探討 PPARα 促效劑對高脂飲食誘發小鼠代謝失調及情緒障礙之影響

Effects of PPARα agonists on high-fat diet induced metabolic disorders and emotional disturbance in mice

李世欽(Shih-Chin Lee)

指導教授 ： 林甫容

國立臺灣大學/生命科學院/生化科技學系/碩士(2017年)

https://doi.org/10.6342/NTU201703648

摘要

邁入 21 世紀的我們所面臨很大的危機就是逐年加劇的肥胖問題，肥胖為許多慢性疾病之危險因子，也是全球重要的公共衛生議題。過氧化物酶體增殖物活化受體α (Peroxisome proliferator-activated receptor alpha, PPARα) 為核受器的一員，可受配體 (ligands) 活化進而調控脂質代謝以達到改善肥胖及代謝症候群的效果。故本實驗旨在以 PPARα 轉錄活性分析平台對中草藥及海洋真菌萃取物做功效評估，並進一步篩選活性高之 PPARα 促效劑應用於飲食誘導肥胖之小鼠模式，觀察是否有改善代謝症候群、非酒精性脂肪肝及情緒失調的現象。
本實驗的第一部分以 PPARα 轉錄活性分析細胞實驗篩選自然界純物質及粗萃物成為 PPARα 促效劑潛力。結果顯示牛樟芝內的三萜類化合物 antcin B、C、H、K 均有具有顯著活化 PPARα 之能力，其中以 antcin B、H、K 活化效果最佳，約為正控制組 (1 µM WY14643) 的 120% 左右；此外自然界天然萃物薄姜木、芋葉括樓、台灣鬼督郵及海洋真菌萃取物 (NTOU4295、NTOU4296) 皆具有顯著活化 PPARα 之效果，活化倍率約為正控制組的 40-60%；牛樟芝內麥角固醇 EK100 雖不具有活化 PPARα 之能力，但先前研究發現其有抗發炎、降血糖及降血脂之功能，故在第二部分動物實驗選擇以 antcin K 以及 EK100 輔以正控制組降血脂藥物 fenofibrate 進行後續實驗，旨於探討給予 PPARα 促效劑於高脂飲食誘發小鼠代謝失調及情緒障礙之影響。
第二部份動物實驗採用 4 週齡大 C57BL/6J 公鼠，以 30% 高脂飼料餵食 8 週誘導肥胖，後續小鼠仍餵食高脂飲食但另以管餵方式給予 20 mg/kg/day BW 之 antcin K以及 EK100 持續 4 週，之後增大劑量為 40 mg/kg/day BW 再持續 4 週，另一組給予 PPARα 促效劑 250 mg/kg/day BW 之 fenofibrate 持續 8 週當正控制組，試驗期共 16 週。結果顯示，高脂飲食成功誘導小鼠肥胖，給予 fenofibrate 可顯著降低小鼠體重、脂肪組織及腓腸肌重量、脂肪細胞大小、血清總膽固醇/三酸甘油酯/游離脂肪酸含量、禁食血糖/胰島素以及胰島素阻抗指標 (HOMA-IR index)，並可增加葡萄糖耐受性與胰島素敏感性、並有較高的個體氧氣消耗量及二氧化碳排出量進而增加能量消耗量。可惜的是給予 antcin K 及 EK100 沒有明顯改善小鼠代謝症候群的現象。有趣的是，以尾部懸吊試驗評估小鼠憂鬱狀況，發現給予 fenofibrate 顯著降低小鼠不動之秒數達到降低憂鬱行為之表現；此外，十字迷宮行為試驗評估抗焦慮狀況，發現給予 antcin K 以及 fenofibrate 可顯著提升小鼠進入開放臂之次數達到改善焦慮行為之表現。另外，給予 fenofibrate 雖有抗肥胖的效果，但與其餘組別相比卻有明顯肝腫大及較高 ALT 活性的情形，分析肝臟表現型，發現高脂飲食組有明顯非酒精性脂肪肝的現象，但給予 fenofibrate 能顯著降低肝臟三酸甘油酯含量、減少油滴之堆積，並能活化肝臟 PPARα 下游基因表現，例如參與脂肪酸氧化 (Cpt1a、Acox1)、脂肪酸運送 (Cd36、Fabp1)、脂肪酸合成 (Acaca、Fasn) 及膽固醇運輸 (Abca1) 等基因提升肝臟整體能量代謝達到改善非酒精性脂肪肝的效果。
綜合以上兩部分的實驗，給予 PPARα 促效劑 fenofibrate 具有顯著改善高脂飲食所誘發小鼠肥胖、代謝失調以及情緒障礙之效果，雖然 antcin K 以及 EK100 並無看到預期改善肥胖之效果，但是 antcin K 在行為試驗上有顯著抗焦慮之效果，詳細機制仍有待更進一步的探討。

