YC-1誘導大鼠脂肪細胞進行脂肪分解之機制研究

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題名:	YC-1誘導大鼠脂肪細胞進行脂肪分解之機制研究 Mechanisms of YC-1 Induced Lipolysis in Rat Adipocytes
作者:	王克銓 Ke-Chuan Wang
貢獻者:	醫學研究所
關鍵詞:	大鼠脂肪細胞脂肪分解 adipocyte lipolysis YC-1
日期:	2005
上傳時間:	2009-09-11 15:40:08 (UTC+8)
摘要:	YC-1 (3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole) 是一種化學合成物，可以活化guanylyl cyclase (GC) 並增加細胞內cGMP與cAMP的濃度。脂肪細胞是體內能量儲存與調控內分泌的重要細胞之一。根據已往的研究指出在促進脂肪細胞進行脂肪分解的途徑中，荷爾蒙先透過與 adrenoceptor的結合活化adenylate cyclase (AC) 使細胞內cAMP濃度上升，並經由cAMP-dependent protein kinase (PKA) 途徑磷酸化hormone-sensitive lipase (HSL) 與perilipin來達到誘導脂肪分解的作用。可是近年來發現除cAMP之外，cGMP也對於脂肪分解以及其他與脂肪分解有關之蛋白質也有影響，但其機制並不十分清楚。因此我們以YC-1 (guanylyl cyclase activator) 作為增加脂肪細胞內cGMP含量之刺激物質，來探討YC-1與脂肪分解之相關機制。本研究以分離出的大鼠初級脂肪細胞為研究對象，經YC-1處理2小時後分別測定游離脂肪酸與甘油之釋放量，以探討YC-1是否與脂肪分解有關。另外透過兩種抑制劑：KT5823 (PKG inhibitor) 與KT5720 (PKA inhibitor) 來研究YC-1誘導脂肪分解之途徑，是否為增加cGMP而活化PKG來調控，還是間接透過其他途徑增加cAMP而活化PKA，因而造成脂肪分解。接著再以ODQ (guanylyl cyclase inhibitor)處理，抑制YC-1與guanylyl cyclase結合而引起的作用，同時並以EIA測定大鼠脂肪細胞內cGMP與cAMP的濃度，藉此來驗證YC-1與脂肪分解的關聯性。我們的研究結果發現，以60 M YC-1刺激2小時有顯著促進脂肪分解的效果，並且由抑制劑的處理證實YC-1誘導脂肪分解是經由PKA途徑而不透過PKG途徑。此外，進一步抑制guanylyl cyclase後發現可以抑制YC-1促進脂肪分解的能力，故在脂肪細胞中YC-1是透過guanylyl cyclase來作用。接著由EIA測定發現YC-1可以增加細胞內cGMP與cAMP的濃度，而且cGMP的濃度與顯著上升的時間點皆高於且優先於cAMP；另以ODQ抑制guanylyl cyclase則會明顯抑制cGMP的產生。另外加入insulin活化PDE3B後，發現可以抑制由YC-1所促進的脂肪分解現象。最後透過western blotting的方式，證實YC-1誘導脂肪分解的途徑與ERK無關，僅與cAMP-PKA途徑有關。由我們的結果證實YC-1與脂肪分解有關，且透過活化guanylyl cyclase後可以藉由增加的cGMP來抑制PDE3B而減少cAMP的水解，間接導致脂肪細胞內cAMP濃度的增加，進而活化PKA而產生脂肪分解的作用。所以本實驗透過YC-1的處理，驗證了在大鼠脂肪細胞中cGMP與脂肪分解的關係，並希望以此cGMP途徑可以提供研究脂肪分解的另一種新的方向。 YC-1 (3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole), a synthetic chemical compound, has been identified as a direct activator of guanylate cyclase (GC), and can increase intracellular cGMP and cAMP level. Adipocyte is one of the important cells to store energy (in the form of triacylglycerol) and regulate endocrine release. Previous studies have demonstrated that -adrenoceptor of adipocytes are responsible to lipolysis by activating adenylate cyclase (AC) and elevating intracellular cAMP content. The rate-limiting hormone-sensitive lipase (HSL) and perilipin are phosphorylated and activated by PKA. Recently, elevation of intracellular cGMP levels is found to be involved in lipolytic process of adipocyte, but the mechanism is not clear. We used YC-1 (an activator of guanylate cyclase) to increase cGMP concentration, and determined whether YC-1 could improve lipolytic reaction in isolated rat adipocyte. Free fatty acid and glycerol were quantified by spectrophotometry after YC-1 was administrated. In addition, we used PKG inhibitor (KT5823) and PKA inhibitor (KT5720) to examine the mechanism of YC-1 induced lipolysis. Pretreatment with ODQ, a guanylyl cyclase inhibitor, determined whether YC-1 could affect guanylyl cyclase on adipocyte directly. In this study, we demonstrated that YC-1 (60 M) treated for 2 hr could induce lipolysis in rat adipocyte. The lipolytic reaction was PKA dependent pathway. Furthermore, we found that ODQ could decrease lipolysis and intracellular cGMP, cAMP content. YC-1 induced lipolysis in rat adipocytes through an ERK-independent mechanism by western blotting analysis. Based on the above observation, we suggested that YC-1 induced lipolysis in rat adipocytes through cAMP-PKA dependent pathway not PKG or ERK pathway. We hope this study may provide much information about the regulation mechanisms of cGMP in adipocytes.
資料類型:	thesis
顯示於類別:	[醫學科學研究所] 博碩士論文

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