POSTERS

peptidase IV-inhibitor, on hepatic inflammation and fibrosis in

models of biliary fibrosis and of NASH.

Methods: Linagliptin was tested in macrophage and myofibroblast

cultures, and administered daily by gavage at 0.5, 5, 10 and

50mg daily per kg BW to Mdr2KO mice from week 7–11 of

age, and to 8 week old C57BL/6 mice fed a methionine and

choline deficient diet (MCD) for 8 weeks. Wildtype mice fed

a supplemented diet served as controls, respectively. Hepatic

collagen was measured biochemically, serum biochemistries were

determined by an autoanalyzer. Fibrosis and inflammation related

transcript levels were quantified from livers by real-time qRT-PCR.

Liver histology was assessed by connective tissue staining and

histochemistry for inflammation and alpha-smooth muscle actin

(alpha-SMA).

Results: In Mdr2KO mice, 10 and 50mg/kg/day of Linagliptin

increased putatively anti-fibrotic MMP-9 and -13, but decreased

procollagen a1(I) and TGFb1, TIMP-1, MMP-8 transcript levels.

However, in vitro macrophage or fibroblast differentiation, and

their production of proinflammatory or profibrogenic transcripts

were unaffected. Mice fed the MCD diet showed a rapid induction

of hepatic steatosis, inflammation and a 2-fold increase in fibrosis

compared to controls, with an up to 10-fold upregulation of

procollagen a1(I), TGFb1, aSMA, MMP-3 and -9, TIMP-1, CCL3 and

TNFa mRNA expression. Linagliptin lowered serum ALT, AST, ALP

and triglycerides, but did not affect hepatic collagen accumulation

in both the Mdr2KO and the MCD model.

Conclusion: In experimental biliary fibrosis (Mdr2KO mice) and in

a surrogate NASH model (MCD) oral Linagliptin was well tolerated

and lowered parameters of (hepatocyte) inflammation. Linagliptin

demonstrated only a modest direct antifibrotic effect at a higher

dose, possibly due to an inhibitory effect on fibroblast activation

protein (FAP) rather than via induction of GLP-1.

1308

A ROLE FOR DNA REPAIR PROTEIN KINASE (DNA-PK) IN THE

PROGRESSION OF SIMPLE STEATOSIS TO STEATOHEPATITIS

(NASH)?

A. Whitehead1, G.L. Patman1, L. Cornell1, D. Televantou1,

N. Curtin1, C.P. Day2, A.D. Burt2, A.-C. Piguet3, J.F. Dufour3,

Q.M. Anstee2, H.L. Reeves1, FLIP Investigators (FP7/2007–2013)

Health-F2–2009–241762. 1Northern Institute for Cancer Research,2Institute of Cellular Medicine, Newcastle University Medical School,

Newcastle Upon Tyne, UK; 3Hepatology, Department of Clinical

Research, University of Berne, Berne, Switzerland

E-mail: h.l.reeves@ncl.ac.uk

Background: NAFLD is the commonest cause of cirrhosis in western

nations and an increasingly common cause of HCC. In advanced HCC

we have previously proposed that rapid DNA repair by DNA-PK is

key to cancer progression and treatment resistance. Recently a role

for DNA-PK in regulating hepatic fat metabolism has been proposed.

DNA-PK phosphorylates and activates USF1 – a transcriptional

regulator of fatty acid synthase (FAS) – in response to insulin. FAS

may be central to the progression of simple steatosis to NASH. Our

aim was to explore a possible role for DNA-PK in NAFLD progression

to NASH.

Methods: We studied hepatic DNA-PK mRNA expression in CD1

mice after 3 months on normal/control; high sucrose; high fat

or, high sucrose/high fat diets (n = 40), compared to a longer

term model (n = 22), in which C3H/He mice developed NASH and

pericellular fibrosis after 9 months on the ‘American lifestyle’

(ALIOS) diet, and human NAFLD liver biopsies. All tissues were

staged using the Kleiner score.

Results: Hepatic DNA-PK fell in mice fed high sucrose or fat diets for

3 months and was reduced in association with mild (S1) steatosis.

