studies

688A AASLD ABSTRACTS HEPATOLOGY, October, 2015
975
Free fatty acid treatment reduces BMP6 signalling in
AML12 hepatocytes
Nishreen Santrampurwala 1,2 , Kim Bridle 1,2 , Janske Reiling 1,3 ,
Laurence J. Britton 1,2 , Lesley-Anne Jaskowski 1,2 , Nathan Subramaniam
1,4 , Darrell H. Crawford 1,2 ; 1 School of Medicine, The University
of Queensland, Greenslopes, QLD, Australia; 2 Gallipoli
Medical Research Foundation, Brisbane, QLD, Australia; 3 NUTRIM
School of Nutrition and Translational Research in Metabolism,
Maastricht University, Maastricht, Netherlands; 4 QIMR Berghofer
Medical Research Institute, Brisbane, QLD, Australia
Introduction: Individuals with non-alcoholic steatohepatitis
(NASH) frequently have increased hepatic iron stores, which
appear to potentiate liver injury. The underlying mechanism of
iron loading in these patients however has remained elusive.
Our group has previously shown a greater severity of injury in
Hfe -/- mice fed a high calorie diet (HCD) compared to wild type
controls. Despite iron loading, mice fed a HCD had markedly
reduced expression of hepcidin (Hamp1), the master regulator
of systemic iron homeostasis. BMP (bone morphogenetic
protein)-SMAD signalling is known to be important in hepcidin
induction; we hypothesised that BMP signalling is altered
in a fatty acid-rich environment. We developed an in vitro
model of NAFLD to examine the consequences of accumulated
fatty acids on the BMP-SMAD signalling pathway. Methods:
The mouse hepatocyte cell line (AML12) was serum-starved
for 4 hours before treatment with free fatty acids (FFA; 2mM
of oleate and palmitate in a 2 to 1 ratio), ferric ammonium
citrate (Fe, 100μM) and/or BMP6 (50ng/ml) for 12-hours.
RNA and protein were extracted for further downstream analysis.
Results: Serum-starved AML12 cells treated with BMP6
had increased expression of Hamp1, Smad7, Id1 and Atoh8
indicating an intact BMP6 signalling pathway. Cells cultured
with FFA however had a blunted response to BMP6 treatment.
This was evident by the significant decrease in expression of
Hamp1, Smad7, Id1 and Atoh8 (p ≤ 0.05) in cells treated with
FFA despite BMP6 treatment. Additionally, we investigated the
levels of phospho-SMAD1/5/8, a BMP6 signalling intermediary,
and found that while phosphorylation of SMAD1/5/8
was stimulated with BMP6 treatment the induction was significantly
blunted with FFA treatment (p ≤ 0.001). Discussion: Iron
loading in NAFLD has the potential to increase oxidative stress
and lipid peroxidation, thus contributing to progressive liver
disease. The mechanisms underlying iron loading in NAFLD
are largely unknown and reduced BMP-SMAD signalling in
response to FFA treatment represents a plausible mechanistic
link, by decreasing hepcidin expression, thus facilitating duodenal
iron absorption via ferroportin.
Disclosures:
Darrell H. Crawford - Advisory Committees or Review Panels: Roche Products
Pty Ltd, Bristol Myers Squibb, Gilead Sciences, Novartis, MSD, Abbvie, Jansen;
Consulting: Roche Products Pty Ltd; Grant/Research Support: Roche Products
Pty Ltd; Speaking and Teaching: Roche Products Pty Ltd, Bristol Myers Squibb,
Gilead Sciences, MSD
The following authors have nothing to disclose: Nishreen Santrampurwala, Kim
Bridle, Janske Reiling, Laurence J. Britton, Lesley-Anne Jaskowski, Nathan Subramaniam
976
Increased sensitivity and compromised time-dependent
adaptation to methoxamine in a rat model of severe
steatosis
Wilhelmus J. Kwanten 1 , Paul Fransen 2 , Joris G. De Man 1 , Benedicte
Y. De Winter 1 , Peter P. Michielsen 1,3 , Sven M. Francque 1,3 ,
Denise Van Der Graaff 1 , Michiel Landen 1 ; 1 Laboratory of Experimental
Medicine and Pediatrics (LEMP) - Gastroenterology &
Hepatology, University of Antwerp, Wilrijk, Belgium; 2 Laboratory
of Physiopharmacology, University of Antwerp, Wilrijk, Belgium;
3 Gastroenterology Hepatology, Antwerp University Hospital
(UZA), Edegem, Belgium
NAFLD causes important intrahepatic vascular alterations and
an increased portal blood pressure, even before the development
of inflammation or fibrosis. Intrahepatic endothelial
dysfunction and a decreased sensitivity to vasoconstriction to
the α1-adrenoceptor agonist methoxamine (Mx) were reported.
AIM To study the underlying mechanisms of the reported hyporesponsiveness
to Mx and potential time-dependent effects.
METHODS Male Wistar rats were fed a methione-choline deficient
diet (MCDD) to induce steatosis or control diet (CD) diet
for 4 weeks (n=4-5/group). Intrahepatic vascular resistance
was studied by in situ isolated liver perfusion (flow: 10-50
ml/min). Dose-response curves to Mx were constructed evaluating
intrahepatic vascular tone. Finally, intrahepatic vascular
tone was assessed as a function of time at a fixed dose
(at Emax, 10-4 M Mx) and flow (30ml/min). RESULTS Basal
intrahepatic resistance didn’t differ between steatotic and
control rats, nor was the response to increasing flow rates.
Dose-response curves to Mx showed a significantly increased
sensitivity to Mx in steatosis compared to controls (-LogEC50:
5.55 vs 5.20; p60min: 19.24>11.25mmHg). Steatotic
livers showed a similar initial contraction, while time-dependent
decrease was delayed and attenuated (5>60min:
19.03>16.88mmHg)(fig 1). Significance was achieved after
20min Mx. When expressed as percentage decrease compared
to the level after 5min Mx, significance was already
achieved after 10min. CONCLUSION Our results show a significant
and time-dependent adaptation of intrahepatic vascular
tone to Mx in control rats. This phenomenon should be taken
into account when interpreting experiments with Mx pre-constriction.
Secondly, although we didn’t observe an increased
intrahepatic vascular resistance, response to Mx was significantly
altered in steatosis, with a significant hyperresponsiviness
and impaired time-dependent adaptation.
Disclosures:
The following authors have nothing to disclose: Wilhelmus J. Kwanten, Paul
Fransen, Joris G. De Man, Benedicte Y. De Winter, Peter P. Michielsen, Sven M.
Francque, Denise Van Der Graaff, Michiel Landen