Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation.
Although insulin normally activates global mRNA translation, it has a specific inhibitory effect on translation of apolipoprotein B (apoB) mRNA. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite global translational stimulation. Recent studies have revealed that insulin functions to regulate apoB mRNA translation through a mechanism involving the apoB mRNA 5' untranslated region (5' UTR). Here, we further investigate the role of downstream insulin signaling molecules on apoB mRNA translation, and the mechanism of apoB mRNA translation itself. Transfection studies in HepG2 cells expressing deletion constructs of the apoB 5' UTR showed that the cis-acting region responding to insulin was localized within the first 64 nucleotides. Experiments using chimeric apoB UTR-luciferase constructs transfected into HepG2 cells followed by treatment with wortmannin, a PI-3K inhibitor, and rapamycin, an mTOR inhibitor, showed that signaling via PI-3K and mTOR pathways is necessary for insulin-mediated inhibition of chimeric 5' UTR-luciferase expression. In vitro translation of chimeric cRNA confirmed that the effects observed were translational in nature. Furthermore, using RNA-EMSA we found that wortmannin pretreatment blocked insulin-mediated inhibition of the binding of RNA-binding factor(s), migrating near the 110 kDa marker, to the 5' UTR. Radiolabeling studies in HepG2 cells also showed that insulin-mediated control of the synthesis of endogenously expressed full length apoB100 is mediated via the PI-3K and mTOR pathways. Finally, using dual-cistronic luciferase constructs we demonstrate that apoB 5' UTR may have weak internal ribosomal entry (IRES) translation which is not affected by insulin stimulation, and may function to stimulate basal levels of apoB mRNA translation.
Sidiropoulos
Konstantinos Gus
KG
Molecular Structure and Function, Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8.
Meshkani
Reza
R
Avramoglu-Kohen
Rita
R
Adeli
Khosrow
K
D001055:Apolipoproteins B / Q000378:metabolism*; D002460:Cell Line; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D058570:TOR Serine-Threonine Kinases
D001055:Apolipoproteins B; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D012269:Ribosomal Proteins; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human