Degradation of cAMP-responsive element-binding protein by the ubiquitin-proteasome pathway contributes to glucotoxicity in beta-cells and human pancreatic islets.
Degradation of cAMP-responsive element-binding protein by the ubiquitin-proteasome pathway contributes to glucotoxicity in beta-cells and human pancreatic islets.
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作者：
Costes S;Vandewalle B;Tourrel-Cuzin C;Broca C;Linck N;Bertrand G;Kerr-Conte J;Portha B;Pattou F;Bockaert J;Dalle S
在2型糖尿病中，慢性高血糖对β细胞有害，导致细胞凋亡和胰岛素分泌受损。转录因子cAMP反应元件结合蛋白（CREB）对β细胞的存活和功能至关重要。我们研究了β细胞长期暴露于高糖是否会影响CREB的功能完整性。使用INS-1 E细胞以及大鼠和人胰岛。通过RT-PCR和Western blotting分析基因表达。细胞凋亡检测裂解caspase-3的出现，DNA片段化，和电子显微镜。INS-1 E细胞以及大鼠和人胰岛长期暴露于高糖导致CREB蛋白表达、磷酸化和与细胞凋亡相关的转录活性降低，β细胞功能受损。高糖处理增加CREB多聚泛素化，而蛋白酶体抑制剂MG-132处理INS-1 E细胞防止CREB含量下降。在INS-1 E细胞中，当CREB蛋白表达被小干扰RNA敲低时出现凋亡，这表明高糖诱导的CREB蛋白含量的丧失有助于β细胞凋亡。用模拟CREB蛋白酶体靶向序列的细胞渗透性肽加载INS-1 E细胞或人胰岛，阻断CREB降解，并保护INS-1 E细胞和人胰岛免受高糖诱导的凋亡。在暴露于高葡萄糖并负载肽的人胰岛中，响应于葡萄糖的胰岛素分泌和胰岛素含量得以保留。这些研究表明，CREB降解的泛素-蛋白酶体途径有助于β细胞功能障碍和死亡后的葡萄糖毒性，并提供了新的见解葡萄糖毒性的细胞机制。
In type 2 diabetes, chronic hyperglycemia is detrimental to β-cells, causing apoptosis and impaired insulin secretion. The transcription factor cAMP-responsive element–binding protein (CREB) is crucial for β-cell survival and function. We investigated whether prolonged exposure of β-cells to high glucose affects the functional integrity of CREB. INS-1E cells and rat and human islets were used. Gene expression was analyzed by RT-PCR and Western blotting. Apoptosis was detected by cleaved caspase-3 emergence, DNA fragmentation, and electron microscopy. Chronic exposure of INS-1E cells and rat and human islets to high glucose resulted in decreased CREB protein expression, phosphorylation, and transcriptional activity associated with apoptosis and impaired β-cell function. High-glucose treatment increased CREB polyubiquitination, while treatment of INS-1E cells with the proteasome inhibitor MG-132 prevented the decrease in CREB content. The emergence of apoptosis in INS-1E cells with decreased CREB protein expression knocked down by small interfering RNA suggested that loss of CREB protein content induced by high glucose contributes to β-cell apoptosis. Loading INS-1E cells or human islets with a cell-permeable peptide mimicking the proteasomal targeting sequence of CREB blocked CREB degradation and protected INS-1E cells and human islets from apoptosis induced by high glucose. The insulin secretion in response to glucose and the insulin content were preserved in human islets exposed to high glucose and loaded with the peptide. These studies demonstrate that the CREB degradation by the ubiquitin-proteasome pathway contributes to β-cell dysfunction and death upon glucotoxicity and provide new insight into the cellular mechanisms of glucotoxicity.