Insights into insulin-mediated regulation of CYP2E1: miR-132/-212 targeting of CYP2E1 and role of phosphatidylinositol 3-kinase, Akt (protein kinase B), mammalian target of rapamycin signaling in regulating miR-132/-212 and miR-122/-181a expression in primary cultured rat hepatocytes.
Several microRNAs (miRNAs) were selected for characterization of their response to insulin signaling based on in silico predictions of targeting CYP2E1 mRNA and previous reports implicating their role in hepatic metabolism and disease. CYP2E1 expression decreases with increasing insulin concentration and has been shown to be regulated by the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. In primary cultured rat hepatocytes, insulin at 0.1, 1.0, and 10 nM elevated miRNA-132 and -212 expression ∼2- and 1.8-fold, respectively, whereas expression of miRNA-181a and -122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to alter significantly the expression of miRNA let-7a. Mechanistic studies using inhibitors of PI3-K, Akt, and mTOR were used to examine the role of the insulin signaling pathway on miR expression and resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels, with a lesser effect observed for miR-181a. Targeting of the rat CYP2E1 3'-untranslated region (UTR) by miR-132 and -212 was demonstrated with an in vitro luciferase reporter assay. These data show that insulin, which regulates CYP2E1 through the PI3-K, Akt, mTOR signaling pathway, also regulates the expression of miRs that target the 3'-UTR of CYP 2E1 mRNA and are involved in the regulation of hepatic metabolism and disease.
Clinical Pharmacology & Toxicology, Children's Hospital of Michigan, Detroit, Michigan (U.S., N.T., T.G., A.D., R.F.N.); and Shriners Hospitals for Children International, Tampa, Florida (R.F.N.).
Upasana
U
Shukla
Nithin
N
Tumma
Theresa
T
Gratsch
Alan
A
Dombkowski
Raymond F
RF
Novak
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D007328:Insulin; D035683:MicroRNAs; D012333:RNA, Messenger; D019392:Cytochrome P-450 CYP2E1; C546845:mTOR protein, rat; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases