天然化合物對脂蛋白解脂酶活性之影響及人類脂蛋白元A5基因替換小鼠之特性

Abstract

第一部分 當血液中三酸甘油脂或膽固醇任何一種或二種在血中之濃度上升時，稱為高脂血症。高脂血症可能會併發動脈硬化、冠心症或腦中風等疾病。脂蛋白解脂酶（lipoprotein lipase, LPL）由血液中脂蛋白元（apolipoprotein , APO） AV及脂蛋白元CII的活化，可以分解血液中乳麋微粒和極低密度脂蛋白所包含的三酸甘油脂，是分解脂肪的重要角色。身體的許多組織中皆會合成LPL，例如脂肪組織、心臟組織、肺臟及腎臟等。當LPL製造後會組成一個二聚體才有活性，之後LPL會穿越內皮細胞到達血管內，由HSPGs及GPIHBP1所呈現。 Peroxisome proliferator-activated receptors（PPARs）是一種核受體，當有配體（ligand）與PPAR結合後，PPAR隨之活化會與retinoid X receptor形成異型二聚體，此二聚體可以結合至DNA上的特定序列peroxisome proliferators response element (PPRE)，啟動下游基因的轉錄。而LPL的起始子區域序列中也包含了PPRE。 先前的研究文獻指出magnolol（厚朴酚）可以作為PPARγ的配體，並且有利於脂肪組織的分化，並且會使得LPL的mRNA表現增加；resveratrol（白藜蘆醇）能刺激sirtuin的活性，而SIRT1可以調節動物體內葡萄糖和胰島素生成及脂肪代謝等；naringenin（柚皮素）可以降低肝臟產生膽固醇，增加脂肪酸氧化，也可以提高PPARγ及PPARα的活性。由於LPL主要表現在脂肪組織內，而脂肪組織內的PPAR以PPARγ為主要，我們測試了此三種天然化合物，magnolol、resveratrol及naringenin，期許此三種藥物可以做為PPRAγ的ligand，進而增加LPL的生成並增加活性，提供預防高脂血症的方向。 利用3T3-L1細胞測試不同濃度的magnolol、resveratrol及naringenin培養六天，第六天分別收集細胞培養液以及細胞溶解物，測試細胞內源性的LPL活性，結果顯示magnolol使LPL活性增加1.04~1.25倍；resveratrol使LPL活性增加1.26~2.72倍；naringenin 則使LPL活性上升1.23~1.67倍。 抽取給予化合物六天後的細胞總RNA，以qPCR定量LPL mRNA表現量，結果顯示magnolol使LPL mRNA增加1.3~1.63倍；naringenin使LPL mRNA增加1.35倍；給予resveratrol 與對照組相較LPL mRNA的表現量並無顯著差異。 當同時加入PPARγ的抑制劑 GW9662或是GW9662及PPARα的抑制劑MK886後，magnolol所增加的LPL活性有被抑制下來，然而，LPL mRNA表現量卻沒有受抑制劑影響而下降。Naringenin在加入GW9662或是GW9662及MK886後也有相同情形，活性有些微抑制，但LPL mRNA表現量卻沒有受抑制劑影響而下降。 目前結果顯示magnolol、naringenin可以促進LPL表現以提高水解活性，resveratrol可以提高LPL活性卻不是透過LPL轉錄此路徑。LPL的活性提高的機制，需要再進一步證實。

第二部分 人體內的脂肪需要被包覆方能在血液中運送，而包覆脂肪的結構則稱為脂蛋白，其外層包含了單層磷脂質、膽固醇及脂蛋白元。脂蛋白元AV主要由肝臟產生，且存在於乳麋微粒、極低密度脂蛋白及高密度脂蛋白。文獻中發現血液中的三酸甘油脂濃度和APOA5的單一核甘酸多型性有關。本實驗室發現在APOA5第四個exon所發生的變異(c.553G>T多型性)，會導致原本的甘胺酸(glycine)變異為半胱胺酸(cysteine)，形成變異型脂蛋白元AV，此與高三酸甘油脂血症有關。 為了探討脂蛋白元AV對於血液循環中三酸甘油脂的影響，建構人類脂蛋白元A5野生型及變異型(G185C)之基因替換小鼠。利用高效率基因重組方法建構標的載體，進一步得到帶有人類脂蛋白元A5基因之嵌合鼠。將公嵌合鼠與野生型C57BL/6品系母小鼠進行交配，出生後之小鼠，取毛色混合者剪尾巴、萃取組織DNA，進行genotyping PCR，以篩選出基因替換之小鼠進行繁殖。 將基因穩定且12周齡的人類脂蛋白元A5變異型（c.553G＞T）基因替換公小鼠犧牲後採血，比較apoa5野生型、小鼠及人類變異脂蛋白元A5雜合型及人類變異脂蛋白元A5同型小鼠間血漿內脂肪濃度的差異。 結果顯示人類變異脂蛋白元A5同型公小鼠與apoa5野生型公小鼠的三酸甘油脂相較有2倍以上提升，可見人類變異脂蛋白元AV對血液中三酸甘油脂的濃度有重要影響。然而，由於人類脂蛋白元A5野生型基因替換小鼠的基因尚為穩定，人類脂蛋白元A5變異型及野生型的差異還需要進一步探討。 初步以magnolol分別餵食apoa5野生型公小鼠、人類脂蛋白元A5 185C variant雜合型及人類脂蛋白元A5 185C variant同型公小鼠，發現apoa5野生型公小鼠餵食magnolol後可使TG下降；人類脂蛋白元A5 185C variant雜合型公小鼠餵食magnolol後TG也有下降；人類脂蛋白元A5 185C variant同型公小鼠餵食magnolol後TG下降不明顯。Magnolol是否能有效降低TG，需更進一步證實。

