Global profiling of genes modified by endoplasmic reticulum stress in pancreatic beta cells reveals the early degradation of insulin mRNAs

Abstract

Aims/hypothesis Pancreatic beta cells respond to endoplasmic reticulum (ER) stress by activating the unfolded protein response. If the stress is prolonged, or the adaptive response fails, apoptosis is triggered. We used a 'homemade' microarray specifically designed for the study of beta cell apoptosis (the APOCHIP) to uncover mechanisms regulating beta cell responses to ER stress. Materials and methods A time course viability and microarray analysis was performed in insulin-producing INS-1E cells exposed to the reversible ER stress inducer cyclopiazonic acid (CPA). Modification of selected genes was confirmed by real-time RT-PCR, and the observed inhibition of expression of the insulin-1 (Ins1) and insulin-2 (Ins2) genes was further characterised in primary beta cells exposed to a diverse range of agents that induce ER stress. Results CPA-induced ER stress modified the expression of 183 genes at one or more of the time points studied. The expression of most of these genes returned to control levels after a 3 h recovery period following CPA removal, with all cells surviving. Two groups of genes were particularly affected by CPA, namely, those related to cellular responses to ER stress, which were mostly upregulated, and those related to differentiated beta cell functions, which were downregulated. Levels of Ins1 and Ins2 mRNAs were severely decreased in response to CPA treatment as a result of degradation, and there was a concomitant increase in the level of IRE1 activation. Conclusions/interpretation In this study we provide the first global analysis of beta cell molecular responses to a severe ER stress, and identify the early degradation of mRNA transcripts of the insulin genes as an important component of this response.

Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis

“…In other cell types, activation of the IRE1α-XBP1s pathway increases production of ERproteins aiming to fold and/or degrade misfolded proteins. Together with previous studies [27,46], our data suggest that the IRE1α-XBP1s pathway has a distinct role in beta cells, relative to the fine-tuning of insulin mRNA expression through transcriptional and post-transcriptional mechanisms, allowing for rapid adjustment of insulin levels under ER-stress conditions [46]. However, due to the deleterious effects of prolonged increased XBP1s level, this mechanism has to be closely regulated in beta cells, resulting in a limited XPB1 splicing as shown by previous studies [2,12] and the present data.…”

“…We and others have previously shown that the IRE1α-XBP1s pathway of the UPR controls insulin expression, mainly through IRE1α-mediated degradation of insulin mRNA [27,45,46]. In the present study we demonstrate another level of regulation of insulin expression by the UPR, via XBP1s-dependent downregulation of Pdx1 and Mafa.…”

“…We studied the impact of blocking XBP1s on the expression of insulin, Pdx1, MafA and Glut2. As previously described [17,27], the expression of these genes was severely decreased upon treatment with CPA or IL-1β+IFN-γ. Knocking-down XBP1s partially restored insulin 2 (Fig.…”

“…XBP1s strongly decreased Mafa and Pdx1 expression, thereby suppressing the cooperative action between these transcription factors and Neurod1 in stimulating insulin transcription [32]. Moreover, Xbp1 knockdown partially prevented cytokines and CPA-mediated decrease in insulin and Pdx1 mRNA expression [18,27], supporting the hypothesis that XBP1s inhibits Pdx1 expression. XBP1s dose-dependently inhibited stimulated but not basal insulin secretion, suggesting that XBP1s interferes with the glucose signalling pathways leading to insulin release.…”

“…Expression values were corrected to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Cytokine or CPA treatment does not modify Gapdh expression in insulin-producing cells under the present experimental conditions [18,27]. The primers used in this study are described in the ESM Table 1.…”