Inhibition of cyclin-dependent kinase 5 activity protects pancreatic beta cells from glucotoxicity
Inhibition of cyclin-dependent kinase 5 activity protects pancreatic beta cells from glucotoxicity
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DOI：
10.1074/jbc.m604690200
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发表时间：
2006-09-29
影响因子：
4.8
通讯作者：
Habener, Joel F.
中科院分区：
文献类型：
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作者：
Ubeda, Mariano;Rukstalis, J. Michael;Habener, Joel F.
2型糖尿病（T2 D）和阿尔茨海默病是退行性疾病，可能具有共同的病理生理机制。阿尔茨海默病患者的神经元功能障碍与细胞周期蛋白依赖性激酶5（CDK 5）及其激活剂p35的过度活性有关。这两种蛋白质都在胰腺的产生胰岛素的β细胞中表达。此外，葡萄糖增强p35基因表达，促进调节胰岛素基因表达的活性p35/CDK 5复合物的形成。在T2 D中，葡萄糖慢性升高、葡萄糖毒性损害β细胞功能。因此，我们推测CDK 5和p35可能是导致这种β细胞损伤的原因，并且抑制CDK 5可能具有有益效果。为了检验这一假设，选择胰腺细胞系INS-1作为已知的葡萄糖毒性体外模型，并使用roscovitine（10 μ M）作为CDK 5抑制剂。INS-1细胞长期暴露于高葡萄糖（20-30 mM）会降低胰岛素mRNA水平和胰岛素启动子报告基因的活性。CDK 5的抑制阻止了胰岛素基因表达的减少。我们使用DNA结合（凝胶位移）试验和Western免疫印迹证明，细胞水平的转录因子PDX-1，通常降低的葡萄糖毒性，保留与CDK 5抑制，是结合的PDX-1的胰岛素启动子。细胞核和细胞质PDX-1蛋白水平的分析显示，CDK 5抑制恢复细胞核PDX-1，而不影响其细胞质浓度，表明CDK 5调节PDX-1的细胞核/细胞质分配。使用Myc标记的PDX-1构建体，我们表明当CDK 5被抑制时，在葡萄糖毒性条件下PDX-1从细胞核到细胞质的易位被阻止。这些研究表明，CDK 5在葡萄糖毒性条件下β细胞功能丧失中起作用，并且CDK 5抑制剂可能对T2 D具有治疗价值。
Type 2 diabetes (T2D) and Alzheimer disease are degenerative diseases that may share common pathophysiologic mechanisms. Neuronal dysfunction in Alzheimer patients has been linked to overactivity of the cyclin-dependent kinase 5 (CDK5) and its activator p35. Both of these proteins are expressed in the insulin-producing beta cells of the pancreas. Further, glucose enhances p35 gene expression, promoting the formation of active p35/CDK5 complexes that regulate the expression of the insulin gene. In T2D, chronic elevations of glucose, glucotoxicity, impair beta cell function. We therefore postulated that CDK5 and p35 may be responsible for this beta cell impairment and that inhibition of CDK5 might have a beneficial effect. To test this hypothesis, the pancreatic cell line INS-1 was selected as a known in vitro model of glucotoxicity, and roscovitine (10 mu M) was used as a CDK5 inhibitor. Chronic exposure of INS-1 cells to high glucose (20-30 mM) reduced both insulin mRNA levels and the activity of an insulin promoter reporter gene. Inhibition of CDK5 prevented this decrease of insulin gene expression. We used DNA binding (gel shift) assays and Western immunoblots to demonstrate that cellular levels of the transcription factor PDX-1, normally decreased by glucotoxicity, were preserved with CDK5 inhibition, as was the binding of PDX-1 to the insulin promoter. Analyses of nuclear and cytoplasmic PDX-1 protein levels revealed that CDK5 inhibition restores nuclear PDX-1, without affecting its cytoplasmic concentration, suggesting that CDK5 regulates the nuclear/cytoplasm partitioning of PDX-1. Using a Myc-tagged PDX-1 construct, we showed that the translocation of PDX-1 from the nucleus to the cytoplasm during glucotoxic conditions was prevented when CDK5 was inhibited. These studies indicate that CDK5 plays a role in the loss of beta cell function under glucotoxic conditions and that CDK5 inhibitors could have therapeutic value for T2D.