指導教授：黃青真臺灣大學：生化科技學系蔣汶龍Chiang, Wen-LungWen-LungChiang2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261602肥胖與代謝症候群是許多疾病的危險因子，包括第二型糖尿病、心血管疾病及癌症等，其全球盛行率逐年攀升，是近年來最重要的公衛議題之一。苦瓜(Momordica charantia L.)是熱帶地區常見的瓜類蔬菜，已有許多研究證實其能有效降低血糖及調節血脂異常。本實驗室先前曾發現添加5%山苦瓜全果凍乾粉(BGP)於高蔗糖飼料，可促進肌肉PGC-1α及TFAM基因表現量增加。此篇研究將更深入探討其是否促進骨骼肌粒線體增殖，藉以增加能量消耗與改善胰島素敏感性。第一部分實驗期為8週，採雙因子試驗設計，試驗因子為BGP及飼料脂質含量。發現BGP均改善低脂高蔗糖或高脂飲食誘導肥胖，降低血糖、脂質堆積及體重增加，提昇血清中三碘甲狀腺素(T3)，且二因子間無交互作用。但對肌肉粒線體相關基因表現量及粒線體含量未造成影響。另外進行代謝體學研究則觀察到苦瓜三萜類化合物出現於肝臟之證據，且膽酸代謝、前列腺素代謝及色胺酸代謝路徑可能受到苦瓜攝取之影響。第二部分以注射T3之動物模式，建立分析粒線體增殖方法並確認其可信度，並依肌纖維組成分別取紅肌及白肌進行分析。T3處理5週後顯著增加小鼠氧氣消耗及二氧化碳排出，且T3主要作用於紅肌，刺激TFAM、COX7a1及UCP2基因mRNA 表現量增加，粒線體含量指標mtDNA copy number與檸檬酸合成酶(CS)活性卻未受影響。第三部分將實驗飼料餵食週數延長至25 週，在高蔗糖飼料中添加5% BGP，同樣顯著降低血中三酸甘油酯、脂質堆積及體重增加量，提高基礎代謝率與胰島素敏感性，且血糖低於餵食chow diet的正常飲食組。儘管血中T3濃度沒有差異，BGP提高肌肉中DIO2基因表現。高糖飲食造成白肌中粒線體相關基因表現下降，BGP則恢復其表現，並在紅肌中相較其他兩組有更高表現。而注射T3 與長期餵食BGP的小鼠皆觀察到紅肌中UCP2基因表現增加。然而在PGC-1α、NRF1及TFAM基因皆提昇的情況下，mtDNA copy number及CS活性未顯著提昇，組織氧氣消耗速率及胞外酸化速率亦未受影響，但紅肌中OCR/ECAR比值顯著較C組高。綜之，BGP確實可降低血糖及三酸甘油酯，改善胰島素敏感性，可能透過促進血中或肌肉組織之T3作用，提高基礎代謝率，以及肌肉中粒線體相關基因表現增加以避免飲食誘導肥胖。Obesity is a risk factor for various chronic diseases, and it has become a worldwide health issue due to the increasing global prevalence. Bitter gourd (Momordica charantia L.) is a common tropical vegetable, which has been shown to prevent metabolic disorder and obesity. Previous studies in our laboratory demonstrated that supplementation of 5% lyophilized wild bitter gourd powder (BGP) to a high sucrose diet up-regulated PGC-1α and TFAM mRNA expression in the muscle. This study thus tested the hypothesis that BGP might increase mitochondrial biogenesis in skeletal muscle, increase energy expenditure and ameliorate insulin resistance. In the first experiment, 4 groups of C57BL/6J male mice were respectively fed high sucrose (HS) or high fat diets supplemented without or with 5% BGP based on a 2X2 factorial design. After 8 weeks of feeding, BGP significantly reduced serum glucose and triglyceride level, adipose mass, and body weight change and increased serum triiodothyronine (T3). But BGP did not affect mitochondria-related gene expression or mitochondria content in skeletal muscle. Through the metabolomic studies, we found supporting evidence that triterpenoid compounds of bitter gourd accumulated in liver of mice fed BGP. In addition, bile acid metabolism, prostaglandin metabolism and tryptophan metabolism might be affected by BGP. In the second experiment, mice were daily injected with triiodothyronin (T3) (50 μg/kg/day) for 5 weeks to establish and validate the methods for evaluate mitochondria biogenesis in skeletal muscle. T3 significantly increased O2 consumption and CO2 production of animals, and stimulated TFAM, COX7a1 and UCP2 gene expression mainly in slow-twitch oxidative skeletal muscle. However, PGC-1α level, mitochondrial DNA (mtDNA) copy number and citrate synthase (CS) activity were not altered. In the third experiment, 3 groups of mice were respectively fed the HS, HS+5% BGP or chow diets for 25 weeks. Similar to results in the first experiment, BGP inhibited diet-induced obesity and reduced serum glucose to a level that was even lower than that of the chow diet-fed mice. In spite of unchanged serum T3 level, BGP up-regulated DIO2 gene expression in both glycolytic and oxidative muscle and UCP2 level in oxidative muscle. High sucrose diet down-regulated mitochondria- related gene expression in glycolytic muscle, but BGP restored it to the normal level. The expression levels of these genes in the oxidative muscle of the BG group were higher than those of the other 2 groups. BG did not significantly change mtDNA copy number, CS activity, tissue oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), but increase the OCR/ECAR ratio. In summary, BGP reduced serum glucose and triglyceride level, improved insulin sensitivity, increase metabolic rate, up-regulated mRNA expressions of DIO2, UCP2 and mitochondria-related gene expression in glycolytic and oxidative muscle.中文摘要................................................... i 英文摘要................................................. iii 縮寫對照表................................................. v 總目錄.................................................... ix 圖目錄................................................... xiv 表目錄.................................................. xvii 第一章、緒論................................................ 1 1.1 前言.................................................. 1 1.2 文獻回顧............................................... 1 1.2.1 肥胖與代謝性疾病....................................... 1 1.2.2 代謝性壓力對肌肉及粒線體之影響........................... 4 1.2.3 代謝體學............................................ 13 1.2.4 苦瓜............................................... 15 1.3 實驗假說與架構......................................... 