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標題: | 藻褐素抗肥胖之機制探討 The Study on Anti-obesity Mechanism of Fucoxanthin |
作者: | Meng-Ting Wu 吳夢婷 |
指導教授: | 黃青真(Ching-jang Huang) |
關鍵字: | 藻褐素,脂肪,代謝率,PGC-1α,粒線體生合成及融合,PPARs,LXRs, Fucoxanthin,Adipose tissue,Metabolic rate,PGC-1α network,Mitochondrial biogenesis and fusion,PPARs,LXRs, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 能量攝取大於消耗是肥胖形成的主因,肥胖係指體內累積過多的脂肪,會增加許多慢性疾病的風險,像是胰島素阻抗、第二型糖尿病、心血管疾病,甚至是癌症等。每年因肥胖相關疾病所造成的醫療費用越來越多,已成為全世界最重要的公衛問題之一。
藻褐素為褐藻特有的類胡蘿蔔素,其結構上具有獨特的 allenic bond 及 5,6-epoxy group。先前研究顯示藻褐素具有降低動物體脂的功能,然而其詳細機制尚未釐清,故本論文旨在探討藻褐素抗肥胖之機制,實驗共分三部分。 第一個部分以小鼠實驗探討藻褐素降體脂之機制。實驗一採用 C57BL/6J 雌鼠,每日腹腔注射藻褐素 (10 mg/kg BW) 或 vehicle (10% EtOH/10% BSA/0.9% NaCl) 共四週,結果顯示腹腔注射藻褐素的小鼠,其體脂未降低。實驗二採用 C57BL/6J 雄鼠,分成四組,分別餵食高蔗糖飼料 (50% 蔗糖,HS) 或高脂飼料 (7%大豆油+23%奶油,HF),兩種飼料再分為不含或含 0.2% 藻褐素的高蔗糖 (HS+F) 或高脂飼料 (HF+F),共餵食五週。結果顯示藻褐素顯著增加小鼠的能量消耗: 動物氧氣消耗量及二氧化碳排出量上升,並顯著減少內臟及皮下脂肪,但肝、心、脾及腎等臟器重量增加,因而體重並未改變。在尿液代謝體分析預測,HS+F 組尿液中 nicotinate D-ribonucleoside 及 pseudouridine 分別顯著高於 HS 組,HF+F 組尿液中 nicotinate D-ribonucleoside、5-acetamidopentanoate、1-methylhypoxanthine、6-succinoaminopurine、1-methylguanosine 及 L-phenylalanyl-L-hydroxyproline 顯著高於 HF。進一步分析基因表現,結果顯示藻褐素促進內臟和皮下脂肪中,褐化 (Cidea、Pgc-1α、Pparα、Errα、Adrb3、Dio2) 及粒線體融合 (Mfn1、Mfn2、Opa1) 基因表現,亦提高內臟脂肪粒線體生合成 (Nrf1、Nrf2) 基因表現,對於產熱 (Ucp1) 基因之影響未達顯著。藻褐素之上述效應,皆未與飲食產生顯著之交互作用,顯示無論高蔗糖或高脂飲食,藻褐素皆有降體脂作用。 另一方面,實驗二之結果也顯示飲食投予藻褐素 (HS+F 及 HF+F 組) 會增加血清三酸甘油酯、總膽固醇、HDL-C、LDL-C 濃度及肝臟膽固醇及游離脂肪酸含量,且與飲食類型無交互作用。進一步分析脂質代謝相關基因,顯示藻褐素增加內臟和皮下脂肪三酸甘油酯分解酵素 (Hsl)、脂肪酸合成酵素 (Acc) 及膽固醇代謝相關核受器及運輸蛋白 (Lxrα、Abca1、Abcg1) 之基因表現,降低白色脂肪及肌肉 Ldlr 基因表現,並顯著增加肝臟脂肪酸合成 (Acc) 及膽固醇合成 (Hmgcr) 基因表現,降低脂肪酸代謝 (Cpt1α、Aco) 和膽固醇代謝 (Lxrβ、Abca1) 相關基因表現。推測血清膽固醇增加可能與白色脂肪組織膽固醇逆運輸相關基因表現增加,且白色脂肪組織和肌肉 Ldlr 基因表現降低有關,可能減少了周邊組織胞吞 LDL 的活性,並且增加 HDL 逆運輸的活性。而肝臟本身之脂質合成基因表現增加及代謝脂肪基因表現減少,可能造成了脂質堆積在血液及肝臟的現象。 第二部分以細胞模式探討藻褐素及藻褐醇在個別組織的作用。實驗一採用HepG2 細胞,以 vehicle、ALA (18:3 n-3)、藻褐素+ALA (18:3 n-3) 分別處理 HepG2 肝細胞 48 小時。結果顯示藻褐素顯著增加細胞中 n-3 (EPA、DPA、DHA) 及 n-6 PUFA (AA) 含量且具劑量效應。接著以 U-13C-ALA 與藻褐素共同處理 0.5、3、6、24 或 48 小時,再次證明藻褐素增加肝細胞 n-3 脂肪酸的合成、D5D 酵素活性指標,對 D6D 酵素活性指標則無顯著影響。已知過氧化體 β-oxidation 為合成 DHA 最後一個步驟,實驗二以 H4IIEC3 肝細胞為平台,以 vehicle、藻褐素、藻褐醇、Wy14643 分別處理 H4IIEC3 細胞 72 小時,結果顯示藻褐素及藻褐醇顯著增加過氧化體 ACO 酵素活性,對 Pparα及 Aco mRNA 表現則無顯著影響。實驗三採用 3T3-L1 前脂肪細胞,藻褐素和藻褐醇分別於 DM2、分化全期、分化完成後等三個階段處理細胞。結果顯示藻褐素及藻褐醇於分化全期處理細胞,顯著增加其三酸甘油酯濃度,於 DM2 添加,則對三酸甘油酯濃度無顯著影響,於分化後處理,僅藻褐醇顯著增加三酸甘油酯濃度。最後於 3T3-L1 分化為成熟脂肪細胞後,處理藻褐素或藻褐醇 12 小時,再分析細胞攝取葡萄糖的能力,結果顯示藻褐素和藻褐醇均稍微增加脂肪細胞汲取葡萄糖的量 (p<0.05)。 第三部分在探討藻褐素及藻褐醇對於調控體內葡萄糖及脂質代謝重要的轉錄因子 PPARs 和 LXRs 之轉錄活性影響。使用 CHO-K1 細胞,短暫共同轉染 chimeric receptor of GAL4-rPPARα (或 rPPARγ, rPPARδ, hLXRα, hLXRβ) LBD 及 (UAS)4-tk-alkaline phosphatase reporter,將藻褐素、藻褐醇單獨或與各受器對應之 ligand 共同處理 48 小時,分析其轉錄活性。