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，Ca2+/calmodulin-dependent protein kinase II (CaM kinase 11)δ2可能通过synapsin 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延长激活的作用并不确定。本研究的目的是通过分析转染：来评估CaM激酶II在2型糖尿病发病或进展中的作用。野生型或多种突变型CaM激酶i82在胰腺b细胞系MIN6细胞中的应用。首先，我们构建了携带WT CaMKII δ2 （WT）、本构活性形式（ACT）、激酶阴性形式（KD）的表达载体pCAGGSneo。然后，为了确定是否观察到由于CaMKII突变而抑制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的荧光素酶报告质粒和renila载体与这些突变体共转染到MIN6细胞中。葡萄糖刺激后，收集这些细胞，细胞裂解物进行荧光素酶测定。当WT和ACT CaM激酶II在5mM葡萄糖条件下过表达时，这些胰岛素启动子活性降低了47%。和53%。另一方面，KD - CaM激酶II过表达可使胰岛素启动子活性增加204%。这些结果提示胰岛素基因启动子活性和胰岛素基因的表达可能是通过磷酸化CREB的丝氨酸-133和丝氨酸-147来激活CaM激酶II 82。

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.