“
“The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach Selleckchem Paclitaxel to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific

peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated R788 clinical trial well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.”
“Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses

cellular glucose uptake by downregulation of cell surface expression of glucose transporter 2 (GLUT2) (D. Kasai et al., J. Hepatol. 50:883-894, 2009). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected

cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of the GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated selleck chemicals when the hepatocyte nuclear factor 1 alpha (HNF-1 alpha)-binding motif was deleted from the GLUT2 promoter. HNF-1 alpha mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased HNF-1 alpha protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1 alpha protein levels. Treatment of HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1 alpha protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1 alpha protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1 alpha protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the region from amino acids 1 to 126 of the NS5A domain I physically interacts with HNF-1 alpha protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via downregulation of HNF-1 alpha expression at transcriptional and posttranslational levels.