miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells

Diabetes. 2008 Oct;57(10):2708-17. doi: 10.2337/db07-1614. Epub 2008 Jun 30.

Abstract

Objective: MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in beta-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3'-phosphoinositide-dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels.

Research design and methods: We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats.

Results: We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.

Conclusions: Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic beta-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and beta-cell growth.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • 3-Phosphoinositide-Dependent Protein Kinases
  • Animals
  • Base Sequence
  • Blotting, Western
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Cells, Cultured
  • Gene Expression / drug effects
  • Glucose / pharmacology*
  • Insulin / pharmacology
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / drug effects*
  • Insulin-Secreting Cells / metabolism
  • Male
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Oligonucleotides, Antisense / pharmacology
  • Phosphorylation
  • Protein Serine-Threonine Kinases / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects

Substances

  • 3' Untranslated Regions
  • Insulin
  • MicroRNAs
  • Oligonucleotides, Antisense
  • RNA, Messenger
  • 3-Phosphoinositide-Dependent Protein Kinases
  • Pdpk1 protein, rat
  • Protein Serine-Threonine Kinases
  • Glucose