Part I Hyperlipidemia is defined as having abnormally elevated levels of triglycerides or cholesterol or lipoproteins in the blood. Hyperlipidemia may be a risk factor of arteriosclerosis, coronary heart disease, stroke and other illnesses. When lipoprotein lipase (LPL) is activated by apolipoprotein AV (Apo AV) and Apo CII, it could catalyze the hydrolysis of triglycerides from chylomicrons and very low density lipoprotein (VLDL). LPL is synthesized in the parenchymal cells of tissues, such as adipose tissue, heart, lung and kidney, etc. Homodimer of LPL has enzyme activity, and is thus secreted and is translocated from the endothelial cells to the intravascular surface, and is finally presented by HSPGs and GHIBP1 on cell the memebrane. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors. When the ligands bind to PPAR, it would become activated. Then PPARs would form heterodimer with retinoid X receptor (RXR), which could then bind to DNA sequence-specific peroxisome proliferator response element (PPRE), and promote the transcription of downstream genes. The PPRE is also present in the promoters of LPL genes. In previous research, magnolol was shown to be a PPARγ ligand. It can promote the differentiation of adipose tissue and increase LPL mRNA expression. Resveratrol could activate sirtuin, and SIRT1 could regulate the production of glucose and insulin, and regulate lipid metabolism in vivo, etc. Naringenin could reduce the production of cholesterol in hepatocytes, and increase β-oxidation. Naringenin is also a ligand for PPARγ and PPARα. LPL is mainly synthesized in the adipose tissue, where PPARγ is abundantly expressed. We hypothesized that, the three compounds, magnolol, resveratrol and naringenin, could be ligands of PPARγ, and increase LPL expression. 3T3-L1 cells were treated with magnolol, resveratrol or naringenin for six days. On the sixth day, the culture medium and cell lysates were collected respectively, and then tested for endogenous LPL activity. The results indicated that magnolol enhanced LPL activity 1.04 ~ 1.25 fold. Resveratrol enhanced LPL activity 1.40 ~ 2.22 fold Naringenin enhanced LPL activity 1.23 ~ 1.67 fold. Real-Time quantitative PCR was used to measure LPL mRNA expression. The results showed that magnolol enhanced LPL mRNA expression 1.3 ~ 1.63 fold. Naringenin enhanced LPL mRNA expression 1.35 fold. However, resveratrol did not significantly enhanced LPL mRNA expression. Addition of the PPARγ inhibitor, GW9662, or GW9662 and PPARα inhibitor, MK886, in the magnolol-treated cells, LPL activity was suppressed. However, LPL mRNA expression was not changed. In naringenin-treated cells, after addition of GW9662 or MK886, the LPL activity was slightly decreased, but LPL mRNA expression was not affected by inhibitors. The results indicated that magnolol and naringenin could promote LPL transcription and enhance the hydrolytic activity. Resveratrol could enhace LPL activity but did not promote LPL transcription. The mechanisms of theses compounds to enhance LPL activity need to be further investigated. Part II Lipid needs to be coated before being transported in the blood and the coated structure is called lipoproteins. The outer layer contains a monolayer of phospholipids, cholesterol and apolipoprotein. Apolipoprotein AV (APOAV) is mainly produced by liver and presents in chylomicron, VLDL and high density lipoprotein (HDL). The levels of triglycerides in blood are related to apoA5 single nucleotide polymorphisms (SNPs). Our laboratory reported that individuals carring a SNP of APOA5 (c.553G＞T), a glycine change to cysteine have higher serum TG levels. To investigate the influence of human APOAV on the circulating triglycerides, the human APOA5 wild type and variant knockin mice were established.A highly efficient recombination-based method was performed to generate the targeting construct vectors, and to create the chimeras of human APOA5 wild type and variant (APOA5 c.553G＞T) knockin mice. The male chimeras were mated with wildtype female C57BL/6 mice and the mice genotype was determined by PCR to select human APOA5 knockin mice for breeding. 12-week-old male human APOA5 variant (c.553G＞T) knockin mice were sacrificed to collect their blood to compare lipid profiles among male C57BL/6 wildtype mice, the male human APOA5 variant heterozygous knockin mice and male human APOA5 variant homozygous knockin mice. The results showed that the triglyceride levels of human APOA5 variant knockin mice is 2-fold higher than those of wildtype mice. It indicates that human APOAV plays an important role in triglyceride metabolism. The differences between C57BL/6 wildtype and variant human APOA5 need to be confirmed after selection of male human APOA5 wildtype knockin mice. The plasma TG levels drcreased in male C57BL/6 wildtype mice after ingestion of magnolol. The same result was also observed in male 185C APOA5 heterozygous knockin mouse. However, the plasma TG concentration was not significantly decreased in male 185C APOA5 homozygous knockin mouse fed magnolol. Whether magnolol could reduce TG in plasma effectively needs to be further confirmed.