Su1604 Lifetime Alcohol Intake and the Patterns of Alcohol Consumption in Patients With Alcoholic Liver Disease

Abstract

G protein receptor coupled increases in cAMP level can protect hepatocytes and cholangiocytes from damage due to various apoptotic insults. Mounting evidence suggests that the cytoprotective effect of cAMP signaling is controlled by protein kinase A (PKA) independent activation of novel cAMP-guanine exchange factor's (cAMP-GEF, also known as exchange proteins activated by cAMP, EPAC). Since G protein coupled signaling has also been shown to attenuate hepatocyte injury associated with intracellular lipid accumulation, the AIM of this study was to determine the effect of cAMP-GEF signaling on hepatocyte lipotoxicity. METHODS: Rat hepatocytes, isolated by standard collagenase perfusion, were treated with 200 uM palmitate to stimulate apoptosis. Some cultures were pre-treated for 1 hr with one of 2 cAMP analogues: chlorophenyl thio-cAMP (CPT-cAMP, 100 uM) which activates both PKA and cAMP-GEF's or the specific cAMP-GEF activator, 4-(4-chloro-phenylthio)-2'-Omethyladenosine-3'-5'-cyclic monophosphate (2-Me-CPT-cAMP, 20 uM)). Apoptosis was monitored morphologically by staining with Hoescht and biochemically by immunblotting for mitochondrial release of cytochrome C and cleavage of caspase 3. As previous studies have shown that the pro-apoptotic Bcl-2 protein, BIM, is necessary for saturated fatty acid toxicity in hepatocytes, the effect of the cAMP analogues on the expression of BIM was determined by immunblotting. RESULTS: There was a significant increase in apoptosis (2.51 +/0.38 fold increase over control, p<0.05) in palmitate treated hepatocytes. In addition, immunoblotting revealed an increase in the amount of the cleavage fragment of caspase 3 and the appearance of cytochrome C in the cytoplasm in palmitate treated cells. Pretreatment with CPT-cAMP or 2-Me-CPT-cAMP reduced the morphologic evidence of palmitate induced apoptosis by 57+/-10% and 57+/8.6% (p<0.05), respectively, and attenuated biochemical evidence of apoptosis. Similar effects of CPT-cAMP and 2-Me-CPT-cAMP suggest that the effect of cAMP is mediated via cAMP-GEF pathway. Palmitate treatment increased BIM expression in whole cell lyastes by 60% +/10%. Both cAMP analogues decreased basal BIM expression by 60% to 80% and ameliorated the palmitate induced increase in BIM expression. In CONCLUSION, these results suggest that cAMP protects hepatocytes from fatty acid-induced apoptosis by inhibiting BIM expression, and this effect of cAMP is mediated via cAMP-GEF pathway