In contrast, in older, fatter ALIOS C3H/He mice, with more S2–3

steatosis and features of NASH, DNA-PK expression increased in

association with impaired glucose tolerance (IGT), measured as the

AUC of intraperitoneal glucose tolerance test (Pearson correlation

0.475; p =0.034); and body weight (0.484; p =0.026). In 28 human

NAFLD biopsies, there was no significant difference in association

with steatosis (stage 1, n = 10; stage 2, n = 7; stage 3 n=6) or fibrosis

(stage 0, n = 13; stage 1 n=7; stage 2 n=8). However, with increasing

inflammation grade 1 (n =16) and grade 2 (n =4), there were highly

significant 3.0 fold (p < 0.001) and 3.3 fold (p < 0.001) increases

in expression (no inflammation;n =8). There was no increase or

correlation with XRCC5 or XRCC6, which form the DDR DNA-PKcs

complex.

Conclusions: DNA-PK expression increases in the presence of NASH

in murine and human disease. This may be in reponse to impaired

glucose tolerance or oxidative stress. In either case, DNA-PK may

be central to up-regulating FAS and promoting NAFLD progression

to NASH.

1309

miR-451 INHIBITS FATTY ACID-INDUCED INCREASES IN NF-úBTRANSACTIVATION THROUGH AMPK IN NON-ALCOHOLIC

STEATOHEPATITIS (NASH)

S.K. Yoon, W. Hur, Y.K. Lee, S.W. Kim, S.W. Hong, S.W. Choi,

S.H. Cho, J.M. Yang, Y.S. Lee. WHO Collaborating Center of Viral

Hepatitis and Department of Internal Medicine College of Medicine,

The Catholic University of Korea, Seoul, Republic of Korea

E-mail: yoonsk@catholic.ac.kr

Background and Aims: Accumulating evidence supports that

microRNAs (miRNAs) are important gene regulators, which can

have critical roles in diverse biological cellular processes including

non-alcoholic fatty liver disease (NAFLD). In the present study, we

investigated the role of miR-451 which was identified as a target

gene for NAFLD, on the mechanism of the inflammatory cytokine

production in NAFLD.

Methods: Microarray and stem-loop RT-PCR were performed to

detect dysregulated miRNAs in mouse models of high fat diet (HFD)-

induced NAFLD. After then, we searched the direct miRNA targets

through performing pair-wise correlation coefficient analysis on

expression levels of mRNAs, and compared the results with

predicted miRNA targets from TargetScan5.1. To validate a candidate

target gene, real time RT-PCR and Western blot were performed

in steatotic HepG2 cells treated with palmitate after transfection

with control, miR-451 mimic or miR-451 antagomirs. We next

investigated whether AMP-activated protein kinase and NF-úB were

downregulated or not, Western blotting and luciferase reporter

assays were carried out in miR-451 mimic-transfected steatotic

HepG2 cells.

Results: We identified 7 new miRNAs-target gene pairs by

bioinformatics analysis and further confirmed their expression

by stem-loop RT-PCR (miR-34a, miR-1224, miR-494, miR-455,

miR-720, miR-451 and miR-19b) in murine models of HFD-

induced NAFLD. Among those genes, we found that miR-451

expression was downregulated in non-alcoholic steatohepatitis

(NASH). We also found that Cab39 is the direct target of miRNA-

451 in steatotic HepG2 cells. Mechanistically, we demonstrated

that AMPK activation through Cab39 as a direct target of

miRNA-451 inhibits NF-úB transactivation induced by fatty

acid palmitate in HepG2 cells. Consequently, overexpression of

miRNA-451 in steatotic HepG2 cells suppressed palmitate-induced

proinflammatory cytokine IL-8 expression.

Conclusions: These results demonstrated the miRNA/mRNA profiles

dysregulated in HFD-induced NAFLD mice model, and suggest that

miRNA-451 may play an important role in the pathogenesis of

NAFLD.

*This research was supported by grants of the Basic Science

Research Program through the National Research Foundation of

Korea (NRF) funded by the Ministry of Education, Science and

Technology (2011–0014620).

S528 Journal of Hepatology 2013 vol. 58 | S409–S566