studies

380A AASLD ABSTRACTS HEPATOLOGY, October, 2015
of dynein in autophagosomes (p=0.002). Interestingly, the
age-dependent differential recruitment is lost under acute starvation.
A similar behavior was also found for the KifC3 motor
in lysosomes. Live cell vesicle tracking revealed differences in
intracellular motility that correlated with these findings. The
motility of autophagosomes was reduced by 16% in cells from
aged mice (p=0.03, p>0.05). In addition, a dynein inhibitor
(EHNA) inhibited autophagosome motility (-24.6%, p=0.02)
exclusively in young mice. Upon serum starvation, no differences
were found in autophagosomal motility pointing out the
recruitment of motors during nutrient deprivation. In agreement
with these results, in vitro assays showed that on average 22%
of the purified organelles moved along microtubules whereas
30% of autophagolysosomes from starved mice were motile
(p=0.04). Conclusions: Our analysis provides for first time
quantitative biochemical and imaging evidence for the impact
of aging in the association of motors to degradative compartments
under basal and nutritional stress-induced autophagy.
Disclosures:
Allan W. Wolkoff - Consulting: Synageva; Grant/Research Support: Merck
The following authors have nothing to disclose: Eloy Bejarano-Fernandez, John
W. Murray, Xintao Wang, Ana Maria Cuervo
334
Acute alcohol binge causes skeletal muscle mitochondrial
intermediate metabolite deficiency
Allawy Allawy 2,3 , Gangarao Davuluri 2 , Avinash Kumar 2 , Bin Gao 4 ,
Ming-Jiang Xu 4 , Megan R. McMullen 2 , Rebecca L. McCullough 2 ,
Srinivasan Dasarathy 1,2 ; 1 Department Of Gastroenterology and
Hepatology, Cleveland Clinic, Cleveland, OH; 2 Department Of
Pathobiology, Cleveland Clinic, Cleveland, OH; 3 Department Of
Internal medicine, Cleveland Clinic, Cleveland, OH; 4 Laboratory
of Liver Diseases. NIAAA, NIH, Bethesda, MD
Introduction Binge drinking is believed to result in more severe
tissue injury than chronic ethanol exposure. There are no data
on the comparing binge drinking and chronic ethanol exposure
on skeletal muscle mitochondrial function. We used multiple
models of in vivo and an in vitro model of skeletal muscle
ethanol exposure to determine its impact on tricarboxylic acid
cycle intermediates. Materials and Methods. Murine C2C12
myotubes were exposed to 100mM ethanol for 6 h and 24 h
and lysates used to quantify TCA cycle intermediates. In vivo
studies were performed in mice exposed to different patterns of
ethanol exposure and included chronic alcohol feeding for 25
or 56 days, ethanol feeding for 10 days followed by a single
binge, twice weekly ethanol feeding for 8 weeks, pair fed
or maltose fed controls. Gastrocnemius muscle was harvested
and TCA cycle intermediates extracted using ethyl acetate,
tertbutyldimethylsilyl derivatives generated and quantified by
gas chromatography-mass spectrometry using protocols established
in our laboratory. All experiments were performed in
3-4 mice in each group and 6 independent experiments in
myotubes. All data are expressed as mean±SD of percentage
change after normalization against the concentrations in the
appropriate controls. Results. In the chronic ethanol exposure
for either 25 or 56 days, no significant changes were observed
in the skeletal muscle concentration of citrate, αketoglutarate,
succinate, fumarate or malate. Interestingly, compared to pair
fed controls (100%), in the single binge model, concentration
of citrate was 49.7±8.9% (p