Upregulation of miR-497 induces hepatic insulin resistance in E3 rats with HFD-MetS by targeting insulin receptor

Mol Cell Endocrinol. 2015 Nov 15;416:57-69. doi: 10.1016/j.mce.2015.08.021. Epub 2015 Aug 20.

Abstract

Objective: The study aims to find regulatory microRNA(s) responsible for down-regulated insulin receptor (InsR) in the liver of HFD-MetS E3 rats with insulin resistance.

Methods: Firstly, hepatic insulin resistance in HFD-MetS E3 rats was evaluated by RT-qPCR, western blotting, immunohistochemistry and PAS staining. Secondly, the candidate miRNAs targeting rat InsR were predicted through online softwares and detected in the liver of HFD-MetS E3 rats with insulin resistance. Then, the expression of InsR, phosphorylated IRS-1 (pIRS-1) at Tyr632, phosphorylated AKTs (pAKTs) at Ser473 and Thr308, phosphorylated GSK-3β (p GSK-3β) at Ser9, phosphorylated GS (pGS) at Ser641 and the glycogen content were detected in CBRH-7919 cells treated with 100 nM insulin for different time periods by western blotting or PAS staining respectively, after transient transfection with miR-497 mimics or inhibitors for 24 h. Lastly, the relation between miR-497 and InsR was further determined using dual luciferase reporter assay.

Results: Elevated miR-497 was negatively related with down-regulated InsR in the liver of HFD-MetS E3 rats with insulin resistance. Comparing with the mNC group, glycogen content and the expression of InsR, pIRS-1 (Tyr632), pAKTs (Ser473 and Thr308) and pGSK-3β (Ser9) decreased significantly in CBRH-7919 cells, while pGS (Ser641) increased significantly, after transient transfection with miR-497 mimics for 24 h and treatment with 100 nM insulin for corresponding time periods, counter to those results in CBRH-7919 cells after similar procedures with miR-497 inhibitors and insulin. In addition, dual luciferase reporter assay further confirmed that miR-497 can bind to the 3'UTR of rat InsR.

Conclusion: Insulin receptor is the target gene of miR-497, and elevated miR-497 might induce hepatic insulin resistance in HFD-MetS E3 Rats through inhibiting the expression of insulin receptor and confining the activation of IRS-1/PI3K/Akt/GSK-3β/GS pathway to insulin.

Keywords: Hepatic insulin resistance; High-fat-diet induced metabolic syndrome (HFD-MetS); Insulin receptor; miR-497.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Diet, High-Fat
  • Disease Models, Animal
  • Down-Regulation
  • Female
  • Glycogen Synthase / chemistry
  • Glycogen Synthase / metabolism
  • Glycogen Synthase Kinase 3 / chemistry
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Insulin / metabolism
  • Insulin Receptor Substrate Proteins / chemistry
  • Insulin Receptor Substrate Proteins / metabolism
  • Insulin Resistance / genetics*
  • Liver / metabolism*
  • Male
  • Metabolic Syndrome / metabolism*
  • MicroRNAs / metabolism*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / chemistry
  • Proto-Oncogene Proteins c-akt / metabolism
  • Rats
  • Receptor, Insulin / genetics*
  • Up-Regulation

Substances

  • 3' Untranslated Regions
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Irs1 protein, rat
  • MIRN497 microRNA, rat
  • MicroRNAs
  • Glycogen Synthase
  • Phosphatidylinositol 3-Kinases
  • Receptor, Insulin
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, rat
  • Proto-Oncogene Proteins c-akt
  • Glycogen Synthase Kinase 3