關鍵字

肥胖 ； PPARα ； PPARα 促效劑 ； 代謝失調 ； 抗焦慮

並列摘要

Prevalence of obesity is rapidly increasing, and has led to increased morbidity and mortality of various chronic diseases. Obesity has become one of most serious public health challenges of the 21st century. Peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that belongs to the nuclear hormone receptor superfamily. Activation of PPARα has been shown to ameliorate obesity and metabolic syndrome through regulation of lipid metabolism. The aim of this study is to identify the natural PPARα agonists from Chinese herbs and marine fungus extracts using in vitro PPARα transactivation system. We further investigate the effects of selected PPAR agonists on metabolic abnormalities, non-alcoholic fatty liver disease (NAFLD) and emotional disturbance in diet-induced obesity mouse model.
In the first part of the study, the PPARα transactivation assay was used to identify the potential PPARα agonists from natural pure compounds and crude extracts. We showed that antcin B, C, H, K, triterpenoid compounds from Antrodia cinnamomea, significantly activated PPARα activity (120% vs. 1 μM Wy14643, 100%). In addition, the natural crude extracts from Vitex quinata (Lour.) F. N. Williams, Trichosanthes homophylla, Ainsliaea reflexa Merr, as well as few marine fungus extracts (NTOU4295, NTOU4296) increased PPARα activity (40-60% vs. 1 μM Wy14643, 100%). In contrast to antcins, EK100, an ergosterol from Antrodia cinnamomea, has limited effects on PPARα activation. Given that EK100 has been shown to exhibit anti-inflammatory, anti-diabetic and anti-hyperlipidemic effects, antcin K, EK100 and fenofibrate (hypolipidemic drugs as positive control) were tested in high-fat diet induced obese mouse model.
4-week-old C57BL/6J mice were fed with 30% high-fat diet to induce obesity for 8 weeks. The mice were treated with antcin K (20 mg/kg/day BW) for 4 weeks and then (40 mg/kg/day BW) for another 4 weeks, EK100 (20 mg/kg/day BW) for 4 weeks and then (40 mg/kg/day BW) for another 4 weeks, fenofibrate (250 mg/kg/day BW) for 8 weeks, or vehicle for 8 weeks afterward. We showed that high-fat diet has successfully induced mice obesity. Treatment of fenofibrate significantly reduced body weight, fat mass, gastrocnemius weight, adipocytes size, serum cholesterol / triglyceride / NEFA and fasting glucose / insulin / HOMR-IR index. In addition, treatment of fenofibrate in mice significantly improved glucose tolerance, insulin sensitivity, increased oxygen consumption and carbon dioxide production. Unexpectedly, antcin K and EK100 have limited effects on prevention of obesity. Interestingly, to further investigate antidepressant activity of PPARα agonists, tail suspension test was performed. We found that treatment of fenofibrate significantly reduced the immobility time compared to high-fat group, indicating less depression-like behavior. Furthermore, we also investigate antianxiety-like activity of PPARα agonists using elevated plus maze. We found that mice treatment with antcin K and fenofibrate significantly increased the time spent in open arms, suggesting less anxiety-like behavior. Although treatment of fenofibrate prevented obesity, the mice exhibited hepatomegaly and increased ALT activity. Besides, hepatic lipid accumulation was significantly decreased in fenofibrate-treated group, suggesting a significant effect on treatment of NAFLD. Exploring the mechanism, the expression of hepatic PPARα target genes, such as fatty acid oxidation (Cpt1a, Acox1), fatty acid transport (Cd36, Fabp1), fatty acid synthesis (Acaca, Fasn) and cholesterol transport (Abca1) was markedly elevated to increased energy metabolism rates in the liver and ameliorated the non-alcoholic fatty liver disease.
Taken together, treatment of PPARα agonist fenofibrate prevented obesity, metabolic disorders, emotional disturbance in high-fat diet fed C57BL/6J male mice. Although antcin K and EK100 treatment has limited effects in this study, the potential anti-anxiety function of antcin K remains to be further investigated.

並列關鍵字

obesity ； PPARα ； PPARα agonists ； metabolic syndrome ； anti-anxiety

參考文獻

Lai, C.I., Chu, Y.L., Ho, C.T., Su, Y.C., Kuo, Y.H., and Sheen, L.Y. (2016). Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. J Tradit Complement Med 6, 48-56.

許珊菁 (2006). 鼠模式中高脂飲食、肥胖與脂質調控基因之表現. In 微生物與生化學研究所 (台北市: 國立臺灣大學), pp. 207.

董又慈 (2016). 山苦瓜改善高脂飲食誘發小鼠代謝異常、脂肪肝與肝相關基因 mRNA 表現. In 生化科技學系 (台北市: 國立臺灣大學), pp. 101.

鄒尚瑀 (2016). 長期餵食山苦瓜對高脂飲食誘導肥胖小鼠代謝異常及白色脂肪組織褐化相關基因表現之效應. In 生化科學研究所 (台北市: 國立臺灣大學), pp. 103.

鍾誠珠 (2009). 代謝症候群動物模式之評估探討. In 微生物與生化學研究所 (台北市: 國立臺灣大學), pp. 159.