Translation regulation of apolipoprotein BmRNA

Apolipoprotein B-100 (apoB) is a protein synthesized by the liver which facilitates the transport of VLDL and LDL in plasma. The link between hepatic insulin signaling and apolipoprotein B (apoB) production has important implications in understanding the etiology of metabolic dyslipidemia commonly observed in insulin resistant states.;The role of PKC, mTOR, and PI-3K signaling molecules was investigated using signaling drugs. Treatment of HepG2 cells with the PKC activator, phorbol 12-myristate 13-acetate (PMA), increased luciferase expression of apoB 5' UTR constructs, and also demonstrated increased newly synthesized apoB-100 protein. These effects were confirmed to be translational in nature based on in vitro translation studies of T7 apoB UTR-luciferase constructs transcribed and translated in vitro in the presence of HepG2 cytosol treated with insulin or signaling modulators. Mobility shift experiments using cytosol treated with either PKC inhibitor (Bis-I) or activator (PMA) showed parallel changes between translation of apoB 5' UTR-luciferase constructs and the binding of a protein doublet migrating around 110 kDa to the apoB 5' UTR.;Using dual luciferase constructs, internal ribosomal entry (IRES) of the 5' UTR of apoB mRNA was examined. The 5' UTR demonstrated 2-fold higher IRES expression relative to an empty construct, and 4-fold less than the positive HRV viral control IRES. Various signaling activators and inhibitors did not have any significant effects on IRES expression. Using several affinity and gel extraction methods, preliminary purification and identification of the trans-acting factors which bind to the 5' UTR using LC-MS analysis of eluted affinity fractions identified several potential 110-kDa RNA-binding proteins.;Deletion constructs of the UTR regions of apoB and immunoblotting revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB 15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using deletion constructs, the 1-96 construct retained the highest level of luciferase expression of all the deletion constructs and localized the insulin-responsive between nucleotides 1-64.