Regulation of Forkhead Box O Transcription Factors by Docosahexaenoic Acid

肥胖與胰島素拮抗、二型糖尿病、高血壓、動脈粥狀硬化、葡萄糖不耐症、血脂異常及慢性發炎息息相關，這些病症統稱為代謝症候群。二十二碳六烯酸（DHA）為一種n-3族多不飽和脂肪酸，美國食品和藥物管理局於2004年宣布日常食物中DHA和EPA可降低罹患冠心病及相關疾病之風險。 本研究目的旨在找出DHA對脂質和醣類代謝途徑之影響，30頭離乳仔豬隨機分成3組分別餵予2％牛油、大豆油或DHA油30天。實驗結束時豬隻經電昏後收集血液及臟器進行後續分析。抽取組織總RNA並設計引子篩選可能受DHA調控之相關基因參與脂質、醣類代謝和慢性炎症之反應。豬隻餵飼2％DHA油30天不影響脂肪組織抗或促發炎因子之表現，包括細胞介白素4（IL4）、IL6、 IL10、IL13和腫瘤壞死因子α（TNFα）。我們也發現一個新的受DHA調控之轉錄因子。餵飼2％DHA於豬隻可顯著降低肝臟和脂肪組織之叉頭轉錄因子（FoxO）包括FoxO1和FoxO3基因表現。同時豬隻餵予2％DHA可顯著降低肝臟和脂肪組織中叉頭轉錄因子輔因子PGC1α表現，但對SIRT表現並無顯著影響。在人類和豬的脂肪細胞處理DHA 24小時可得類似之結果。且豬隻餵予2％DHA可顯著降低FoxO下游基因包括參與肝臟VLDL組裝之微粒體三酸甘油酯轉移蛋白（MTTP）、參加糖質新生之葡萄糖-六-磷酸酶（G6Pase）、參與三酸甘油酯分泌之載脂蛋白CⅢ（apoCⅢ）和胰島素生長因子結合蛋白1（IGFBP1）之基因表現。餵予DHA之豬隻同時降低血漿中總膽固醇及三酸甘油酯之含量。綜合上述，DHA可能透過抑制FoxO基因表現進而影響豬隻脂質和醣類代謝。伴隨著降低三酸甘油酯和脂蛋白VLDL組裝之相關基因表現可能可解釋DHA對預防或改善代謝綜合症候群之益處。Obesity is closely linked to insulin resistance or type 2 diabetes mellitus. Obesity is also associated with hypertension, atheroschlerosis, glucose intolerance, dyslipidemia, and chronic inflammation, also known as the metabolic syndrome. Docosahexaenoic acid (DHA) is one of the n-3 polyunsaturated fatty acids and USA FDA had announced on 2004 that DHA and EPA could reduce the risk of coronary heart disease in conventional foods.o decipher beneficial effects of DHA on lipid and glucose metabolism, thirty weaned pigs were divided into three groups and fed ad librium for thirty days with a diet plus either 2% tallow, soybean or DHA oil, respectively. At the end of experiment, pigs were sedated and killed by electrical stunning. Viscera and blood samples were collected. Tissues were homogenized and total RNA was extracted for mRNA determination. Primers were designed for screening several genes involved in lipid metabolism and chronic inflammatory markers which may be regulated by supplementation of DHA oil. Supplementation of DHA on pig diets didn’t influence the expression of anti- or pro-infiltration cytokines, including interleukin 4 (IL4), IL6, IL10, IL13 and Tumor necrosis factor α (TNFα) in adipose tissues. Herein, we demonstrated a new transcription factor which DHA regulates. The class O of forkhead box transcription factors (FoxO) including FoxO1 and FoxO3 were decreased in the liver and adipose tissues of DHA-supplemented pigs compared with tallow-supplemented pigs. Pig diets containing DHA also decreased the cofactor of FoxO, PPARγ coactivator-1α (PGC1α), expression in both liver and adipose tissues. Similar results could also be found in human and pig primary adipocytes treated with DHA for 24 hours. Pigs fed with diets supplemented with DHA also down-regulated FoxO-target genes such as microsomal triglyceride transfer protein (MTTP), a protein which is involved in hepatic VLDL assembly. Expression of Glucose-6-phosphatase (G6Pase) participated in gluconeogenesis, apolipoprotein CⅢ (apoCⅢ) involved in TG secretion and insulin-like growth factor binding protein 1 (IGFBP1) was also reduced. Pigs supplemented with DHA also reduced plasma total cholesterol and triglyceride concentrations. n conclusion, DHA may partially act through down-regulation of FoxO function to influence lipid and glucose metabolism. Down-regulation of genes related triglyceride metabolism and VLDL assemble may implicate the prevention and beneficial role of DHA on metabolic syndrome.中文摘要 I錄 IV目錄 V目錄 VIntroduction 1art 1: Obesity, inflammation, and macrophage infiltration 1dipose tissue macrophage 2acrophage polarization 4art 2: Forkhead box O and metabolic regulation 6iver 7dipose tissue 9xperimental design 11aterials and methods 12nimals and Diets 12ample and data collection 12lasma glucose, triacylglycerol and cholesterol determination 13solation of porcine stromal/vascular cells 13ell culture and differentiation of porcine adipocytes 14eal-time quantitative PCR analysis 14ata Analysis 15esults 18iscussion 31eferences 3