Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic beta-cells.

In pancreatic beta-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0.5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1.5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, beta-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining beta-cell integrity and survival during conditions of nutritional stress.

The selective recruitment of mRNA to the ER and an increase in initiation are important for glucose-stimulated proinsulin synthesis in pancreatic beta-cells.

Glucose acutely stimulates proinsulin synthesis in pancreatic beta-cells through a poorly understood post-transcriptional mechanism. In the present study, we demonstrate in pancreatic beta-cells that glucose stimulates the recruitment of ribosome-associated proinsulin mRNA, located in the cytoplasm, to the ER (endoplasmic reticulum), the site of proinsulin synthesis, and that this plays an important role in glucose-stimulated proinsulin synthesis. Interestingly, glucose has greater stimulatory effect on the recruitment of proinsulin mRNA to the ER compared with other mRNAs encoding secretory proteins. This, as far as we are aware, is the first example whereby mRNAs encoding secretory proteins are selectively recruited to the ER and provides a novel regulatory mechanism for secretory protein synthesis. Contrary to previous reports, and importantly in understanding the mechanism by which glucose stimulates proinsulin synthesis, we demonstrate that there is no large pool of 'free' proinsulin mRNA in the cytoplasm and that glucose does not increase the rate of de novo initiation on the proinsulin mRNA. However, we show that glucose does stimulate the rate of ribosome recruitment on to ribosome-associated proinsulin mRNA. In conclusion, our results provide evidence that the selective recruitment of proinsulin mRNA to the ER, together with increases in the rate of initiation are important mediators of glucose-stimulated proinsulin synthesis in pancreatic beta-cells.

Glucose-stimulated protein synthesis in pancreatic beta-cells parallels an increase in the availability of the translational ternary complex (eIF2-GTP.Met-tRNAi) and the dephosphorylation of eIF2 alpha.

In pancreatic beta-cells, glucose causes a rapid increase in the rate of protein synthesis. However, the mechanism by which this occurs is poorly understood. In this report, we demonstrate, in the pancreatic beta-cell line MIN6, that glucose stimulates the recruitment of ribosomes onto the mRNA, indicative of an increase in the rate of the initiation step of protein synthesis. This increase in the rate of initiation is not mediated through an increase in the availability of the initiation complex eIF4F, because glucose is unable to stimulate eIF4F assembly or, in the absence of amino acids, modulate the phosphorylation status of 4E-BP1. Moreover, in MIN6 cells and isolated islets of Langerhans, rapamycin, an inhibitor of the mammalian target of rapamycin, only partially inhibited glucose-stimulated protein synthesis. However, we show that glucose stimulates the dephosphorylation of eIF2 alpha in MIN6 cells and the assembly of the translational ternary complex, eIF2-GTP.Met-tRNAi, in both MIN6 cells and islets of Langerhans. The changes in the phosphorylation of eIF2 alpha are not mediated by the PKR-like endoplasmic reticulum eIF2 alpha kinase (PERK), because PERK is not phosphorylated at low glucose concentrations and overexpression of a dominant negative form of PERK has no significant effect on either glucose-stimulated protein synthesis or the phosphorylation of eIF2 alpha. Taken together, these results indicate that glucose-stimulated protein synthesis in pancreatic beta-cells is regulated by a mechanism largely independent of the activity of mammalian target of rapamycin, but which is likely to be dependent on the availability of the translational ternary complex, regulated by the phosphorylation status of eIF2 alpha.