Diabetes Mellitus (DM) increases the risk of developing chronic non-alcoholic liver disease, non-alcoholic steatohepatitis, and hepatocellular carcinoma. DM elevates CYP2E1, which is critically important in human health, because it increases oxidative stress, catalyzes the metabolism of carcinogens, therapeutic agents, eicosanoids, and fatty acids, reflects an altered phenotype, and is a biomarker of progressive hepatic disease development with loss of CYP2E1 expression reflecting an aggressive hepatic tumor phenotype. The mechanism(s) by which DM regulates CYP2E1 expression are unknown. Our research shows that increasing insulin decreases CYP2E1 gene transcription by ~90%, activates Akt, and increases FOXO1, 3a and 4 transcription factor phosphorylation and inactivation. Inhibition of Akt decreases FOXO1, 3a and 4 phosphorylation and reverses the insulin-mediated downregulation of CYP2E1 mRNA expression. At the translational level, insulin decreases CYP2E1 mRNA half-life by ~50%, increases CYP2E1 microRNAs -336 and -352 by 1.5- to 3-fold,.and shifts CYP2E1 mRNA from the 60S-80S untranslated fraction to the actively translated polysome fraction, which is inhibited by inhibitors of PI3K and mTOR. Transient transfection with miR-336 and -352 suppresses CYP2E1 mRNA levels. Evidence suggests that insulin regulates CYP2E1 gene transcription through Akt and translation through mTOR. In contrast, glucagon antagonizes insulin effects through cyclic nucleotides and PKA. Thus, the overall goal of this research is to understand the mechanism(s) by which insulin simultaneously regulates the transcriptional and translational expression of CYP2E1. The hypothesis of this research is that Akt and mTOR regulate CYP2E1 gene transcription and mRNA translation, respectively, which is antagonized by glucagon through PKA at the level of transcription and/or translation.
The specific aims are: 1) To establish the mechanism(s) of insulin activation of Akt on CYP2E1 gene transcription;2) To examine the mechanism(s) by which insulin activation of mTOR regulates CYP2E1 mRNA translation and miRNA translation repression or degradation;3) To establish whether glucagon, through PKA, and/or AMPK signaling, regulates CYP2E1 gene transcription;and 4) To determine the mechanism by which glucagon, through PKA and/or AMPK signaling, regulates CYP2E1 translation. Activation and inhibition of Akt and mTOR will be used, in combination with mechanistic molecular approaches, to identify the mechanism(s) which regulate CYP2E1 expression in primary cultured rat and human hepatocytes, and animals in vivo. The use of an insulin mimetic, atorvastatin and metformin are relevant to Akt signaling, insulin resistance, and clinical treatment of diabetes, while the inhibitors of Akt and mTOR are relevant to the progression and treatment of cancer. Understanding CYP2E1 regulation, as a sentinel of Akt, mTOR signaling, in the insulin regulation of gene transcription, miRNAs and mRNA translation is critical to understanding the progression of hepatic disease culminating in HCC and the role of CYP2E1 as a biomarker of this process.
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