Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein.
Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. 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 the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.
Sidiropoulos
Konstantinos Gus
KG
Clinical Biochemistry Division, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Pontrelli
Louisa
L
Adeli
Khosrow
K
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D020121:5' Untranslated Regions; D001055:Apolipoproteins B; D007328:Insulin; D012333:RNA, Messenger; D016601:RNA-Binding Proteins