A decrease in cellular energy status stimulates PERK-dependent eIF2α phosphorylation and regulates protein synthesis in pancreatic β-cells

Edith Gomez,Mike L Powell,Alan Bevington,Terence P Herbert

doi:10.1042/bj20071367

Abstract

In the present study, we demonstrate that, in pancreatic beta-cells, eIF2alpha (eukaryotic initiation factor 2alpha) phosphorylation in response to a decrease in glucose concentration is primarily mediated by the activation of PERK [PKR (protein kinase RNA activated)-like endoplasmic reticulum kinase]. We provide evidence that this increase in PERK activity is evoked by a decrease in the energy status of the cell via a potentially novel mechanism that is independent of IRE1 (inositol requiring enzyme 1) activation and the accumulation of unfolded nascent proteins within the endoplasmic reticulum. The inhibition of eIF2alpha phosphorylation in glucose-deprived cells by the overexpression of dominant-negative PERK or an N-terminal truncation mutant of GADD34 (growth-arrest and DNA-damage-inducible protein 34) leads to a 53% increase in the rate of total protein synthesis. Polysome analysis revealed that this coincides with an increase in the amplitude but not the number of ribosomes per mRNA, indicating that eIF2alpha dephosphorylation mobilizes hitherto untranslated mRNAs on to polysomes. In summary, we show that PERK is activated at low glucose concentrations in response to a decrease in energy status and that this plays an important role in glucose-regulated protein synthesis in pancreatic beta-cells.

Highlights

Glucose stimulated insulin secretion from pancreatic β-cells is mediated by a series of steps initiated by the metabolism of glucose, which results in an increase in the cellular ATP/ADP ratio
To investigate whether the eIF2α kinase PKR-like endoplasmic reticulum kinase (PERK) is activated by glucose deprivation, MIN6 cells were mock infected or infected with a recombinant adenovirus over-expressing a dominant-negative mutant of PERK (AdPERKΔC). 48h post-infection, the cells were incubated in Krebs-Ringer bicarbonate buffer (KRB) without glucose or containing 20mM glucose in the presence or absence of thapsigargin as a positive control
These results demonstrate that PERK is activated upon glucose deprivation and that PERK activation is primarily responsible for eIF2α phosphorylation in glucose deprived cells

Summary

Introduction

Glucose stimulated insulin secretion from pancreatic β-cells is mediated by a series of steps initiated by the metabolism of glucose, which results in an increase in the cellular ATP/ADP ratio. An important rate limiting step in protein synthesis is the assembly of the translational ternary complex, made up of the methionyl initiator tRNA (Met-tRNAi) attached to eukaryotic initiation factor 2 (eIF2) in its GTP bound state (eIF2-GTP:Met-tRNAi) (for review see [14]). In islets of Langerhans and in the pancreatic β-cell line MIN6, that the availability of the translational ternary complex is increased in response to glucose [11] and that this was likely mediated through the dephosphorylation of eIF2α. The eIF2α kinase that phosphorylates eIF2α at low glucose concentration in pancreatic β-cells and its role in glucose regulated protein synthesis is unknown

Methods

Results

Conclusion