Cooperativity between the Preproinsulin mRNA Untranslated Regions Is Necessary for Glucose-stimulated Translation

Barton Wicksteed,Terence P Herbert,Cristina Alarcon,Melissa K Lingohr,Larry G Moss,Christopher J Rhodes

doi:10.1074/jbc.m011214200

Abstract

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic beta-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5'-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3'-UTR appeared to suppress translation. However, together the 5'- and 3'-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3'-UTR led to reduced mRNA levels compared with the presence of the SV40 3'-UTR, consistent with the presence of mRNA stability determinants in the 3'-UTR that stabilize the preproinsulin mRNA in a pancreatic beta-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic beta-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA.

Highlights

Pancreatic islet ␤-cells secrete insulin in response to increases in circulating nutrients, the most physiologically relevant of which is glucose [1]
Virus Construction and Evaluation—To examine the role of the untranslated regions of the rat preproinsulin II mRNA, a series of gene constructs were designed that would express mRNA molecules that mirror the endogenous rat preproinsulin II mRNA as closely as possible, except for defined alterations (Fig. 2). These mRNAs encode a His-tagged proinsulin flanked by the preproinsulin 5Ј- and 3Ј-UTRs (5HisPI3), the preproinsulin 5Ј-UTR and SV40 3Ј-UTR (5HisPI), the preproinsulin 3Ј-UTR and a synthetic 5Ј-UTR that is based upon a previously characterized sequence that is A/T-rich and predicted to have little secondary structure (HisPI3; see Ref. 17), or the synthetic 5Ј-UTR and the SV40 3Ј-UTR (HisPI)
These results showed that the initiation of transcription of the endogenous preproinsulin mRNA in INS-1 cells occurred 55 bp from the initiation codon

Summary

Introduction

Pancreatic islet ␤-cells secrete insulin in response to increases in circulating nutrients, the most physiologically relevant of which is glucose [1]. To replenish secreted insulin and maintain optimal intracellular insulin stores there is a corresponding rapid and specific stimulation of proinsulin biosynthesis [1,2,3] Under normal circumstances this occurs by increasing translation of the existing preproinsulin mRNA and is mostly controlled at the initiation phase of the translational mechanism. The translation of the ferritin mRNA is regulated by intracellular iron levels acting through an element in the 5ЈUTR [13], known as an iron response element, that includes a cis-acting stem-loop secondary structure (dG ϭ Ϫ15.4 kcal/ mole) This element up-regulates ferritin mRNA translation in response to increased cellular iron through an interaction with a trans-acting iron-binding protein. Glucose-regulated Proinsulin Translation cific translation in response to glucose, which has been directly tested in this study

Methods

Results

Conclusion