Department of Medical Cell Biology, Uppsala University, Biomedicum, Uppsala, Sweden.
Biochem Biophys Res Commun. 2011 Sep 9;412(4):693-8. doi: 10.1016/j.bbrc.2011.08.030. Epub 2011 Aug 16.
The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5'-UTR of the human insulin mRNA.
本研究旨在探讨在基础条件下、高葡萄糖刺激条件下和氮氧化应激条件下，人胰岛是否存在非帽子依赖型胰岛素 mRNA 翻译。我们还旨在将非帽子依赖型胰岛素 mRNA 翻译与 IRES 反式作用因子多嘧啶 tract 结合蛋白（PTB）与胰岛素 mRNA 5'-UTR 的结合相关联。为此，将人胰岛在低（1.67 mM）或高葡萄糖（16.7 mM）存在的情况下孵育 2 小时。通过添加 1 mM DETA/NO 诱导氮氧化应激，并使用 hippuristanol 抑制帽子依赖性 mRNA 翻译。通过放射性标记和免疫沉淀测定胰岛素生物合成率。通过磁性微珠下拉程序评估 PTB 与胰岛素 mRNA 5'-UTR 的亲和力。我们观察到，在 1.67 mM 葡萄糖存在的情况下， hippuristanol 抑制了约 70%的胰岛素 mRNA 翻译。在孵育于 16.7 mM 葡萄糖的胰岛中，相应的值为 93%。DETA/NO 处理显著降低了高葡萄糖孵育胰岛中胰岛素的翻译，抑制率为 85%，而在低葡萄糖浓度下则降低了 50%。DETA/NO 暴露的胰岛的胰岛素生物合成率进一步被 hippuristanol 抑制，在 16.7 mM 葡萄糖时抑制率为 55%，但在 1.67 mM 葡萄糖时则没有。因此，与对照胰岛相比，DETA/NO 处理的胰岛中 hippuristanol 诱导的胰岛素生物合成抑制作用不那么明显。我们还观察到，PTB 特异性地与体外胰岛素 mRNA 5'-UTR 结合，并且这种结合与不同条件下非帽子依赖型胰岛素生物合成的速率很好地对应。总之，我们的研究表明，在高葡萄糖浓度下，胰岛素生物合成主要依赖于帽子，但在氮氧化应激条件下，非帽子依赖型胰岛素的生物合成可以占所有胰岛素生物合成的 40-100%。这些数据表明，胰岛β细胞能够通过一种不依赖帽子和 eIF4A 的机制维持基础胰岛素合成，可能是通过 PTB 与人类胰岛素 mRNA 的 5'-UTR 结合介导的。
