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研究生:劉榮勝
研究生(外文):Jung-Sheng Liu
論文名稱:Okadaicacid誘發大鼠脂肪細胞脂肪分解時細胞內perilipin與beta-actin的含量變化
論文名稱(外文):Effects of okadaic acid-induced lipolysis on lipid droplet-associated perilipin and beta-actin in rat adipocytes
指導教授:馮琮涵馮琮涵引用關係
指導教授(外文):Tsorng-Harn Fong
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:58
中文關鍵詞:脂肪細胞beta-actin脂肪分解okadaic acidperilipin
外文關鍵詞:adipocytesbeta-actinlipolysisokadaic acidperilipin
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中文摘要
(Abstract in Chinese)
已有研究證實okadaic acid處理脂肪細胞,可以誘發hormone-sensitive lipase (HSL)的分佈位置改變,由平滑內質網轉移至脂肪滴表面,進而促進脂質分解。但是其對於其他重要的脂肪滴表面蛋白質(如perilipins與beta-actin)的影響卻尚未有研究報告。所以本實驗擬探討在okadaic acid處理脂肪分解反應發生時,分析perilipins及beta-actin這兩種重要的脂肪滴表面蛋白質的含量變化,並探討可能的作用機轉。本實驗取材自大白鼠的脂肪組織,分離出脂肪細胞,加入藥物isoproterenol以及okadaic acid處理,並以測量甘油的釋放量作為評估脂質分解的效果。結果顯示isoproterenol的正反應對照組以及okadaic acid的實驗組,隨著藥物處理的時間逐漸增加,甘油的釋放量都逐漸上升。兩種藥物同時處理時,甘油的釋放量則有顯著的加成作用,顯示兩種藥物可能經由不同訊息傳遞途徑引發脂質分解。藥物處理後分離出脂肪細胞內的脂肪滴,並且利用電泳(SDS-PAGE)與免疫轉漬方法分析脂肪滴表面蛋白質中的perilipins以及beta-actin的含量,結果發現以isoproterenol或是okadaic acid處理皆會導致脂肪滴表面的perilipins含量下降;但是脂肪滴表面的beta-actin含量卻無明顯改變。此外,利用PKA的抑制劑(KT 5720), PKC的抑制劑(calphostin C)與PKG的抑制劑(KT 5823),皆無法有效抑制okadaic acid所引發的脂解作用;然而以vanadate (protein phosphotyrosine phosphatase的抑制劑)處理卻可以有效地抑制okadaic acid誘發的脂解反應。推測okadaic acid可能不是經由已知的PKA, PKC, PKG等途徑,而是經由抑制protein phosphatase type 1 (PP1)與type 2A (PP2A)的途徑,分別活化perilipins與HSL,進而誘發perilipins脫離脂肪滴表面,同時促進HSL轉移至脂肪滴表面加速脂質分解。然而vanadate抑制okadaic acid誘發脂質分解的機轉仍待研究深入探討。

關鍵字:脂肪細胞, beta-actin, 脂肪分解, okadaic acid, perilipin
Abstract
Previous studies have reported that okadaic acid stimulated lipolysis by improving translocation of hormone-sensitive lipase (HSL) from endoplasmic reticulum to surface of intracellular lipid droplets. However, the effects of okadaic acid on lipid droplet-associated proteins (perilipins and beta-actin) remain unknown. The purpose of this study is to investigate the changes of perilipins and beta-actin after stimulation of okadaic acid. A adipocytes were isolated from rat epididymal fat pads and treated with isoproterenol and/or okadaic acid. Lipolysis was estimated by the measurement of glycerol release. Both isoproterenol (as a positive control) and okadaic acid were found to stimulate lipolysis via a time-dependent manner. The augment of lipolysis was observed in the presence of combination of the two drugs. Therefore, we suggested isoproterenol and okadaic acid might induce lipolysis by different pathways. After drug treatment, intracellular lipid droplets were purified and then lipid droplet-associated perilipins and beta-actin were analyzed by SDS-PAGE and Western blot. Perilipins were decreased in response to isoproterenol or okadaic acid, but beta-actin was no significantly changed neither stimulated or unstimulated cells. In addition, administration of KT 5720 (a PKA inhibitor), calphostin C (a PKC inhibitor) or KT 5823 (a PKG inhibitor) cannot effectively inhibit okadaic acid-induced lipolysis. However, vanadate (a protein phosphotyrosine phosphatase inhibitor) significantly inhibited okadaic acid-mediated lipolysis. Our data suggested that okadaic acid might activate perilipins and HSL by inhibition of type 1 and type 2A protein phosphatases, respectively, but not by activation of PKA, PKC, and PKG. Activated perilipins detach from lipid droplets and activated HSL translocate to lipid droplets, and then accelerate lipolysis. However, the mechanism of inhibition of vanadate on okadaic acid-induced lipolysis still needs to be clarified.

Key words: adipocytes; beta-actin; lipolysis; okadaic acid; perilipin
目 錄
致謝………………………………………………….……………..….….Ⅰ
目錄…………………………………………………………….…..….….Ⅲ
中文摘要(Abstract in Chinese)…………………………………..….…..Ⅴ
英文摘要(Abstract in English)………………………………….………Ⅶ
縮寫表(Abbreviations)……………………………..…………….…..….Ⅸ
第一章 緒論(Introduction)……………………………….…………........1
1.1. 脂質(Fat)…………………………………………………..….2
1.2. 脂肪細胞(Adipocyte)…………………………………...….....2
1.3. 脂肪分解(Lipolysis) …………………………………….........3
1.4. 脂肪分解相關蛋白(Lipolysis associated proteins)…….…...4
(1) Hormone sensitive lipase (HSL)………………………..........4
(2) 脂肪滴表面蛋白質(Perilipin) ……………………….……...5
(3) 貝它型肌動蛋白(beta-actin) ……………………….…….…6
1.5. 藥物okadaic acid…………………………………….…..….7
1.6. 研究目的…………………………………………..…….........8
第二章 材料與方法(Materials and Methods)…………………….....….9
2.1. 分離脂肪組織與脂肪細胞……………………………..…..10
2.2. 藥物isoproterenol與okadaic acid的處理方式………....11
2.3. MTT Assay以測定細胞存活率……………………….....12
2.4. 甘油濃度之測定…………………………………..…….….13
2.5. 脂肪滴表面蛋白質的分離與電泳分析..…………………..14
2.6. 脂肪滴的免疫螢光染色…………………………. …….….16
2.7. 統計分析……………………………………………………16
第三章 結果(Results)……………………………………………….…..17
3.1. Isoproterenol誘發脂肪分解……………………………….17
3.2. Okadaic acid誘發脂肪分解………………………………..17
3.3. 脂肪滴表面perilipins與beta-actin含量的變化………...18
3.4. 脂肪滴表面perilipins與beta-actin分布位置的變化…...19
3.5. Okadaic acid促進脂質分解的訊息傳遞途徑……………..20
第四章 討論(Discussion)………………………………………………..21
第五章 未來研究方向(Future Directions)……………….…………….27
圖表(Figures) …………………………….………………………….......29
參考文獻(References) …………………………………………………..42
參考文獻 (References)

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