Molecular Mechanism of Pancreatic β-cell Glucose Toxicity in Anti-oxidative Enzyme Overexpressing Transgenic db/db Obese Diabetic Mice.

葡萄糖毒性在控制不良的2型糖尿病患者β细胞功能的进行性恶化中起主要作用。氧化应激可能在一定程度上解释了这一现象，因为在慢性高血糖状态下，包括β细胞在内的许多组织中的活性氧（ROS）增加。我们已经报道过氧化应激降低了胰岛素促进因子的活性，抗氧化剂的管理在糖尿病小鼠中显示出有益的效果。为了评估氧化应激在糖尿病胰腺β-细胞功能障碍中的作用，我们试图建立谷胱甘肽过氧化物酶（GSHPx）仅在胰腺β-细胞中过表达的转基因小鼠。我们构建的转基因含有胰岛素启动子驱动的GSHPx基因，从而实现了GSHPx在胰腺β细胞中的选择性表达。在获得的转基因小鼠胰岛中，通过免疫组化染色证实了GSHPx在β-细胞中的特异性过表达。然后将转基因小鼠与C57BL/Ks db/-小鼠杂交，获得胰β-细胞中过表达GSHPx的db/db肥胖糖尿病小鼠。然而，反映细胞内氧化应激的抗血红素氧化酶免疫组化染色在对照db/db小鼠和过表达GSHPx的db/db小鼠之间没有观察到差异。这一结果表明，过表达的GSHPx没有足够的活性来减少胰腺中的氧化应激。考虑到已知c端非编码病变与GSHPx基因翻译有关，我们假设c端非编码病变。转基因损伤不起作用。因此，我们设计了具有更长的c端非编码区的新质粒，并插入flag标签，以便对全长蛋白的翻译进行监测。我们还研究了氧化应激在葡萄糖毒性中的作用机制。特别是我们看到转录因子PDX-1（胰腺和十二指肠同源盒因子-1）的参与。因为PDX-1表达的减少常常与胰岛素含量或β细胞数量的减少同时发生。事实上，当抗氧化处理改善糖尿病小鼠的β细胞功能和葡萄糖耐量时，它也增加了细胞核中PDX-1的表达量。因此，我们研究了氧化应激导致PDX-1核-细胞质易位从而降低PDX-1活性的可能性。在β细胞衍生细胞系HIT-T15中，在常规条件下培养细胞时，过表达的绿色荧光蛋白（GFP）标记的PDX-1主要定位在细胞核中。然而，当添加50μM H_2O_2对细胞施加氧化应激时，PDX-1从细胞核中分散，主要存在于细胞质中，而添加抗氧化剂（50μM n -乙酰半胱氨酸）则逆转了这一现象。因此，氧化应激导致PDX-1从细胞核转移到细胞质。另一方面，c-Jun氨基末端激酶1 （JNK1）的过表达可以在没有氧化应激的情况下诱导PDX-1的细胞质易位。此外，在h_2o_2处理的HIT-T15细胞中，主导负JNK1的过表达几乎完全阻止了氧化应激诱导的PDX-1细胞质易位，这表明JNK/应激激活蛋白激酶（SAPK）途径是易位的中介。因此，我们目前的观察表明，PDX-1的细胞内分布可以通过激活JNK/SAPK被ROS修饰，因此是β-细胞葡萄糖毒性的潜在靶点。少

Glucose toxicity plays a major role in progressive deterioration ofβ-cell function in poorly controlled type 2 diabetes mellitus. Oxidative stress may in part explain this phenomenon because, under chronic hyperglycemia, reactive oxygen species (ROS) are increased in many tissues includingβ-cells. And we have reported that oxidative stress reduced insulin promotor activity and that administration of antioxidant showed beneficial effect in diabetic mice. To evaluate the role of oxidative stress in dysfunction of pancreatic β-cells in diabetes mellitus, we have tried to produce transgenic mice in that glutathione peroxidase (GSHPx) are overexpressed only in pancreatic β-cells. The transgene we constructed has the GSHPx gene driven by the insulin promoter so that selective expression of GSHPx in pancreatic β-cells would be achieved. In pancreatic islets of obtained transgenic mice, the specific overexpression of the GSHPx inβ-cell was verified by the immunohistochemical staining using ant … More i-GSHPx antibody. Then we obtained db/db obese diabctic mice overexpressing GSHPx in pancreaticβ-cells by crossing our transgenic mice to C57BL/Ks db/- mice. However, no difference was observed in anti-heme oxigenase immunohistochemical staining, which reflect intracellular oxidative stress, between control db/db mice and db/db mice overexpressing GSHPx. This result suggested that overexpressed GSHPx did not have activity enough to reduce the oxidative stress in the pancreas. Considering that C-terminal non-coding lesion is known to be responsible for GSHPx gene translation, we assumed that the C-terminal non-coding. lesion of the transgene did not work. Therefore, we designed new plasmid that has a longer C-terminal non-coding region and inserted the Flag-tag to allow monitoring of the translation of full-length protein.We have also investigated the mechanism how oxidative stress is implicated in glucose toxicity. Especially we saw the involvement of transcription factor PDX-1 (pancreas and duodenum homeobox factor-1). Because the reduction of PDX-1 expression is often observed simultaneously with the decrease in insulin content or β-cell numbers. Indeed, when antioxidant treatment improved β-cell function and glucose tolerance in diabetic mice, it also increased the amount of PDX-1 expressed in nuclei. Accordingly, we investigated the possibility that oxidative stress causes the nucleus-cytoplasm translocation of PDX-1 and thereby reduces the PDX-1 activity. In a β-cell derived cell line HIT-T15, overexpressed GFP (green fluorescent protein)-tagged PDX-1 was localized mostly in nuclei when cells were cultured under a regular condition. However, when oxidative stress was charged upon the cells by adding 50μM H_2O_2, PDX-1 was dispersed from nuclei and mainly found in cytoplasm and the addition of antioxidant (5OmM N-acetyl L-cysteine) reversed this phenomenon. Thus, it was shown that oxidative stress causes translocation of PDX-1 from nucleus to cytoplasm. On the other hand, overexpression of c-Jun amino-terminal kinase 1 (JNK1) could induce cytoplasmic translocation of PDX-1 without the presence of oxidative stress. Also, the overexpression of a dominant negative JNK1 almost totally prevented the oxidative stress-induced cytoplasmic translocation of PDX-1 in the H_2O_2-treated HIT-T15 cells suggesting the implication of JNK/stress-activated protein kinase (SAPK) pathway as a mediator of the translocation. Thus, our present observations indicate that the intracellular distribution of PDX-1 can be modified by ROS through activation of JNK/SAPK and therefore is a potential target of the β-cell glucose toxicity. Less