Rapid changes of mRNA-binding protein levels following glucose and 3-isobutyl-1-methylxanthine stimulation of insulinoma INS-1 cells

Mol Cell Proteomics. 2009 Mar;8(3):393-408. doi: 10.1074/mcp.M800157-MCP200. Epub 2008 Oct 14.

Abstract

Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic beta-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the beta-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in beta-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated beta-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-3-isobutylxanthine / pharmacology*
  • Amino Acid Motifs
  • Animals
  • Binding Sites
  • Blotting, Western
  • Computational Biology
  • Conserved Sequence
  • Electrophoresis, Gel, Two-Dimensional
  • Glucose / pharmacology*
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Humans
  • Insulinoma / metabolism*
  • Insulinoma / pathology*
  • Mass Spectrometry
  • Mice
  • Muscle Proteins / metabolism
  • Neoplasm Proteins / metabolism
  • Polypyrimidine Tract-Binding Protein
  • Proteomics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Rats
  • Reproducibility of Results
  • Secretory Vesicles / drug effects
  • Secretory Vesicles / metabolism
  • Untranslated Regions / genetics

Substances

  • Heterogeneous-Nuclear Ribonucleoproteins
  • Muscle Proteins
  • Neoplasm Proteins
  • Ptbp1 protein, rat
  • RNA, Messenger
  • RNA-Binding Proteins
  • Untranslated Regions
  • Polypyrimidine Tract-Binding Protein
  • Glucose
  • 1-Methyl-3-isobutylxanthine