c9,t11,t13-共軛次亞麻油酸對肝臟脂質代謝之影響

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題名:	c9,t11,t13-共軛次亞麻油酸對肝臟脂質代謝之影響 Effects of c9,t11,t13-conjugated linolenic acid on hepatic lipid metabolism
作者:	鄒泊諺;Po-Yen Tsou
貢獻者:	營養學系碩士班
關鍵詞:	c9;t11;t13-CLN;PPARα;三酸甘油酯;AMPK;cis9;trans11;trans13-conjugated linolenic acid;Peroxisome proliferator- activated receptor α;triglycerides;AMPK
日期:	2014-01-23
上傳時間:	2014-10-02 09:44:31 (UTC+8)
出版者:	中國醫藥大學
摘要:	本實驗室先前動物研究發現苦瓜籽油 (BMSO)有降肝脂功效，但功能成分與作用機制並不清楚。由於BMSO富含c9, t11, t13-共軛次亞麻油酸(cis9, trans11, trans13- conjugated linolenic acid; c9, t11, t13- CLN)，因此本論文將研究c9, t11, t13- CLN是否能夠降低肝細胞TG堆積並探討其作用機制。實驗一利用動物實驗證實BMSO 降肝脂功效，並初步提供肝臟PPARα活化證據。將C57BL/6J小鼠分別餵食低脂(5% soybean oil ; LF)、高脂(22.5% soybean oil ; HS)及高脂添加BMSO(11.5% soybean oil +11.5% BMSO；HBM)飼料10天或 8週。結果8週時HBM組肝臟TG顯著低於HS組，與LF無差異；且不論短期或長期均可見HBM組肝臟PPARα活化指標Acyl-CoA oxidase酵素活性增加，表示BMSO降低肝臟TG可能與PPARα活化有關。實驗二使用HepG2嘗試建立c9, t11, t13-CLN降低肝細胞TG之模式並探討其作用機制。MTT顯示細胞耐受c9, t11, t13-CLN最高劑量為25 μM，c9, t11, t13-CLN效果與含相同碳及雙鍵數目之LN (18:3, n-3) 相較，結果顯示CLN並無顯著降低18:3引起肝細胞TG堆積；脂肪酸β氧化指標活性，不論是24或48小時，各組間皆無顯著差異；但CLN有降低de novo lipogenesis關鍵酵素Fatty acid synthase (FASN)活性趨勢。為確認HepG2是否為PPARα responsive細胞株，將HepG2處理已知PPARα活化物clofibrate後測定活性，結果確認HepG2為 PPARα non-responsive。實驗三使用PPARα responsive細胞株 H4IIEC3 再次建立c9, t11, t13-CLN降低肝細胞TG之模式並探討其作用機制。H4IIEC3處理不同濃度clofibrate，可劑量反應性增加活性。將細胞處理不同比例的18:3與c9, t11, t13-CLN (脂肪酸總濃度控制100 μM) 24及72小時，CLN劑量反應性抑制細胞內TG堆積、促進ACOX活性與抑制FASN活性，qRT-PCR證實CLN抑制脂質新生與促進脂肪酸氧化，涉及轉錄因子PPARα活化與下調SREBP-1、ChREBP及LXRα基因表現。此外，CLN可活化AMPK，促進ACC磷酸化失活。本研究證實了c9, t11, t13-CLN可能是BMSO降肝脂功能成分之一，其機制涉及PPARα與AMPK活化，因此促進脂肪酸氧化及抑制脂質新生作用達降低肝臟TG之功效。 The anti-steatosis effect of bitter melon seed oil (BMSO) has previously been shown in animals studies, without knowing the underlying mechanisms and the functional components. Since BMSO is enriched in cis9, trans11, trans13-conjugated linolenic acid (c9, t11, t13-CLN) fatty acid, also known as α-eleostearic acid, this study aimed at investigating whether the TG accumulation in hepatocytes can be attenuated by c9, t11,t13-CLN, and the working mechanism was studied as well. Experiment 1 was set to demonstrate the BMSO has anti-steatosis effect which was associated with PPARα activation. C57/B6J mice were separated into 3 groups to receive a low-fat control (5% soybean oil; LF), a high-fat control (22.5% soybean oil; HS) or a high-fat experimental (11.5% soybean oil +11.5% BMSO, HBM) diet, respectively, for 10 days or 8 wk. As a result, the liver TG content of HBM group was significantly lower than that of the HS group, with a level similar to that of the LF group. The marker for PPARα activation, i.e. Acyl-CoA oxidase (ACOX) activity, showed that the HBM group had the highest PPARα activation in livers regardless treatment time. In Experiment II, we used HepG2 to establish a cell culture model to show the TG-lowering effect of c9, t11, t13-CLN. The MTT studies showed the tolerable concentrations of c9, t11, t13-CLN for HepG2 was ?T25 μM. Considering c9, t11, t13-CLN is a fatty acid, the effect of c9, t11, t13-CLN on cellular TG and lipid metabolism was compared to those of its non-conjugated counterpart α-linolenic acid (LN), which has the same number of carbon atoms and double bonds as CLN. Results showed c9, t11, t13-CLN had no effect on the TG accumulation caused by LN. Compared with LN, the ACOX activity was not affected by c9, t11, t13-CLN, when the cells were treated with fatty acids for 24 or 48 hr. However, the enzyme activity of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, tented to be lowered by c9, t11, t13-CLN. To verify whether HepG2 is a PPARα responsive cell line, HepG2 was treated with a well-known PPARα activator, i.e. clofibrate, and ACOX activity was measured. Our results showed HepG2 is a PPARα non-responsive cell line, and this might compromise the TG-lowering effect of c9, t11, t13-CLN seen in this study. Subsequently, we conducted Experiment III. By using a PPARα responsive cell lines, H4IIEC3, a cell culture mode to show c9, t11, t13-CLN-mediated TG lowering effect and the plausible working mechanism were investigated. When H4IIEC3 was treated with clofibrate at different concentration for 48 and 72 hr, ACOX activity increased in a dose dependent manner. When cells were treated with a combination of LN and CLN (total concentration fixed at 100 μM) at different ratio, the intracellular TG was significantly reduced, dose dependently, by CLN. Treatment of CLN significantly increased the ACOX activity and reduced the FASN activity. Data of qRT-PCR confirmed the CLN-mediated upregulation of genes associated with fatty acid catabolism and doweregulation of genes associated with lipogenesis, might be linked with alterations in transcription factors, such as an increase in PPARα transcriptional activity and repressed SREBP-1c, ChREBP and LXRα gene expression. In addition, CLN activated AMPK and increased ACC phosphorylation, which resulted in ACC inactivation. Summary, this study demonstrate that c9, t11, t13-CLN is one of the functional components of BMSO-mediated TG lowering effect in liver. The hypo-lipidative effect of c9, t11, t13-CLN is associated with PPARα and AMPK activation, thus leading to an increased fatty acid catabolism and reduced lipogenesis, and consequently, lowering TG content in liver.
顯示於類別:	[營養學系暨碩士班 ] 博碩士論文

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