山苦瓜酸水解萃物影響3T3-L1前脂肪細胞褐化相關基因mRNA的表現量

Abstract

近年來因為生活水平的改善，飲食偏向高糖高脂的食物以及體能活動的不足，使得能量供給大於需求，導致過重及肥胖的盛行率逐漸攀升。肥胖的防治主要策略是降低能量攝取，增加能量消耗。最近研究顯示，白色脂肪「褐化」後，可消耗熱量，減少脂肪組織累積，達到減肥的效果。因此如何刺激白色脂肪褐化是近年來相當熱門的話題。本實驗室先前研究指出，小鼠攝食山苦瓜，可促進白色脂肪中「棕色脂肪」及粒線體相關基因表現，並增加能量代謝。在3T3-L1 脂肪細胞模式中發現，山苦瓜的乙酸乙酯萃取物的促褐化效果明顯優於水萃物。而乙酸乙酯萃物中的三萜類化合物多為不帶糖基或帶短鏈糖基的形式，屬於極性較低的化合物；而水萃物中多為帶長鏈糖基的形式。因此本實驗擬以3T3-L1 脂肪細胞為平台，探討山苦瓜萃物酸水解去糖基後對於褐化的影響 實驗樣品在脂肪細胞分化的不同時期進行處理。phase 1：分化初期開始樣品處理，直到分化成熟，phase 2：分化成熟前四天，樣品處理四天，phase 3：分化成熟後，樣品處理四天。樣品包括：山苦瓜萃物酸水解後正己烷萃物 (Hex)、山苦瓜萃物未酸水解之正己烷萃物 (Hex(un))及山苦瓜中單一化合物：熊果酸(ursolic acid)、齊墩果酸 (oleanolic acid)、9c, 11t, 13t-CLN。結果顯示Hex 在phase 2，以低濃度0.2 μg/mL Hex 處理時，Pgc1α (約1.1 倍)、Cidea (約1.5 倍)以 及Nrf1 (約1.2 倍)的基因表現皆顯著高於控制組；在phase 3，同樣以低濃度0.2μg/mL Hex 處理時，Pgc1α (約1.1 倍)、Ucp1 (約2 倍)及Cidea (約1.2 倍)的基因表現皆有顯著增加。Hex(un)在phase 2 時，Ucp1 在0.2 μg/mL 及5 μg/mLHex(un)的濃度下，基因表現量會顯著增加 (約2 倍及2.5 倍)，而Cidea 在0.2μg/mL 及1 μg/mL 的濃度下有顯著增加的情況 (約1.2 倍)，Tfam 則是僅在0.2μg/mL 的濃度下有顯著增加 (約1.1 倍)；而在phase 3 的時候，只在1 μg/mL 的濃度時，Pgc1α 和Ucp1 的表現量有上升的情形 (約1.3 倍及1.8 倍)。而山苦瓜中的單一化合物，脂肪細胞在phase 1 以OA 進行處理，在5 μM 的濃度下，Ucp1和Cidea 的基因表現皆有顯著升高 (約1.5 倍及2 倍)，而Tfam 的基因表現則是在三種處理濃度之下皆有明顯較高 (p <0.05)。UA 在phase 3 刺激脂肪細胞，只能夠有效地促進Ucp1 的基因表現(約2 倍)，其它褐化相關基因皆沒有上升的情況。CLN 則是在三種時期及三種濃度之下，皆沒有觀察到Ucp1 的基因表現上升。本研究亦嘗試使用海馬能量代謝測定儀，測定脂肪細胞褐化後之能量代謝變化。結果顯示，將脂肪細胞直接培養於海馬培養盤，會有溶氧量讀值偏低的情形，似無法正確測量細胞之氧消耗。綜合以上所述，在本實驗之細胞模式下，經酸水解與未經酸水解的苦瓜低極性萃物具有不同的促褐化效果。UA 及OA 這兩種五環的三萜類化合物皆可部份促進褐化相關基因的表現，可推測三萜類確實具有促褐化的潛力。

The prevalence of obesity in developed and developing countries has been increasing over the past decades, presumably due to excessive energy intake and less energy consumption. The main strategy of obesity prevention is to decrease energy intake and to increase consumption. Recent studies indicate that “browning” of white adipose tissue (WAT) can dissipate energy as heat and thus can decrease fat accumulation and ameliorate the obesity condition, which results in weight loss. Previous studies showed that mice fed bitter gourd powder had lower body weight and adipose mass and higher mRNA expression of Pgc1α, Ucp1 and Nrf1 in WAT, Pgc1α and Nrf1 or Tfam in skeletal muscle and brown adipose tissue, suggesting the browning of WAT. In 3T3-L1 cell model, ethyl acetate extract (EAE) of BGP but not water extract (WE) has been shown to trigger Ucp1 mRNA expression. We hypothesize that eglucosylated triterpenoids of BGP can trigger browning. In this study, polar fraction of BGP was treated with HCl (acid hydrolysis) and was extracted with n-hexane and was used to treat 3T3-L1 preadipocytes during or after differentiation: phase 1: during differentiation, phase 2: 4 days before maturation, treatment for 4 days, phase 3: after maturation. The samples included BG extract with acid-hydrolysis (Hex), BG extract without acid-hydrolysis (Hex(un)), ursolic acid (UA), oleanolic acid (OA) and 9c, 11t, 13t-CLN. The results indicated that 0.2 μg/mL Hex was able to increase Pgc1α, Cidea, and Nrf1 mRNA expressions at phase 2, and at phase 3, 0.2 μg/mL Hex could increase Pgc1α, Ucp1 and Cidea mRNA expressions. At phase 2, Ucp1 mRNA expression could be increased at 0.2 μg/mL and 5 μg/mL Hex(un); however, Tfam mRNA expression could only be increased at 0.2 μg/mL Hex(un). At phase 3, Pgc1α and Ucp1 mRNA expressions could only be increased at 1 μg/mL Hex(un). OA increased Ucp1, Cidea and Tfam mRNA expressions at phase 2. UA increased only Ucp1 mRNA expression at phase 3. CLN had no effect on Ucp1 mRNA expression at three phases and at three concentrations. We also tried to use XFe24 Flux analyzer to measure oxygen consumption rate (OCR) of 3T3-L1 adipocytes after browning. The data showed that when we induced differentiation in XF24 microplates, we could always have low readings of dissolved oxygen, which may be the reason why we couldn’t measure correct OCR of cells.In conclusion, in this cell model, acid-hydrolyzed or unhydrolyzed extract of bitter gourd had different browning activities. Both UA and OA, oleanane-type, could increase some brown-related gene expressions. Hence, triterpenoids presumably can promote browning.