結果顯示藻褐素及藻褐醇均顯著拮抗 PPARs 和 LXRs 之 ligand 的轉錄活性且具劑量效應,藻褐醇的拮抗效果又比藻褐素明顯,藻褐醇的下一個代謝物 amarouciaxanthin A 之 PPARs 和 LXRs 轉錄活性值得進一步分析。 綜此,本研究結果顯示藻褐素須經消化道代謝、吸收始有降體脂的效果。並開啟探討飲食投予藻褐素抗肥胖機制的新方向,透過增加脂肪組織能量消耗及粒線體生合成/融合之 mRNA 表現,增加動物整體的代謝率,而降低白色脂肪塊重量。從動物及細胞實驗的結果可推測,飲食攝取藻褐素或其代謝產物對 PPARs/LXRs 影響似乎具組織特異性,詳細的調控機制值得進一步探討。 Obesity, defined as excess accumulation of adipose, is a worldwide endemic health problem. Obesity and its related disorders are associated with increased morbidity, mortality and healthcare costs. Fucoxanthin (FX) is a major carotenoid in brown algae and has an unusual allenic structure. FX has a suppressive effect on adipose accumulation in genetically diabetic KKAy mouse, Wistar rat and diet-induced obese C57BL/6J mouse. However, the mechanism for how FX suppresses adipose accumulation is still unclear. This study thus aimed to explore the effect and mechanism of FX on body fat regulation. In the first part, the anti-obesity mechanism was studied in mice. C57BL/6J female mice were ip injected with FX (10 mg/kg/day) or vehicle for 4 weeks. FX did not reduce body fat through ip injection, suggesting the importance of intestinal digestion and absorption. Using a 2x2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased O2 consumption and CO2 production, and reduced white adipose tissue (WAT) mass. The anti-obesity effect was further associated with significant up-regulation of mRNA expressions of PPARγ coactivator-1α (Pgc-1α), cell death-inducing DFFA-like effector a (Cidea), peroxisome proliferator-activated receptor (Ppar) α, PPARγ, estrogen-related receptor α (Errα), β3-adrenergic receptor (Adrb3), and type 2 deiodinase (Dio2) in inguinal WAT (iWAT) and epididymal WAT (eWAT) in FX fed mice. Mitochondrial biogenic genes, nuclear respiratory factor 1 (Nrf1) and Nrf2, were also up-regulated in eWAT of FX fed mice. Noticeably, FX up-regulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2, and optic atrophy 1 (Opa1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In addition, FX increased predicted urinary metabolites including nicotinate D-ribonucleoside and pseudouridine in HS+F group compared to HS group and nicotinate D-ribonucleoside, 5-acetamidopentanoate, 1-methylhypoxanthine, 6-succinoaminopurine, 1-methylguanosine and L-phenylalanyl-L-hydroxyproline in HF+F group compared to HF group. The increases in the Ucp1 mRNA expression in WATs and BAT in FX fed mice were, nevertheless, not statistically significant. On the other hand, FX also significantly increased serum triglycerides, total cholesterol, LDL and HDL cholesterol. The