Glucose regulated protein synthesis in pancreatic β-cells
The pancreatic beta-cell rapidly releases insulin in response to glucose and in order to maintain insulin stores within the beta-cell, there is a rapid and specific increase in proinsulin (PI) synthesis (10-20 fold within 60min). This rapid increase in PI synthesis is mediated entirely at the post-transcriptional level, likely through an increase in the rate of protein synthesis. As the regulation of glucose-stimulated proinsulin synthesis is poorly understood, the first objective of this thesis was to investigate the mechanism of glucose-regulated proinsulin synthesis in pancreatic beta-cells. This thesis shows that contrary to previous reports, there is no pool of 'free' preproinsulin (PPI) mRNA at low glucose concentrations and that glucose does not stimulate the rate of de novo initiation. However, glucose does stimulate the recruitment of ribosomes onto ribosome-associated PPI mRNA, indicative of an increase in the rate of initiation of translation. Additionally, glucose stimulates the recruitment of PPI, PC2 and CPH mRNAs to the ER. Moreover, PPI mRNA is preferentially recruited to the ER over the other secretory protein mRNAs. This preferential recruitment of PPI mRNA may be an important mechanism for the specific up-regulation of PI synthesis at high glucose concentrations. Indeed, data presented here shows that the recruitment of PPI mRNA to the ER plays an important role in glucose stimulated PI synthesis. In addition to an increase in PI synthesis, glucose stimulates a rapid increase in the synthesis of more than 260 unidentified beta-cell proteins. It is likely that this rapid up- regulation of beta-cell proteins is important in beta-cell function. Therefore, microarray analysis of polysomal mRNAs was used to identify beta-cell proteins that were translationally regulated in response to increases in glucose concentration. Interestingly, the majority of polysomal mRNAs whose levels changed between low and high glucose encoded proteins important in transcription and oxidative stress.