| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1998: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Research Abstract |
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
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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
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