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

过表达抗氧化酶的转基因 db/db 肥胖糖尿病小鼠胰腺 β 细胞葡萄糖毒性的分子机制。

• 批准号: 10470231
• 负责人: YAMASAKI Yoshimitsu
• 金额: $ 3.26万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470231/
• 关键词: diabetes mellitus; antioxidative enzyme; transgenic mouse; glucose toxicity; oxidative stress; transcription factor; stress activated protein kinase

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

葡萄糖毒性在控制不佳的2型糖尿病中β细胞功能进行性恶化中起主要作用。氧化应激可能部分解释了这种现象，因为在慢性高血糖症下，包括β细胞在内的许多组织中的活性氧（ROS）增加。我们已经报道了氧化应激降低了胰岛素促进剂的活性，并且抗氧化剂的施用在糖尿病小鼠中显示出有益的效果。为了评价氧化应激在糖尿病胰腺β细胞功能障碍中的作用，我们尝试制备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。我们构建的转基因具有由胰岛素启动子驱动的GSHPx基因，从而实现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，通过免疫组织化学染色证实GSHPx在β细胞中特异性过表达， ...更多信息 i-GSHPx抗体。然后将我们的转基因小鼠与C57 BL/Ks db/-小鼠杂交，获得了胰腺β细胞中过表达GSHPx的db/db肥胖糖尿病小鼠。然而，在反映细胞内氧化应激的抗血红素氧合酶免疫组织化学染色中，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。该结果表明，过表达的GSHPx没有足够的活性来降低胰腺中的氧化应激。考虑到已知C端非编码损伤负责GSHPx基因的翻译，我们假设C端非编码损伤。转基因的损伤不起作用。因此，我们设计了一个新的质粒，有一个更长的C-末端非编码区，并插入Flag-tag，以允许监控全长蛋白的翻译。我们还研究了氧化应激如何参与葡萄糖毒性的机制。特别是我们看到了转录因子PDX-1（胰腺和十二指肠同源盒因子-1）的参与。因为PDX-1表达的减少通常与胰岛素含量或β细胞数量的减少同时观察到。事实上，当抗氧化剂治疗改善糖尿病小鼠的β细胞功能和葡萄糖耐量时，它也增加了细胞核中表达的PDX-1的量。因此，我们研究了氧化应激导致PDX-1的核质易位从而降低PDX-1活性的可能性。在β细胞来源的细胞系HIT-T15中，当在常规条件下培养细胞时，过表达的GFP（绿色荧光蛋白）标记的PDX-1主要定位在细胞核中。当加入50μM的H_2O_2对细胞进行氧化应激时，PDX-1从细胞核中分散出来，主要分布在细胞质中，而加入抗氧化剂（50 mM的N-乙酰L-半胱氨酸）可逆转这一现象。因此，显示氧化应激导致PDX-1从细胞核易位至细胞质。另一方面，c-Jun氨基末端激酶1（JNK 1）的过表达可以诱导PDX-1的胞质易位，而不存在氧化应激。此外，显性负性JNK 1的过表达几乎完全阻止了H_2O_2处理的HIT-T15细胞中氧化应激诱导的PDX-1胞质易位，提示JNK/应激激活蛋白激酶（SAPK）途径可能是该易位的介导者。因此，我们目前的观察表明，活性氧可以通过激活JNK/SAPK来修饰PDX-1的细胞内分布，因此是β细胞葡萄糖毒性的潜在靶点。少

项目成果

Kaneto H, et al.: "Oxidative stress induces p21 expression in pancreatic islet cells : possible implication in beta-cell dysfunction"Diabetologia. 42(19). 1093-1097 (1999)

Kaneto H 等人：“氧化应激诱导胰岛细胞中的 p21 表达：可能与 β 细胞功能障碍有关”Diabetologia。

Kishimoto M,et al: "Sensitive,selective gas chromatographic-mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol -priodoping activity." J.Chromatogr.Biomed.668. 1-10 (1997)

Kishimoto M 等人：“使用三氟乙酰基衍生物和稳定同位素进行灵敏、选择性气相色谱-质谱分析，用于研究组织山梨醇 -priodoping 活性。”

Kaneto H,et al: "Expression of HB-EGF-like during pancreas development : a potential role of PDX-1 in the transcriptional activati" J.Biol.Chem.272 46. 29137-29143 (1997)

Kaneto H 等人：“胰腺发育过程中 HB-EGF 样的表达：PDX-1 在转录激活中的潜在作用”J.Biol.Chem.272 46. 29137-29143 (1997)

Fujitani Y, et al.: "Identification of a portable repression domain and an E1A-responsive activation domain on pax4 : a possible role of pax4 as a transcriptional repressor in the pancreas"Molecular and Cellular Biology. 19(12). 8281-8291 (1999)

Fujitani Y 等人：“pax4 上便携式抑制结构域和 E1A 响应激活结构域的识别：pax4 作为胰腺中转录抑制因子的可能作用”分子和细胞生物学。

Kaneto H. et al.: "Benefical effects of antioxidant in diabetes : possible protection of pancreatic beta-cells against glucose toxicity"Diabetes. 48(12). 2389-2406 (1999)

Kaneto H. 等人：“抗氧化剂对糖尿病的有益作用：可能保护胰腺 β 细胞免受葡萄糖毒性”糖尿病。