轉錄因子 FoxO6 在 3T3-L1 脂肪細胞分化中蛋白質表現量的變化;changes in the protein expression of the forkhead transcription factor FoxO6 during differentiation of 3T3-L1 fat cells

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Title:	轉錄因子 FoxO6 在 3T3-L1 脂肪細胞分化中蛋白質表現量的變化;changes in the protein expression of the forkhead transcription factor FoxO6 during differentiation of 3T3-L1 fat cells
Authors:	林松賢;Lin,Song-sian
Contributors:	生命科學系
Keywords:	FoxO6;沒食子酸酯型唲茶素酸酯;脂肪細胞分化;FoxO6;EGCG;3T3-L1;adipocyte differentiation
Date:	2014-07-14
Issue Date:	2014-10-15 14:38:52 (UTC+8)
Publisher:	國立中央大學
Abstract:	Forkhead box class O (簡稱 FoxO) 轉錄因子成員包含 FoxO1 、FoxO3 、 FoxO4 和 FoxO6 。已有很多文獻分別指出 FoxO 各成員在細胞中，調控著多種不同的功能，包括分化、能量代謝、細胞的修復與細胞週期等。在本論文中我們以地塞米松、胰島素及 1-甲基-3-異丁基黃嘌呤為分化劑，將3T3-L1前脂肪細胞分化成脂肪細胞的系統，探討該細胞分化過程中 FoxO6 蛋白質表現量的變化，在這為期十四天的分化過程中，我們發現其蛋白質表現量上升了一倍，並且在細胞質與細胞核中，觀察到其表現量分別上升了 0.5 倍與 11 倍。這些數據顯示 FoxO6 會隨著脂肪細胞分化的情況而改變其蛋白質表現。而由於有文獻指出綠茶兒茶素，特別是其中的表沒食子酸酯型唲茶素酸酯(簡稱 EGCG)，能夠減少分化所造成的 FoxO1 磷酸化，並且增加其轉錄活性，因此在這裡我們想探討是否在分化期間 EGCG 也會改變 FoxO6 或是其他 FoxO 成員蛋白質的表現量。我們發現以 5 µM 濃度的 EGCG 處理，會增加脂肪分化中第二天、第六天與第十二天時，細胞內 FoxO6 蛋白質的量。反之，以 25 µM 濃度的 EGCG 處理，則會傾向於抑制分化中第八天與第十天時，細胞內 FoxO6 蛋白質的量。這些結果證明 EGCG 對 FoxO6 蛋白質表達的影響，會與處理劑量及細胞分化階段相關。不過，我們也發現到以 5 µM 濃度的 EGCG 處理，雖然在分化中第十二天時，亦會增加 FoxO1 的蛋白表現量，但卻會減少 FoxO1 蛋白在分化中第二天時的表現量，並對第六天時的 FoxO1 蛋白表現量不造成影響。而在 FoxO3 蛋白方面，以 5 µM 濃度的 EGCG 處理，雖然在分化中第二天與第十二天時，也會增加其表現量，但卻會減少其在分化中第六天時的表現量。而以 25 µM 濃度的 EGCG 處理，則分別減少分化中第八天時 FoxO1 與分化中第十天 FoxO3 的蛋白表現量，卻對第十天時 FoxO1 與第八天時 FoxO3 的蛋白表現量沒有影響。這些實驗結果證明 EGCG 對不同種 FoxO 成員的影響是具有選擇性的，並且這種選擇性的影響會隨著細胞分化階段的不同以及 EGCG 處理的劑量和時間的不同而變。;Forkhead box class O (FoxO) transcription factors contained FoxO1, FoxO3, FoxO4, and FoxO6, and they have been respectively reported to regulate diverse cellular functions, including differentiation, energy metabolism, cell survival, and cell cycle. In this study using the preadipocyte-adipocyte differentiation system of 3T3-L1 cells induced by dexamethasone, insulin and 1-methyl-3-isobutylxanthine, we examined changes in the FoxO6 protein expression during the adipogenic process. We found that FoxO6 increased its protein levels by about 100% during the 14-day period of fat cell differentiation. Also, levels of cytosolic and nuclear FoxO6 were increased by 50% and 1100%, repectively. This suggests the differentiation-dependent protein expression of FoxO6. Because green tea catechins, especially (-)-epigallocatechin gallate(EGCG), were reported to reduce FoxO1 phosphorylation and increase its transcriptional activity, we studied herein whether EGCG altered protein levels of FoxO6 and other FoxO members during adipocyte differentiation. EGCG at 5 µM tended to increase FoxO6 protein levels at Day 2, 6, and 12 differentiating cells, while, at 25 µM, it tended to decrease FoxO6 protein expression at Day 8 and 10 differentiating cells. This suggests the dose- and differentiation stage-dependent effect of EGCG on FoxO6 protein expression. However, treatment with 5 µM EGCG decreased FoxO1 levels at Day 2 cells, unaltered FoxO1 levels at Day 6 cells, and increased FoxO1 at Day 12 cells, respectively. EGCG at 5 µM increased FoxO3 levels at Day 2 cells, decreased FoxO3 levels at Day 6 cells, and increased FoxO3 levels at Day 12 cells. Treatment with 25 µM EGCG decreased FoxO1 levels at Day 8 cells and unaltered FoxO1 levels at Day 10 cells, while it unaltered FoxO3 levels at Day 8 cells and decreased FoxO3 protein expression at Day 10 cells. These data suggest that EGCG selectively affects particular types of FoxO family members and the effect varies with the duration and dosage of EGCG treatment and with different differentiation stages of fat cells.
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