ASMase)-induced ceramide generation regulates
hepatic steatosis and liver fibrogenesis. Our previous observations
in NASH indicated the association between decreased
SAMe and ASMase activation. Thus, we investigated the role
of ASMase in the NASH model of MAT1A null mice fed a
diet deficient in choline (CD). Methods: MAT1A -/- mice were
fed a CD diet for 2 weeks with or without amitriptyline (5mg/
Kg) to inhibit ASMase or after injection with an adenoviral
vector expressing ASMase shRNA (ASMSH) to silence hepatic
ASMase. Moreover, we generated double null mice deficient
in ASMase and MAT1A to examine their susceptibility to CD
feeding for 7 days. Serum and liver samples were processed
to assess ALT levels, ASMase activity, H&E, Oil Red, myeloperoxidase
staining, inflammation and RT-PCR and Western Blot
analyses. Results: MAT1A -/- mice fed CD diet and treated with
amitriptyline or injected with ASMSH exhibited 40-60% lower
mRNA levels of inflammatory markers such as TNFα, IL-1β and
IL-6 and less MPO positive cells. Liver damage monitored by
serum ALT and H&E analyses was reduced in MAT1A KO mice
after amitriptyline treatment or ASMase silencing (ASMSH).
Furthermore, markers of endoplasmic reticulum stress PDI,
CHOP, BiP due to CD diet feeding as well as overexpression of
lipogenic genes SREBP-1c, FAS, HMGCoA, and SREBP2 and
fibrosis, Col1A and TGFb, were markedly reduced (50-60%)
in MAT1A null mice after amitriptyline treatment or ASMase
silencing. These findings correlated with reduced hepatic steatosis
monitored by oil-red staining and lower triglycerides
levels. Similar results regarding steatosis, inflammation, fibrosis
and liver injury were observed in double knockout mice
MAT1A -/- /ASMase -/- fed a CD diet. Conclusions: Modulation
of ASMase activity, through pharmacological o genetic intervention,
protects MAT1A null mice for developing steatohepatitis,
highlighting the role of ASMase in the progression of
steatohepatitis
Disclosures:
The following authors have nothing to disclose: Cristina Alarcón-Vila, Vicent
Ribas, Susana Nuñez, Carmen Garcia-Ruiz, Jose Fernandez-Checa
900
Genetic Ablation of Phosphatidylcholine Transfer Protein
(PC-TP) in ob/ob Mice Improves Glucose Homeostasis
Without Increasing Energy Expenditure: Evidence for the
Direct Role for PC-TP in Hepatic Insulin Resistance
Tibor I. Krisko 1,2 , Katherine B. Leclair 1 , David E. Cohen 1 ; 1 Medicine,
Brigham and Women’s Hospital, Boston, MA; 2 Medicine, VA
Medical Center, Boston, MA
Background: Hepatic insulin resistance is a hallmark of non-alcoholic
fatty liver disease (NAFLD). PC-TP is a specific phosphatidylcholine
binding protein that is highly expressed in liver and
oxidative tissues. Genetic ablation or small molecule inhibition
of PC-TP increases both hepatic insulin sensitivity and energy
expenditure in high fat fed mice. We have demonstrated that
PC-TP suppresses insulin signaling in the liver. However, its
direct contribution to hepatic insulin resistance remains unclear
owing to the capacity of PC-TP to also reduce energy expenditure.
Aim: We utilized leptin-deficient obese (ob/ob) mice,
which exhibit both glucose intolerance and defective thermogenesis,
to dissect the direct contribution of PC-TP to the regulation
of glucose metabolism. Methods: Mice deficient in both
leptin and PC-TP were prepared by crossing Pctp -/- mice on a
C57BL/6J genetic background with ob/ob mice of the same
background. Tolerance tests to glucose (GTT) were performed
by measuring blood glucose concentrations at baseline and
following intraperitoneal administration of glucose. We measured
energy expenditure by indirect calorimetry as functions
of variations in ambient temperatures ranging from thermoneutrality
(30°C) to cold stress (15°C) using a comprehensive
laboratory animal monitoring system, which also recorded
physical activity and food intake. Body composition was deter-