Determine the molecular mechanism of insulin gene expression for the regeneration of pancreatic islets

据报道，有许多因素会影响胰岛细胞中胰岛素的合成。虽然葡萄糖是胰岛β细胞胰岛素基因表达的最重要的调节因子，但葡萄糖刺激胰岛素基因转录以响应葡萄糖浓度变化的机制尚不清楚。在这项研究中，我们研究了钙离子/钙调蛋白(CaM)依赖的蛋白激酶(CaM-K)级联在胰岛素转录激活中的作用。RT-PCR、Western blotting和免疫组织化学染色分析表明，CaM-K激酶-α(CaM-KKalpha)和CaM-KIV定位于大鼠胰岛β细胞及其细胞系INS-1。INS-1细胞暴露于11.2 mmol/L葡萄糖后，在刺激后2分钟内胰岛素启动子活性增加，CaM-KIV活性上调。我们在INS-1细胞中研究了CaM-KIV的结构活性形式(CaM-KIVc)和显性-阴性突变体(CaM-KIVdn)对胰岛素启动子活性的影响。仅CaM-KIVc就足够了，上游的CaM-KK被增强以上调INS-1细胞中的胰岛素启动子活性。此外，共转染CaM-KIVdn在很大程度上抑制了胰岛素启动子的葡萄糖上调活性。综上所述，这些结果表明，CaM-KK/CaM-KIV级联可能在葡萄糖上调的胰岛素基因转录激活中发挥重要作用。我们还确定了PREB和IB1作为转录因子在胰岛素基因转录中的作用。

A number of factors have been reported to affect insulin synthesis in beta-cells. Although glucose is the most important regulator of insulin gene expression in pancreatic beta-cells, the mechanisms whereby glucose stimulates insulin gene transcription in response to changes in glucose concentration have not been clarified yet. In this study, we examined the role of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM-K) cascade in transcriptional activation of insulin. RT-PCR, Western blotting, and immunohistochemical staining analysis revealed that CaM-K kinase-alpha (CaM-KKalpha) and CaM-KIV were localized in rat pancreatic beta-cells and their cell line, INS-1. Exposure of INS-1 cells to 11.2 mmol/l glucose elicited an increase of insulin promoter activity as well as upregulation of CaM-KIV activity within 2 min after stimulation. We investigated the influence on insulin promoter activity of the constitutively active form (CaM-KIVc) or dominant-negative mutant (CaM-KIVdn) of CaM-KIV in transfected INS-1 cells. CaM-KIVc alone was sufficient, and the upstream kinase, CaM-KK, was enhanced to upregulate the insulin promoter activity in INS-1 cells. Furthermore, cotransfection of CaM-KIVdn suppressed to a significant degree the glucose-upregulated activity of the insulin promoter. Taken together, these results indicated that the CaM-KK/CaM-KIV cascade might play an important role in glucose-upregulated transcriptional activation of the insulin gene. We have also determined the role of PREB and IB 1 as a transcriptional factor for insulin gene transcription.