studies

856A AASLD ABSTRACTS HEPATOLOGY, October, 2015
The following authors have nothing to disclose: Zhuan Li, Jie Zhao
1312
PDE4 inhibition attenuates alcohol induced hepatic oxidative
stress by increasing antioxidant enzyme expression
Diana Avila 1 , Jingwen Zhang 1 , Craig J. McClain 1,2 , Shirish
Barve 1 , Leila Gobejishvili 1 ; 1 Department of Medicine/GI, University
of Louisville, Louisville, KY; 2 Robley Rex VAMC, Louisville, KY
Alcohol metabolism leads to generation of free radicals and
oxidative stress with a resultant formation of lipid peroxidation
products, which contribute to the development of alcoholic
liver disease (ALD). Additionally, alcohol decreases antioxidant
capacity of liver, specifically nuclear factor erythroid 2–
related factor 2 (Nrf2) dependent enzymes. cAMP increasing
agents have been shown to effectively mitigate oxidative stress
both in vivo and in vitro. Our previous work has shown that
alcohol increases the expression of PDE4, cAMP degrading
enzyme, in macrophages and hepatic Kupffer cells. Hence,
we hypothesized that compromised cAMP levels due to alcohol
might contribute to the decreased antioxidant capacity
of liver, and restoring cAMP signaling could prevent alcohol
induced oxidative stress. To test this hypothesis, we employed
two approaches to block degradation of cAMP by phosphodiesterases
(PDEs): genetic (by using PDE4B knockout mice)
and pharmacological inhibition of PDE4 (Rolipram) in a mouse
model of ALD. C57BL/6 and Pde4b knockout (pde4b -/- ) male
mice were pair-fed Lieber-DeCarli liquid diet containing either
alcohol (AF) or isocaloric maltose dextrin (PF) for 4 weeks.
Additional groups of mice were treated with Rolipram at 5
mg/kg, 3 times a week for 4 weeks. Mice were sacrificed
at 1, 2 and 4 weeks after starting alcohol. Liver cAMP levels
were measured by cAMP ELISA kit; Hepatic inflammation and
oxidative stress was examined by immunohistochemical staining
of liver tissue with 4HNE (4-Hydroxynonenal) and F4/80
(monocyte/macrophage marker) antibodies. Effect of alcohol
on activation of Nrf2 was evaluated by examining nuclear
Nrf2 levels. Further, expression of Nrf2-dependent hepatic antioxidant
enzymes, superoxide dismutase 1 and 2 (SOD1/2),
Catalase and glutathione peroxidase (GPx) was analyzed by
Western blot. Alcohol feeding resulted in a significant increase
in hepatic PDE4 expression and a decrease in cAMP levels.
Liver 4HNE and F4/80 staining was significantly increased in
alcohol fed WT mice demonstrating increased oxidative stress,
but was significantly attenuated by PDE4 inhibition. Alcohol
reduced Nrf2 activation in wild type mice but not in Rolipram
treated and PDE4B knockout mice. Catalase and SOD2 levels
did not seem to be affected by alcohol, but SOD1 and GPx1/2
were decreased in alcohol fed wild type mice. Notably, in comparison
with wild type, Pde4b knockout and Rolipram treated
alcohol fed mice had higher levels of SOD1/2, and GPx1/2.
In summary, these data indicate that alcohol effect on hepatic
cAMP levels contribute to compromised antioxidant capacity of
the liver, which could be restored by PDE4 inhibition.
Disclosures:
Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco,
Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline;
Speaking and Teaching: Roche
Shirish Barve - Speaking and Teaching: Abbott
The following authors have nothing to disclose: Diana Avila, Jingwen Zhang,
Leila Gobejishvili
1313
Hepatocyte-specific deletion of the essential circadian
clock transcription factor BMAL1 enhances chronic ethanol-induced
depletion of hepatic glycogen in mice
Uduak S. Udoh, Telisha Swain, Shannon Bailey; Pathology, University
of Alabama at Birmingham, Birmingham, AL
The molecular circadian clock plays a central role in regulating
fundamental physiological and metabolic processes and allows
organisms/organs/cells to anticipate and adapt to changes in
their 24 hr environment. Previously, we found that chronic alcohol
consumption disrupts the liver molecular clock mechanism
and daily rhythms in liver glycogen metabolism. Herein, our
goal was to determine the effect chronic alcohol consumption
has on hepatic glycogen metabolism using a model of hepatocyte
clock dysfunction; i.e., the hepatocyte-specific BMAL1
knockout (HBK) mouse. For these studies, single-housed male
HBK and wild type (WT) littermates were kept under a 12:12
hr light-dark cycle and fed either the Lieber-DeCarli control
diet or the ethanol-containing (3% w/v) diet using a pair-fed
isocaloric study design for 5 weeks. Livers were collected every
4 hr for a 24 hr period at Zeitgeber time (ZT) 3, 7, 11, 15,
19, and 23 where ZT 0 = lights on (beginning of less active
phase) and ZT 12 = lights off (beginning of more active phase),
and used for glycogen determination and gene expression.
Chronic alcohol consumption significantly decreased glycogen
content in livers of WT mice at all times of the day. The diurnal
rhythm in glycogen was also altered in livers of WT ethanol-fed
mice as compared to the rhythm measured in livers of WT control-fed
mice [Mesor/Mean: WT-Control (40.30) vs. WT-Ethanol
(24.87), p