18 第二章、初探小鼠攝入8週山苦瓜對肌肉粒線體含量與相關基因之影響暨代謝體學研究....................................................... 20 2.1 前言................................................. 20 2.2 材料與方法............................................ 21 2.2.1 試驗大綱............................................ 21 2.2.2 動物飼養............................................ 22 2.2.3 飼料配製............................................ 22 2.2.4 氧消耗量、二氧化碳排出量與呼吸商測定..................... 23 2.2.5 代謝物收集.......................................... 24 2.2.6 口服葡萄糖耐受性測試.................................. 24 2.2.7 動物犧牲............................................ 24 2.2.8 血液生化分析......................................... 24 2.2.8.1 血清葡萄糖........................................ 24 2.2.8.2 血清三酸甘油酯..................................... 25 2.2.8.3 血清膽固醇........................................ 26 2.2.8.4 血清胰島素........................................ 26 2.2.8.5 血清四碘甲狀腺素................................... 26 2.2.8.6 血清三碘甲狀腺素................................... 27 2.2.9 代謝體學............................................ 27 2.2.9.1 樣品製備.......................................... 27 2.2.9.2 液相層析質譜分析.................................... 28 2.2.9.3 分析結果資料擷取.................................... 30 2.2.10 Real-time PCR法分析基因表現與粒線體DNA複本數........... 31 2.2.11 統計分析........................................... 33 2.3 結果................................................. 33 2.3.1 體重變化、攝食量、飼料與能量利用率....................... 33 2.3.2 組織絕對與相對重量.................................... 34 2.3.3 氧消耗量、二氧化碳排出量及呼吸商......................... 34 2.3.4 餵食5週、8週之血液生化分析............................. 34 2.3.5口服葡萄糖耐受性測試................................... 34 2.3.6 肌肉粒線體含量與相關基因表現量.......................... 35 2.3.7 代謝體學............................................ 35 2.4 討論................................................. 58 2.5 結論................................................. 61 第三章、骨骼肌粒線體增生之動物模式建........................... 62 3.1 前言................................................. 62 3.2 材料與方法............................................ 64 3.2.1 試驗大綱............................................ 64 3.2.2 動物飼養............................................ 64 3.2.3 氧消耗量、二氧化碳排出量與呼吸商測定..................... 65 3.2.4 動物犧牲............................................ 65 3.2.5 粒線體分離與檸檬酸合成酶之酵素活性測定.................... 66 3.2.6 血液生化分析......................................... 67 3.2.7 Real-time PCR法分析基因表現與粒線體DNA複本數............ 67 3.2.8 穿透式電子顯微鏡切片分析............................... 70 3.2.9 統計分析............................................ 71 3.3 結果................................................. 71 3.3.1 體重變化、飼料及能量攝取與利用情形....................... 71 3.3.2 絕對與相對組織重量.................................... 71 3.3.3 氧氣消耗、二氧化碳排出及呼吸商.......................... 71 3.3.4 血液分析............................................ 72 3.3.5粒線體相關基因表現、mtDNA複本數與CS活性分析............... 72 3.3.6 穿透式電子顯微鏡組織切片分析............................ 72 3.4 討論................................................. 86 3.5 結論................................................. 89 第四章、探討小鼠飼以添加山苦瓜之高蔗糖飼料25週對肌肉粒線體增殖之影響. 90 4.1 前言................................................. 90 4.2 材料與方法............................................ 91 4.2.1 試驗大綱............................................ 91 4.2.2 動物飼養............................................ 92 4.2.3 飼料配製............................................ 92 4.2.4氧消耗量、二氧化碳排出量與呼吸商測定...................... 93 4.2.5 動物犧牲............................................ 94 4.2.6 組織氧氣消耗速率與胞外酸化速率分析....................... 94 4.2.7 粒線體分離與檸檬酸合成酶之酵素活性測定.................... 95 4.2.8 血液分析............................................ 95 4.2.9 Real-time PCR法分析基因表現與粒線體DNA複本數............ 95 4.2.10 穿透式電子顯微鏡切片分析.............................. 96 4.2.11統計分析............................................ 96 4.3 結果................................................. 96 4.3.1 體重變化、攝食量、飼料與能量利用率....................... 96 4.3.2 組織絕對與相對重量.................................... 97 4.3.3 氧消耗量、二氧化碳排出量及呼吸商......................... 97 4.3.4 血液生化分析......................................... 97 4.3.5 粒線體相關基因表現、mtDNA複本數與CS活性分析.............. 98 4.3.6 肌肉組織氧氣消耗速率與胞外酸化速率分析................... 98 4.4 討論................................................ 112 4.5 結論................................................ 116 第五章、綜合討論與結論...................................... 117 5.1 綜合討論............................................. 117 5.2 總結論............................................... 122 第六章、參考文獻........................................... 123 附錄一、預測代謝物與標準品資料庫之M/z at RT比較................ 140 附錄二、Chow diet (LabDiet) 飼料成分表..................... 142 附錄三、不同品系山苦瓜乙酸乙酯萃物對PPARδ之轉染活性............. 1438292878 bytesapplication/pdf論文公開時間：2017/08/21論文使用權限：同意有償授權(權利金給回饋本人)苦瓜骨骼肌粒線體甲狀腺素[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261602/1/ntu-103-R01b22005-1.pdf