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標題: | 線蟲飲食轉換調節之三酸甘油酯酶的脂肪分解作用 ATGL-1-Dependent Lipolysis during Dietary Change in C. elegans |
作者: | Yen-Ling Chang 張宴菱 |
指導教授: | 吳益群(Yi-Chun Wu) |
關鍵字: | DA1877,脂肪代謝,脂肪分解作用,三酸甘油酯?, DA1877,fat metabolism,lipolysis,adipose triglyceride lipase (ATGL), |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 在生物體中,能量的調節對於發育各個時期是相當關鍵的。能量的累積與消耗經由脂肪代謝過程來進行,而脂肪代謝受到錯綜複雜的基因調控。這些調控脂肪的基因對於不同的營養素攝入如何反應目前尚未完全了解。油滴作為脂肪儲存的胞器存在於線蟲的腸道及表皮組織當中,油滴裡的中性脂肪(三酸甘油酯)透過脂肪分解作用(lipolysis)被降解。脂肪分解作用的第一步驟是由酵素三酸甘油酯酶(adipose triglyceride lipase; ATGL)催化釋放出一條脂肪酸鏈。在本研究裡,利用線蟲解析脂肪分解作用(lipolysis)在飲食改變體內的脂肪恆定 (lipid homeostasis)過程及現象中所扮演的角色。不同的細菌飲食已知造成線蟲當中的脂肪含量變化。我們首先建立BODIPY 493/503活體染色法以及DHS-3螢光蛋白標定線蟲為飲食研究中可用的脂肪分析工具,發現Comamonas DA1877餵食下的線蟲相比於標準食物Escherichia coli OP50餵食下的線蟲能觀察到腸道與表皮組織裡皆具有數量較少且尺寸較小的油滴。值得注意的是,atgl-1突變時造成DA1877飲食下的油滴明顯變大,OP50飲食下油滴則稍微變大,顯示atgl-1對於不同飲食的線蟲具有不同的貢獻,而此差異造成了不同飲食下線蟲體內脂肪含量變化的原因。我們進一步了解飲食如何造成ATGL-1有不同的貢獻,經由轉錄報導品種及定量反轉錄聚合酶鏈式反應(quantitative RT-PCR)證實atgl-1的基因轉錄表現在兩種食物餵食下是相同的。然而,根據西方墨點法的分析,ATGL-1蛋白表現量在DA1877餵食下的線蟲比起OP50餵食下較低,這可能是ATGL-1因應兩種飲食下的油滴大小所造成的效應。此外,因為ATGL-1坐落於油滴的表面,經由顯微鏡觀察發現在OP50和DA1877飲食中ATGL-1表現量的差異並非來自於蛋白坐落形式的不同。抑制蛋白酶體(proteasome)的實驗也說明不是ATGL-1降解速率(turnover rate)改變所造成。另一方面,AMPK被證實在DA1877飲食下會抑制ATGL-1蛋白表現。有趣的是,我們發現ATGL-1對於飲食轉換快速產生反應,推測其控制的脂肪分解能幫助生物因應飲食差異以達到新的脂肪動態平衡。此外,經由脂肪分解作用釋放的脂肪酸及其用途也被證明受到飲食的調控。本研究為飲食如何經由控管良好的脂肪分解作用進一步影響體內脂肪含量變化提供新的見解。 Energy regulation is crucial for developmental processes in the organisms. Accumulation and exploitation of energy through fat metabolism involves an intricate network of genes. How these fat-regulatory genes respond to different nutrients remains unclear. Lipid droplets as fat storage organelles are found in the intestine and hypodermis of C. elegans. Neutral lipids, mainly triacylglycerols, constitute lipid droplets which are decomposed by lipolysis. The initial rate limiting step of lipolysis is catalyzed by the enzyme adipose triglyceride lipase (ATGL) to release a single free fatty acid from substrate. In this study, we proposed that different bacterial diet may regulate fat storage through ATGL-1 dependent lipolysis in C. elegans. We first established BODIPY 493/503 staining protocol and ensured DHS-3::GFP labeling worms are suitable for dietary studies. By using these approaches, the results revealed that Comamonas DA1877-fed worms showed decreased lipid droplets in size and number in both of intestine and hypodermis compared to standard E. coli OP50-fed worms. Notably, atgl-1 mutation enlarged lipid droplets clearly on DA1877 but slightly on OP50, indicating that atgl-1 contributes differerently to diets-mediated lipid storage change. To understand how diets resulted in different ATGL-1 activities, we found the transcriptional level of atgl-1 is identical in both diets by reporter strain and quantitative RT-PCR analysis. Surprisingly, by microscopic observation, we demonstrated that ATGL-1 protein level is reduced in the worms fed DA1877 compared to OP50 while ATGL-1 localization patterns on lipid droplets were very similar between two diets-fed animals. These results which were also supported by Western blotting suggested that the ATGL-1 expression is adjusted to correlate with lipid droplet size regardless of the diets. We further demonstrated that the difference of ATGL-1 expression level on OP50 and DA1877 was not caused from changes of ATGL-1 turnover rate by proteasome inhibition experiments. The protein degradation pathway other than proteasome, such as lysosomal proteolysis could explain the differences. On the other hand, AMPK was identified to inhibit ATGL-1 expression in DA1877 diet. Moreover, we found that ATGL-1 is rapidly responsive to dietary switch for new lipid homeostasis achievement. Furthermore, in a pulse-chase experiment, the release of lipid droplets-derived fatty acids and subsequent location were demonstrated to be modulated by diets. The study here would provide new insights into how nutrient options affect lipid content alteration through well-regulated lipolysis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77843 |
DOI: | 10.6342/NTU201703885 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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