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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/66774


    Title: 抑制環狀核苷酸磷酸二酯酶 3 (PDE3)對 3T3-L1 脂肪細胞內蛋白質表現之影響;Effects of phosphodiesterase 3 (PDE3) inhibition on protein expression in 3T3-L1 adipocytes
    Authors: 王柏翔;Wang,Bo-xiang
    Contributors: 生命科學系
    Keywords: 環狀核苷酸磷酸二酯酶 3;脂肪細胞;蛋白質;phosphodiesterase 3;3T3-L1;adipocytes;protein
    Date: 2015-01-27
    Issue Date: 2015-03-16 15:12:11 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 環狀核苷酸磷酸二酯酶3 (phosphodiesterase 3;PDE3)在脂肪細胞中扮演重要的角色,藉由水解cAMP降低細胞內cAMP濃度可調節脂肪分解、脂肪生成與葡萄糖攝取等作用以維持細胞內能量平衡。已知增加脂肪細胞內cAMP的濃度會促進脂肪分解,相反的,降低cAMP則會阻礙脂肪分解並增加脂肪合成作用,然而在PDE3影響下會參與調控脂肪生成或代謝的蛋白質仍不甚清楚。本研究利用二維電泳與質譜技術分析鑑定脂肪細胞中受到PDE3調控的蛋白質。我們首先以PDE3抑制劑cilostazol與胰島素共同處理3T3-L1脂肪細胞18小時,之後萃取細胞內蛋白質並使用二維電泳分離,其膠體經銀染後顯示,有三個蛋白質表現強度上升,此電泳的果與胰島素單獨處理細胞之二維電泳分析結果比較顯示,有兩個蛋白質表現的上升是受到cilostazol調控所致。接著由質譜技術與胺基酸序列比對分析後,此兩個蛋白質身分為annexin a2 (ANX A2)與nucleoside diphosphate kinase b (NDPK-B),第三個蛋白質點無法被質譜方法鑑定出。我們進一步以real-time PCR分析兩個蛋白質的mRNA表現,在胰島素存在下,cilostazol也會促使其基因的表現上升。另外,我們也使用β-adrenergic receptor的刺激劑isoproterenol (ISO)與cilostazol共同處理脂肪細胞8小時,二維電泳與質譜分析結果顯示thioredoxin-1 (TXN-1)的表現呈明顯上升,相同的,在ISO存在下,cilostazol對TXN-1 mRNA表現也有一致的提升。根據文獻報導,TXN-1與NDPK-B均有抑制脂肪生成的作用,由此我們推測,cilostazol抑制脂肪生成可能是經由促進此二蛋白質表現所致。此外,ANX A2在細胞內可能與CLUT4運送有關,因此我們推測在胰島素刺激下,cilostazol促進ANX A2的表現可能會增加GLUT4的運送,但此作用仍有待進一步實驗證明。;Cyclic nucleotide phosphodiesterase 3B (PDE3B), a PDE3 family isozyme that hydrolyzes cAMP with high affinity, plays an important role in the regulation of energy homeostasis in adipocytes mainly through regulation of lipolysis, lipogenesis, gluconeogenesis, and glucose uptake. It is known that increasing intracellular cAMP concentration by inhibition of PDE3B triggers rapid lipid degradation, whereas decreasing cAMP level leads to antilipolytic action and lipogenesis. However, the proteins that are regulated by PDE3B in these lipid metabolic processes remain largely undefined. In this study, we used two-dimensional (2-D) gel electrophoresis and mass spectrometry to identify the proteins expressed in mouse 3T3-L1 adipocytes in the presence of the PDE3 inhibitor cilostazol. The initial results showed that three proteins were upregulated in 3T3-L1 cells after treatment with cilostazol and insulin for 18 h. Comparing to the 2-D gel results derived from the cells treated with insulin alone indicates that two of the three proteins were induced in expression by cilostazol. Following the mass spectrometry analysis, these proteins were identified as annexin a2 (ANX A2) and nucleoside diphosphate kinase b (NDPK-B). The third protein upregulated in the presence of cilostazol and insulin was not identified by the method. Further analysis by real-time PCR revealed that ANX A2 and NDPK-B mRNA expression was also upregulated by cilostazol. In a separate experimental setting, 3T3-L1 adipocytes were treated with cilostazol and the β-adrenergic receptor agonist isoproterenol (ISO) for 8 h, followed by 2-D gel analysis. The results showed that in the presence of ISO one protein was upregulated by cilostazol, and mass spectrometry identified it as thioredoxin-1 (TXN-1). This induction in TXN-1 protein also was associated with an increase in mRNA expression. Both TXN-1 and NDPK-B have been shown to inhibit lipogenesis in adipocytes. Thus, we rationalize that inhibition of PDE3B to induce TXN-1 and NDPK-B expression is a mechanism underlying the blockage of lipogenesis by cilostezol. In addition, ANX A2 is implicated in GLUT4 translocation in adipocytes, pointing to a potential role of PDE3 inhibitors in regulation of glucose transport via enhancing ANX A2 expression. This proposed effect, however, awaits further experimental attestation.
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