POSTERS

P286

EFFECTS OF LIVER-SPECIFIC NO-DONOR, V-PYRRO/NO ON LIVER

STEATOSIS IN MICE FED HIGH FAT DIET

E. Maslak1, A. Jasztal1, K. Kochan1,2, B. Sitek1, P. Zabielski3,

A. Chabowski3, K. Kus1, M. Walczak1,4, B. Proniewski1,

M. Baranska1,2, L. Keefer5, S. Chłopicki1,6. 1Jagiellonian Centre for

Experimental Therapeutics (JCET), 2Faculty of Chemistry, Jagiellonian

University, Krakow, 3Department of Physiology, Medical University of

Bialystok, Białystok, 4Department of Pharmacokinetics and Physical

Pharmacy, Jagiellonian University, Krakow, Poland; 5Chemistry

Section, Laboratory of Comparative Carcinogenesis, National Cancer

Institute at Frederick, Frederick, MD, United States; 6Department of

Experimental Pharmacology, Jagiellonian University, Krakow, Poland

E-mail: edyta.maslak@jcet.eu

Background and Aims: Nitric oxide is hypotensive and thus NO-

based therapy of NAFLD that assumes precise targeting of NO to

the liver, without systemic effects, is appealing. The aim of this

study was to assess the effects of novel liver-selective NO donor,

V-PYRRO/NO in comparison with metformin on hepatic steatosis in

mice.

Methods: Six-week old C57BL/6J male mice were fed for 15 weeks

the control or high fat diet (60 kcal% fat) and additionally treated for

the last 5 weeks of experiment with V-PYRRO/NO and metformin.

Results: V-PYRRO/NO and metformin significantly blunted mice

body weight increase and inhibited liver steatosis development

induced by high fat diet. Reduction of fat content in liver

after V-PYRRO/NO and metformin determined by histopathological

staining were confirmed by GC/MS method demonstrating reduced

liver contents of triacylglycerol, diacylglycerol and ceramide.

Moreover, V-PYRRO/NO and metformin decreased SFA and

increased UFA in liver lipid droplets when compared to HFD

group. Surprisingly, V-PYRRO/NO substantially improved glucose

tolerance in mice fed high fat diet while the effect of metformin

was modest. In addition, V-PYRRO/NO increased plasma nitrite

and NOHb concentrations and liver nitrite concentration in treated

mice.

Conclusions: V-PYRRO/NO acts as a liver-specific NO donor

prodrug affording anti-steatotic effects and may represent more

efficient novel approach to prevent liver steatosis with subsequent

development of insulin resistance then metformin.

Acknowledgments: This study was supported by European Union

from the resources of the European Regional Development Fund

under the Innovative Economy Programme (grant coordinated by

JCET-UJ, No POIG.01.01.02–00–069/09).

P287

HEPATIC C6-CERAMIDE NANOLIPOSOMAL UPTAKE AFFECTS MCD-

INDUCED NASH IN VIVO VIA ALTERATIONS IN PROLIFERATIVE,

FIBROTIC AND OXIDATIVE STRESS SIGNALING PATHWAYS

F. Zanieri1, S. Omenetti1, D. Cavallaro1, S. Galastri1, S. Madiai1,

T.V. Luong2, S.S.S. Velandy3, T. Fox3, M. Kester3, K. Rombouts4,

M. Pinzani4. 1Department of Experimental and Clinical Medicine

and Center of Excellence for the Study at Molecular and Clinical

Level of Chronic, Degenerative and Neoplastic Diseases to Develop

Novel Therapies, DENOthe, University of Florence, Florence, Italy;2Cellular Pathology, Royal Free London NHS Foundation Trust and

University College London, London, United Kingdom; 3Department of

Pharmacology, Penn State University College of Medicine, Hershey, PA,

United States; 4University College London (UCL), Division of Medicine,

Institute for Liver & Digestive Health, London, United Kingdom

E-mail: francescazanieri@yahoo.it

Background and Aims: Ceramides are members of the sphingolipid

family that comprise cell membranes and exert biological effects

by interacting with several pathways involved in insulin resistance,

oxidative stress, inflammation and apoptosis. The role of ceramides

in the pathogenesis of nonalcoholic steatohepatitis (NASH) is still

evolving. In this study the effect of exogenous liposomes containing

C6-Ceramide (Lip-C6) was evaluated in a NASH model.

