Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin

BMC Cell Biol. 2010 Jul 26;11:59. doi: 10.1186/1471-2121-11-59.

Abstract

Background: Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic beta-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual beta-cell apoptosis. A double-stable pancreatic beta-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.

Results: Expression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival.

Conclusions: The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in beta-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / genetics
  • Cell Line
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / pathology
  • Gene Expression Profiling
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Insulin / genetics
  • Insulin / metabolism*
  • Insulin-Secreting Cells / metabolism*
  • Insulin-Secreting Cells / pathology
  • Mice
  • Mice, Mutant Strains
  • Microarray Analysis
  • Proinsulin / genetics
  • Proinsulin / metabolism*
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Engineering
  • Protein Folding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transgenes / genetics
  • Unfolded Protein Response / genetics

Substances

  • Ins1 protein, mouse
  • Insulin
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Proinsulin
  • Proteasome Endopeptidase Complex