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Title: | 不同營養分對3T3-L1脂肪細胞胰島素敏感性之影響 The effect of different nutrients on insulin sensitivity in 3T3-L1 adipocytes |
Authors: | Chi-Jung Chwang 章起榕 |
Advisor: | 丁詩同(Shih-Torng Ding) |
Keyword: | 脂肪細胞,胰島素敏感性, adipocytes,insulin sensitivity, |
Publication Year : | 2005 |
Degree: | 碩士 |
Abstract: | 胰島素敏感性的下降時,葡萄糖運送子 4(glucose transporter 4, GLUT4)位於細胞膜濃度降低,導致細胞無法有效地利用葡萄糖。移動至細胞膜上則是經由 PI3-kinase pathway 來進行。
本研究的目的在建立大鼠脂肪細胞與脂肪前身細胞的初代培養環境,並研究營養分影響脂肪細胞胰島素抗性的發生。利用豬的脂肪前身細胞培養系統為基礎,已成功地建立大鼠脂肪細胞初代培養系統及脂肪前身細胞之分化系統。 添加 thiazolidinediones(TZD)、葡萄糖、葡萄糖胺(glucosamine)、油酸(oleic acid)及二十二碳六烯酸(docoxahexaenoic acid)等不同的營養分於 3T3-L1 脂肪細胞,以研究此等營養分對胰島素抗性的影響。3T3-L1 脂肪細胞分別培養於含有 5 mM 葡萄糖之 DMEM(Dulbecco’s Modified Eagle Medium),並添加 5% FBS(fetal bovine serum)做為胰島素來源及不同之營養分 48 小時。藉由測定培養液中的甘油含量,來比較 3T3-L1 脂肪細胞的解脂能力。 此外,我們偵測幾個參與 PI3-kinase pathway 的基因 mRNA 在細胞的表現量,分別是胰島素受體(insulin receptor, IR)、胰島素受體受質 1(insulin receptor substrate 1, IRS-1)、PI3-K及 GLUT4。試驗中亦檢測兩個脂肪細胞所分泌並參與能量平衡的蛋白質,瘦體素(leptin)及脂肪細胞結合素(adiponectin)其 mRNA 的表現量。此外,並利用西方點墨法測定細胞膜上含有的 GLUT4 蛋白質含量,用以評估 GLUT4 在各營養分刺激後位移的情形。 試驗中,TZD 的刺激顯著降低降低細胞培養液中甘油的含量,而葡萄糖的刺激則增加了甘油在培養液中的含量,顯示在體外培養的環境下,TZD 的刺激能增加胰島素抑制細胞解脂的能力,而高濃度的葡萄糖則降低了胰島素的功能。葡萄糖胺的刺激則增加了細胞膜上 GLUT4 的含量,但是葡萄糖胺是否能增加胰島素的功能仍須更進一步的試驗釐清。本試驗中,各營養分對參與 PI3-kinase pathway 的基因 mRNA 表現量並無顯著影響,然而除了葡萄糖胺之刺激對脂肪細胞結合素 mRNA 含量沒有影響,其餘營養分刺激,皆使脂肪細胞結合素 mRNA 含量上升。結果顯示,試驗中所使用之營養分藉由影響脂肪細胞結合素的表現而改變脂肪細胞的生理功能。 本試驗亦利用女貞子、紅景天、荔枝核、木香及朴子草等中草藥之萃取物處理脂肪細胞。結果發現,數種中草藥之萃取物對於脂肪細胞解脂能力及 GLUT4 在細胞膜上的含量,並無顯著之影響。其中利用紅景天萃取物培養脂肪細胞,會降低脂肪細胞中 IR、IRS-1 及 GLUT4 mRNA 的濃度,推測利用紅景天培養脂肪細胞,會藉由減少參與 PI3-kinase pathway 的各基因表現量,來降低 PI3-kinase pathway 中訊息的傳導。然而試驗中所使用之中草藥萃取物,參與 PI3-kinase pathway 的詳細機制仍待釐清。 Type II diabetes accounts for 90 to 95% of diabetes patients. Muscle and adipose tissue in such individuals have a decreased insulin sensitivity. Decrease in insulin sensitivity reduces translocation of glucose transporter 4(GLUT4) protein to cell membrane upon insulin stimulation. Therefore, cells can not uptake glucose effectively. Insulin modulates animal physiology through a phosphatidylinositol-3-kinase pathway(PI3-kinase pathway) and a mitogen-activated protein kinase pathway(MAP-kinase pathway). The major regulation of GLUT4 translocation from intracellular vesicle to cell membrane is generated from PI3-kinase pathway. The purpose of this study were to establish primary cell culture systems for rat adipocytes and preadipocytes and to study the effect of different nutrients on insulin sensitivity in rodent adipocytes. In the current study, thiazolidinedione(TZD), glucose, glucosamine(GlcN), oleic acid(OA), and docosahexaenoic acid(DHA)were used to treat 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in DMEM containing 5 mM glucose plus 5% fetal bovine serum(FBS)as insulin source with different nutrients for 48 hours. Concentration of glycerol in the cultured medium was measured to evaluate the ability of lipolysis in 3T3-L1 adipocytes. In addition, we measured the mRNA concentrations of insulin receptor(IR), insulin receptor substrate 1(IRS-1), PI3-K and GLUT 4, genes that are participated in the PI3-kinase pathway. We also measured mRNA concentrations of adiponectin and leptin, two secretive proteins in adipocytes involving energy balance. Western blot was used to determine the concentration of GLUT4 protein on cell membrane in order to estimate the degree of GLUT4 translocation. In our study, TZD treatment significantly reduced the concentration of glycerol in cultured medium whereas glucose treatment increased the glycerol concentration, indicating that TZD may increase insulin function to reduce lipolysis and high concentration of glucose may reduce insulin function to increase lipolysis in vitro. GlcN treatment increased the GLUT4 protein level on the cell membrane. Whether GlcN can increase insulin function requires further experiment to demonstrate. TZD, glucose, OA and DHA increased the steady state adiponectin mRNA concentration suggesting that theses nutrients may influence cellular function though manipulating the expression of aiponectin. Several Chinese herbs extracts, Ligustri Fructus, Rhodiola, Lychee Seed, Costus Root, and India Abutilon were used to treat 3T3-L1 adipocytes. We found no significant effect for these herbs on lipolysis and GLUT4 protein concentration on the cell membrane. Rhodiola reduced IR, IRS1, and GLUT4 mRNA concentrations, suggesting that Rhodiola may downregulate the PI3-kinase pathway signal transduction by reducing the mRNA concentrations of the genes involved in this pathway. However, the mechanism of these herbs extracts on PI3-kianse pathway in adipocytes requires further demonstration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36634 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 動物科學技術學系 |
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