Methods: NASH was induced by feeding mice for 9 weeks a

methionine-and choline-deficient (MCD) diet, or control diet (CD),

followed by a single tail-vein injection of Lip-C6. The effect of Lip-

C6-treatment was investigated by measuring ALT/AST, histology,

Q-PCR and protein analysis. Possible changes in hepatic ceramide

magnitude/species specificity and sphingosines were measured by

employing MS-based lipidomics.

Results: MCD-Lip-C6 treatment did not exacerbate MCD-induced

NASH when analyzing ALT/AST, steatosis, lobular inflammation,

ballooning, apoptosis and fibrosis. Protein analysis showed that

Lip-C6-treatment affects the antioxidant system KEAP1-Nrf2-

NQO1 in MCD-fed mice. MCD-fed mice showed a reduction in

p-JNK, caspase-3 and cleaved PARP when compared to CD-fed

mice which were not affected by Lip-C6-treatment. A strong

phosphorylation of AMPK was induced in Lip-C6-treated MCD-fed

mice. Of particular note, Lip-C6-treatment restored homeostatic

levels of phospholipids/sphingolipids, increasing specific species

of phosphatidylcholines and phosphatidylethanolamines and

reducing profibrotic, proinflammatory sphingolipid metabolites in

MCD-fed mice.

Conclusions: These results demonstrate that a single injection of

C6-ceramide nanoliposomes affects different signaling pathways in

NASH, possibly by restoring changes in membrane lipid content

induced by NASH.

P288

PIOGLITAZONE AMELIORATES NUTRITIONAL FIBROTIC

STEATOHEPATITIS THROUGH THE TOLL-LIKE

RECEPTOR 4-DEPENDENT SIGNALING PATHWAY IN MICE

Y. Nan, J. Du, R. Wang, S. Zhao, X. Niu. Hebei Medical University,

Shijiazhuang, China

E-mail: nanyuemin@163.com

Background and Aims: PPARg specific agonist pioglitazone is an

anti-inflammatory agent in non-alcoholic steatohepatitis (NASH).

This study aimed to investigate the effects of pioglitazone on toll-

like receptor 4 (TLR4)-dependent signaling pathway in MCD diet

induced nutritional fibrotic steatohepatitis mice.

Methods: Male C57BL/6J mice were fed with MCD diet for 8 weeks

to develop hepatic fibrosing steatohepatitis and were administered

by PPARg agonist pioglitazone or antagonist GW9662, respectively.

Liver injury was estimated by serum ALT, AST levels and histological

examination. The hepatic mRNA and protein expressions of PPARg,TLR4, IKK-b, NF-úB, JNK1, AP-1 as well as inflammatory cytokines

and pro-fibrotic genes were analyzed by real-time PCR and western

blot.

Results: The MCD diet resulted in steatosis, inflammatory and

fibrosis in mice liver along with increase of ALT and AST. Compared

with the controls, the mRNA and protein levels of PPARg were

down-regulated in MCD diet mice (P =0.05), accompanied with

higher levels of TLR4, MYD88, IKK-b, NF-úB, JNK1, AP-1 and

up-regulated inflammatory cytokines. Pioglitazone administration

significantly attenuated the severity of liver injury, depressed the

expressions of TLR4 signaling pathway related genes, lowered

expression of pro-inflammatory and pro-fibrotic cytokines. A

contrary effect was observed in mice treated with GW9662

(P < 0.05).

Conclusions: These findings revealed that the TLR4 signaling

pathway might play a pivotal role in the progression of nutritional

fibrotic steatohepatitis in mice. TLR4 may be a drug target involved

in the ameliorative effects of pioglitazone, which might exert the

anti-inflammatory and anti-fibrotic effects by modulating the TLR4-

dependent IKK-b/NF-úB and /JNK/AP-1 signaling pathway.

Journal of Hepatology 2014 vol. 60 | S67–S214 S161