ABSTRACT
The hepatic secretion of apolipoprotein B [apoB], containing lipoproteins, is known to be regulated by insulin, and the overproduction of these atherogenic lipoproteins occurs in insulin-resistant states. Protein tyrosine phosphatase 1B [PTP-1B] is a key regulator of hepatic insulin signaling and is also upregulated in insulin resistance. We aimed to investigate the role of PTP-1B in regulating apoB mRNA translational efficiency mediated by 5'/3' untranslated regions [UTRs] under conditions of insulin stimulation. Human hepatoma HepG2 cells were transfected with a vector carrying the firefly luciferase reporter gene and either a chimeric apoB mRNA encoding the 5'/3' untranslated region [5' LUC3' -pGL3] or a null sequence of length equivalent to apoB 5' UTR [LUC-pGL3]. The transfected cells were then infected with adenovirus carrying the mouse PTP-1B gene [AdPTP1B] in the absence or presence of insulin, and the cellular luciferase activity was determined. The RNA extracts from cells were transfected with constructs carrying 5'/3' apoB UTR, or a null sequence was also translated in vitro in a rabbit reticulocyte translation system. The luciferase activity of the cells transfected with constructs containing the apoB UTR sequences [5' LUC3'] was significantly higher than that of the control constructs carrying a null sequence [p<0.01, n=12]. Similar results were observed following in vitro translation studies showing a significantly higher expression of the 5'/3' UTR constructs [p<0.001, n=6]. Treatment with 100 nM insulin led to a significant reduction in the luciferase activity of the constructs carrying apoB 5'/3' UTR [p<0.0001, n=12]. The down regulation of the apoB mRNA translation mediated by insulin was mediated by the apoB 5'/3' UTR sequences since insulin did not affect the control constructs containing a null sequence. The infection of HepG2 cells expressing 5' LUC3' or control constructs with AdPTP-1B attenuated the inhibitory effect of insulin and led to higher levels of luciferase activity compared to the Ad beta-gal infected control cells [p<0.05, n=12]. However, the activity was lower than that in the control cells infected with 5' LUC3' -pGL3 but not treated with insulin [p<0.05, n=12]. Our data suggest that PTP-1B can potentially modulate apoB synthesis by blocking insulin-mediated inhibition of the apoB mRNA translation via its 5'/3' UTR sequences. We hypothesize that the PTP-1B-mediated attenuation of the insulin action can lead to the upregulation of the apoB mRNA translation and contribute to a lipoprotein overproduction in conditions such as insulin resistance