Regulation of pancreatic・cell function by Ca2+/calmodulin-dependent protein kinase II delta.-Analysis using CaM-KIIN, noble inhibitory protein, and constitutive active CaM kinase II-

据报道，Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaM kinase 11）δ2可能通过突触蛋白I的磷酸化参与葡萄糖诱导的胰岛素分泌。然而，糖尿病患者长期高血糖诱导的长期激活的影响并不确定。本研究旨在通过对胰腺B细胞株MIN 6细胞中野生型和各种突变型CaM激酶Ⅱ 82的表达分析，探讨CaM激酶Ⅱ在2型糖尿病发病和发展中的作用。首先构建了携带野生型CaMKII δ2（WT）、组成型活性型（ACT）和激酶阴性型（KD）的表达载体pCAGGSneo。然后，为了确定所观察到的是否由于CaMK II突变体而抑制CREB依赖性胰岛素启动子转录，使用HilyMax转染试剂将一系列含有胰岛素启动子和renila载体的荧光素酶报告质粒与这些突变体共转染到MIN 6细胞中。用葡萄糖刺激后，收获细胞，用荧光素酶法检测细胞裂解物，发现在5 mM葡萄糖条件下过表达WT和ACT CaM激酶II，这些胰岛素启动子活性下降了47%。分别为53%。另一方面，KD CaM激酶II过表达使胰岛素启动子活性增加至204%。这些结果表明，CaM激酶II 82通过磷酸化CREB的Serine-133和Serine-147来激活，从而抑制胰岛素基因启动子活性和胰岛素基因表达的可能性。

Ca2+/calmodulin-dependent protein kinase II (CaM kinase 11)δ2 has been reported to be involved in the glucose induced insulin secretion possibly by the phosphorylation of synapsin I. However, the effect of prolonged activation that was induced by the long-term hyperglycemia in diabetes was not uncertain. The purpose of this study are to evaluate the role of CaM kinase II in the onset or progression of type 2 diabetes by analysis of transfect:.on of wild type or various mutated CaM kinase II 82 in pancreatic b cell line, MIN6 cells. At first, We constructed the expression vectors pCAGGSneo carrying WT CaMKII δ2 (WT), constitutive active form (ACT), kinase negative form (KD). Then, to determine if the observed to repress CREB dependent insulin promoter transcription that was due to CaMKII mutants, a series of luciferase reporter plasmids containing the insulin promoter and renila vector were co-transfected with these mutants into MIN6 cells using HilyMax transfection reagent. After stimulation by Glucose, these cells were harvested, and the cell lysates were subjected to luciferase assay.When WT and ACT CaM kinase II were overexpressed under 5mM Glucose condition, these insulin promoter activities were decreased by 47%. and 53% respectively. Other way, KD CaM kinase II overexpression increased insulin promoter activity to 204%. These results suggest the possibility that insulin gene promoter activity and the expression of insulin gene are inhibited by the activation of CaM kinase II 82 via phosphorylation of Serine-133 and Serine-147 of CREB.