up-regulated lipolytic (Hsl) and cholesterol reverse transport (Lxrα、Abca1 and Abcg1) genes, and down-regulated Ldlr mRNA in WATs as well as up-regulated lipogenic (Acc) and cholesterogenic (Hmgcr) genes, together with down-regulated fatty acid oxidation (Cpt1α and Aco) and cholesterol metabolism (Lxrβ and Abca1) genes expressions in liver might contribute to the higher serum lipids in FX-fed mice. Cell models were used in the second part of the study. HepG2 cells were treated with alpha-linolenic acid (ALA, 18:3 n-3, 100 μM) with FX (0~100 μM) or vehicle for 48 hr. FX increased EPA, docosapentaenoic acid (DPA, 22:5 n-3), DHA and AA but decreased eicosatetraenoic acid (ETA, 20:4 n-3) in HepG2 cells in a dose-dependent manner. Similar results were obtained when ALA was replaced by U-13C-labeled ALA (100 μM) to trace fatty acid metabolism. The delta-5 desaturase (D5D) but not delta-6 desaturase (D6D) activity index was increased. On the other hand, FX and and its metabolite, fucoxanthinol (FUOH) elevated the acyl-CoA oxidase (ACO) activity, but not the mRNA expression in H4IIEC3 hepatocytes. In addition, FX and FUOH promoted TG accumulation in 3T3-L1 preadipocyte under treatment during the whole differentiation period but not within the DM2 or after maturatuion. Furthermore, 12hr treatment of FUOH increased glucose uptake of mature 3T3-L1 adipocyte. The inconsistency between the in vitro and in vivo experiments implied the bioactive compound might be the metabolites from FUOH, i.e., amarouciaxanthin A. PPARs and LXRs are master regulators of glucose and lipid metabolism. Transactivation assay was employed to examine FX and FUOH on PPARs and LXRs in CHO-K1 cells were transiently co-transfected with vectors of chimeric receptors containing the ligand-binding domain of PPAR or LXR and reporter and treated with FX or FUOH. Both FX and FUOH showed antagonistic activity to PPARs and LXRs and the suppressive effect of FUOH were more pronounced than that of FX. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These effects are associated with the up-regulation of PGC-1α network involved in energy expenditure, lipid metabolism/transport and mitochondrial biogenesis/fusion in eWAT and iWAT. The concomitant increases in serum and liver lipids together with observations in the in vitro studies led to a speculation that FX, FUOH and its further metabolites might act, at least in part, as tissue selective PPARs/LXRs modulators. Further investigations are worth to confirm the speculation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58201 |
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