Abstract
BackgroundThe Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic β-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line.ResultsThe IRE1 endoribonuclease inhibitors 4μ8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1.ConclusionsAlthough maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic β-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis.
Highlights
The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice
We find that the IRE1 pathway is required for maximal induction of most unfolded protein response (UPR) target genes, but unexpectedly does not sensitize the cells against chronic endoplasmic reticulum (ER) stress-induced apoptosis
To define the role of the IRE1 pathway in the UPR in this model system we used a recently described IRE1 inhibitor 4μ8c that inhibits the endoribonuclease activity of IRE1 and prevents splicing of the XBP1 mRNA in response to ER stress [24]
Summary
The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic β-cells and the cell activates the unfolded protein response (UPR). The UPR is an adaptive program that in metazoans is mediated by three ER stress response sensors, PERK, IRE1 and ATF6 These are ER-localized transmembrane proteins that sense the protein products produced by different cell types. ER stress has been implicated in contributing to pancreatic β-cell dysfunction and death resulting in the development of diabetes. This is evident in rodents and human patients with certain mutations in the insulin gene that cause misfolding of proinsulin in the ER [14,15] and in rodents and patients with mutations in the PERK gene [16,17]. Understanding how pancreatic β-cells respond to ER stress may prove beneficial in developing strategies to improve cell function and survival as potential treatment options for the disease
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