Glucose- and hypoxia-regulated insulin gene expression

Sammanfattning: Although extensively studied, there are still many unanswered questions regarding the exact regulation of insulin gene expression. This is important to further investigate since it will hopefully help us understand the pathophysiology of some types of diabetes. The insulin mRNA has a long half-life and it is therefore likely to be regulated through specific mechanisms. The aim of the present work was to evaluate the importance of the 3´untranslated region (UTR) of the insulin mRNA in the regulation of insulin mRNA stability. The number of insulin mRNA molecules/rat β-cell was first determined to be 20 000 - 100 000, depending on the glucose concentration of the culture medium. However, the insulin mRNA level was neither increased by glucose nor insulin when stimulated for only one hour. Glucose withdrawal for 16 h caused only a small decrease in insulin mRNA levels. This decrease was of the same magnitude in the presence of a transcriptional inhibitor, indicating that the insulin mRNA stability significantly contributes to the regulation of insulin gene expression. To study the mechanisms by which glucose regulates insulin mRNA stability, we probed for protein binding to the rat and human 3´UTR insulin mRNA (ins-PRS) using the electrophoresis mobility shift assay. Specific binding was observed and the protein was identified as the polypyrimidine tract-binding protein (PTB). Introduction of the ins-PRS in to the 3´UTR of a reporter gene mRNA did not significantly affect the mRNA stability. However, mutation of the ins-PRS and disruption of PTB binding resulted in a marked decrease in expression of the reporter gene mRNA. The binding activity was increased in islets in response to glucose, hypoxia, sphingomyelinase (an inducer of endogenous ceramide) and in vitro by the addition of the reducing agent dithiothreitol (DTT). The hypoxia-induced binding activity was counteracted by exogenous administration of hydrogen peroxide, while the glucose-induced PTB binding activity was inhibited by rapamycin, which is an inhibitor of mTOR and p70s6 kinase. The insulin mRNA levels were increased in response to hypoxia, equal to the increase in response to glucose. In conclusion, PTB binding to the ins-PRS is an important, but not sufficient, event in regulation of insulin mRNA stability. Binding activity is increased in response to glucose and hypoxia, possibly mediated through pathways involving the redox status and/or reactive oxygen species (ROS), ceramide and mTOR/p70s6k.