Analysis of protein kinases relating to glucotoxicity

糖毒性是2型糖尿病病理生理学的重要组成部分，然而，糖毒性的分子机制仍不完全清楚。在这项研究中，我们分析了糖毒条件下INS-1细胞中与胰岛素合成相关的蛋白激酶。当用多PK抗体进行Western blotting分析INS-1细胞中蛋白激酶的表达模式时，在糖中毒条件下，随着胰岛素分泌的减少，63kd的蛋白激酶显著减少。根据天然激酶/溴化氰片段的分子大小和等电点，推测该63kd的蛋白激酶为CaMKIV。糖毒条件下降低的CaMKIV水平在改变培养基至正常葡萄糖浓度后恢复到原来的水平。重组CaMKIV在糖毒条件下与INS-1细胞裂解物孵育后，以钙依赖的方式降解，并被钙蛋白酶抑制剂保护。此外，CaMKIV在糖尿病大冢长埃文斯德岛肥胖大鼠的胰岛中减少，而非糖尿病长埃文斯德岛肥胖大鼠的胰岛CaMKIV不减少。本研究提示CaMKIV的异常调节是高糖引起的β细胞功能障碍的一个组成部分。

Glucotoxicity is a critical component of the pathophysiology of type 2 diabetes mellitus ; however, the molecular mechanisms of glucotoxicity are still not fully understood. In this study, we analyzed the protein kinase that correlated with insulin synthesis in INS-1 cells under glucotoxic conditions. When expression patterns of protein kinases in INS-1 cells were analyzed by Western blotting with Multi-PK antibodies, a kinase of 63 kd was significantly reduced concomitant with the decrease of insulin secretion under glucotoxic conditions. Judging from the molecular size of a native kinase/cyanogen bromide fragment and pI value, the 63-kd protein kinase was deduced to be CaMKIV. The decreased CaMKIV levels under glucotoxic conditions recovered to original levels after changing the medium to a normal glucose concentration. Recombinant CaMKIV was degraded in a Ca^<2+>-dependent manner by incubation with cell lysates from INS-1 cells under glucotoxic conditions, and degradation was protected by calpain inhibitor. Furthermore, CaMKIV was reduced in the pancreatic islets of diabetic Otsuka Long-Evans Tokushima fatty rats, whereas that of nondiabetic Long-Evans Tokushima Otsuka rats was not. This study suggests that the abnormal regulation of CaMKIV is a component of β-cell dysfunction caused by high glucose.