脂質代謝途徑失調時，常導致心血管疾病、糖尿病與肥胖等疾病的發生。第二型糖尿病的病徵除了血糖濃度異常高以外，還常伴隨著肥胖的現象，近年來許多研究顯示，罹癌風險與肥胖成正相關，由此可知糖尿病、肥胖與癌症間存在關聯性。脂質新生(Lipogenesis)為乙醯輔酶A (Acetyl-CoA)轉換為脂肪酸(Fatty acids)再合成為三酸甘油脂的過程；許多酵素參與脂質新生過程，其中Acetyl-CoA carboxylase1 (ACC1)會催化Malonyl-CoA的合成，為脂肪合成中的限速酶(Rate-limiting enzyme)，而Fatty Acid Synthase (FAS) 可將Malonyl-CoA合成為長鏈脂肪酸，亦是脂肪合成中重要的酵素之一。Sterol Regulatory Element-Binding Protein 1 (SREBP1)作為ACC1與FAS的轉錄因子，可調控其基因表現而影響脂質新生。DNA甲基化在不改變DNA序列的情況下即能影響基因表現量，亦是調控基因表現的重要機制之一。釐清糖尿病、肥胖與癌症間互相影響的機制並探討DNA甲基化是否調控脂質新生相關基因的表達為本研究的目標。高濃度胰島素與胰島素抗性亦為第二型糖尿病病徵，胰島素的功能除了調節血糖之外，亦是促進脂質新生的重要賀爾蒙，而脂質為癌細胞增生不可或缺的養分，因此我們比較正常細胞與癌細胞中，高濃度胰島素是否會藉由影響啟動子(promoter)區域的DNA甲基化而調控脂質新生相關基因的表現，另外還使用第二型糖尿病治療用藥metformin與AMP-activated protein kinase (AMPK)的活化劑AICAR來探討當AMPK被活化時，除了透過磷酸化ACC1而影響脂質新生以外，是否也會藉由影響DNA甲基化的機制而使脂質合成基因表現量改變。將正常乳腺細胞MCF10A、乳癌細胞MCF7與肝癌細胞HepG2用高濃度胰島素後，ACC1、FAS與SREBP1蛋白質表現量上升，而在metformin與AICAR處理下則蛋白質表現量下降，將細胞處理DNA甲基轉移酶(DNMTs)抑制劑5’-Azacytidine (5’-Aza)後，則發現ACC1、FAS與SREBP1蛋白質表現量有上升之趨勢，我們分析三者基因啟動子區域上可能受到甲基化調控的位點，並利用重亞硫酸鹽定序法分析甲基化情形，結果顯示在實驗的三株細胞中，ACC1啟動子區域本身的甲基化程度不高，而在metformin與AICAR處理下ACC1甲基化的程度也未有明顯改變，觀察其他種類癌細胞，ACC1啟動子區域本身甲基化的程度亦不高。反之，在此三株細胞中，FAS啟動子區域本身甲基化程度則較明顯，我們發現MCF10A細胞在高濃度胰島素刺激下，FAS啟動子區域甲基化程度有顯著下降，另一方面則觀察到 HepG2細胞於metformin處理下FAS啟動子區域甲基化程度則明顯增加。此外，我們分析了餵食高脂飼料促使肥胖的小鼠肝臟與白色脂肪組織中關於脂質新生相關蛋白的表現量及基因啟動子區域的甲基化情形，實驗結果顯示肥胖小鼠肝臟中ACC1、FAS與SREBP1蛋白質表現量上升，而在白色脂肪組織中三者蛋白質表現量皆下降，然而比較正常飲食與高脂飲食，重亞硫酸鹽定序分析結果皆顯示小鼠肝臟與白色脂肪組織的SREBP1啟動子區域甲基化之程度無明顯改變。綜合以上結果得知高濃度胰島素與metformin、AICAR會影響MCF10A、MCF7與HepG2細胞中FAS、ACC1與SREBP1的蛋白質表現量，而重亞硫酸鹽定序分析結果則顯示在MCF10A細胞中高濃度胰島素的作用以及在HepG2細胞中metformin的處理下會明顯改變FAS啟動子區域甲基化的程度。由細胞實驗及重亞硫酸鹽初步結果顯示FAS啟動子區域確實會受到DNA甲基化的修飾，進而改變FAS的表現量，表示DNA甲基化為影響脂質新生過程的機制之一。

關鍵字：脂質新生、ACC1、FAS、SREBP1、DNA甲基化

Accumulating data indicate that obesity and diabetes are positively correlated with a higher risk of cancer. Obesity is known for increased risk of developing insulin resistance and type II diabetes (T2D) leading to high level of blood insulin. Insulin is one of the critical hormones controlling fatty acids metabolism which is essential for tumor growth. Lipogenesis is the process that converts acetyl-CoA into fatty acids and triglycerides. Acetyl-CoA carboxylase1 (ACC1) catalyzes the formation of malonyl-CoA from acetyl-CoA. Fatty acid synthase (FAS) converts malonyl-CoA into long chain fatty acids. Sterol regulatory element-binding transcription factor 1 (SREBP1) is a vital transcription factor for ACC1 and FAS. DNA methylation is an important mechanism to modulate gene expression. We examined the effects of high dose insulin, metformin (anti-T2D drug) and AICAR (AMPK activator) on lipogenic gene expression. In addition, we also investigated whether DNA methylation is involved in the regulation of lipogenesis. Our data showed that insulin upregulated FAS, ACC1 and SREBP1 protein expression whereas metformin and AICAR decreased them. Next, we found that FAS, ACC1 and SREBP1 protein expression was enhanced by inhibition of DNA methyltransferase activities. Bisulfite sequencing assay was used to examine the methylation status of FAS and ACC1 promoter regions in MCF10A, MCF7 and HepG2 cells. We found that the basal methylation level on ACC1 promoter regions was low but higher on FAS promoter regions. Insulin decreased methylation level of FAS promoter regions in MCF10A cells. On the other hand, metformin increased methylation level of FAS promoter regions in HepG2 cells. In contrast, metformin or AICAR did not significantly affect the methylation level on ACC1 promoter regions. Moreover, in high-fat diet (HFD)-induced obese mice, protein expression of FAS, ACC1 and SREBP1 was increased in the liver but decreased in white adipose tissue. However, bisulfite sequencing results revealed that the basal level of methylation on mice SREBP1 promoter regions was low and the changes of methylation level were not significant between normal and obese groups. These findings suggested that FAS expression upon insulin and metformin treatments were regulated by modifying methylation status on FAS promoter regions.
Key words：Lipogenesis, ACC1, FAS, SREBP1, DNA methylation

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