The role of PKC-delta was evaluated using a PKC activator (PMA, 100 nM), PKC inhibitors KU 57788 (5 uM chelethryine, 100 uM H-7 or 0.5 uM calphostin), siRNA to PKC delta, and wild type (WT) or constitutively active (CA) PKC delta plasmid constructs. Activation of PKC
delta was monitored by immunoblotting for Thr 505 phosphorylation (HUH7Ntcp) or for total PKC delta in the mitochondria (rat hepatocytes). Phosphorylation of JNK and Akt and the amount of total BIM were determined by immunoblotting. RESULTS: GCDC treatment increased total PKC delta expression in rat hepatocyte mitochondria by 1.70 +/- 0.22 fold and induced a 5.71 +/-1.2 fold increase in the phosphorylation of PKC delta in HUH7-Ntcp cells. Within 2 hrs GCDC induced apoptosis in 16% +/- 4.5 % of rat hepatocytes and 10.5% +/- 2.3% of HUH7-Ntcp cells and resulted XL184 price in cleavage of caspase 3.Treatment of hepatocytes or HUH7-Ntcp cells with PMA decreased GCDC apoptosis by 71% +/-3.4% and 92% +/1 6.7%, respectively. In rat hepatocytes, PKC inhibitors increased GCDC induced apoptosis from 24% to 92%. Knock down of PKC delta increased GCDC apoptosis by 2.7 +/-0.98
fold, while WT- and CA-PKC-delta decreased apoptosis by 35% +/2.5% and 54% +/- 5.6%, respectively. Knock down of PKC delta increased pro-apoptotic JNK phosphorylation and total BIM levels by 2.4 and 2.3 times, respectively and decreased anti-apoptotic Akt phosphorylation by 52% +/-6.7%. GCDC apoptosis was accompanied by mitochondrial translocation of BIM and knock down
of BIM decreased GCDC induced apoptosis in HUH7-Ntcp cells by 51% +/- Exoribonuclease 3.6%. CONCLUSION: Taken together, these results suggest that activation of PKC-delta by GCDC induces a cytoprotective pathway that results in inhibition of JNK activation, activation of Akt and down-regulation of BIM. Disclosures: The following people have nothing to disclose: Cynthia R. Webster, Mohammed S. Anwer “
“Background and Aim: Gallstone formation is characterized by the abnormal regulation of cholesterol trafficking and solubilization. The prevalence of gallstone disease (GSD) differs between ethnic groups sharing the common environment. These differences can be explained by a genetic predisposition to gallstone formation. Studies have identified single nucleotide polymorphisms (SNP) D19H and T400K in the cholesterol transporter gene ATP-binding cassette, subfamily G, member 8 (ABCG8) in patients with cholesterol gallstones. The aim of this study was to analyze the relationship between D19H and T400K polymorphisms in the ABCG8 gene and GSD in an Indian population, and the effects of these polymorphisms on cholesterol levels in sera and bile. Methods: A total of 226 patients with GSD were analyzed for their lipid profile in plasma and bile.