- 學位論文
高脂飲食飼養鼠在缺乏甘氨酸N-甲基轉移酶將導致類血管生成素蛋白8高度表現
Loss of Glycine N-Methyltransferase Associates with Angiopoietin-Like Protein 8 Expression in High Fat-Diet-Fed Mice
摘要
研究背景： 隨著人類文明的發展，人類的生活習性已從狩獵、畜牧、農耕演進至工業化生活。在緊張競爭的同僚互比貢獻度環境裡，早睡早起已然成為遙不可及的期許。在高壓過勞的體制下，犧牲的睡眠時間與運動習慣，往往只能從飲食中得到撫慰補償，卻因而衍生出許多多潛藏的文明病，以習以為常之姿蔓延在二十世紀忙忙碌碌的文明人身上。在過去，心寬體胖是一種嚮往的悠哉閒適，時至今日，肥胖卻成為健康隱形殺手的代名詞，不單是因為潮流所趨，更有諸多文獻證實肥胖和代謝症候群與心血管、腎臟、肝臟等重大器官疾病息息相關[1,2]，因此近年來，研究脂肪及醣類代謝成為熱門焦點。在肌肉、肝臟組織及脂肪細胞中的蛋白質已被證實與諸多代謝疾病有關[3-5]，其中包括發炎細胞素、賀爾蒙及生長激素[6-8]，尤其由脂肪、肌肉及肝臟生成的激素已被證實對多項代謝疾病有關[9,10]。面對肥胖造成的健康災難與日俱增，瞭解因高脂飲食導致的分子代謝異常原因以及肌肉細胞、肝臟組織及脂肪細胞中的蛋白質在脂質代謝中扮演的角色實在刻不容緩。諸多研究顯示肥胖易造成代謝障礙及胰島素阻抗，胰島素過分表現則促使Angptl8濃度上升，導致血液中三酸甘油酯代謝減少而造成血脂累積過高。而GNMT可促進磷酸酯酶與張力蛋白同源物(Phosphatase and tensin homolog,PTEN)去抑制Akt訊號傳遞，但是關於Angptl8與GNMT兩者之間的分子關聯性，目前尚未被了解。 方法： 我們使用20隻6週大C57BL/6J雄性鼠隨機分入正常餵食群(ND)與高脂餵食群(HFD)各10隻，經十二週後，統計分析群組間體重增加、進食量與餵食效率(feed efficiency ratio)的關係。實驗分為三大方向，第一群為正常餵食與高脂餵食小鼠間的差異，第二群為正常餵食小鼠的肝細胞在各種不同變數介入下蛋白質表現量的差異，第三群為一般小鼠(wide type)與基因控制小鼠(GNMT-knock out)肝細胞間的差異。於實驗終點時，採檢實驗鼠心臟血液、肝臟細胞及脂肪細胞解密Angptl8及GNMT蛋白質及mRNA表現量之間的關聯性。另外針對正常鼠肝細胞施以PI3K抑制劑(LY294002) 、Akt抑制劑(MK2206) 、kinase-dead Akt plasmid(dn-Akt)及 myrAkt，觀察其被胰島素刺激後磷酸化Akt (p-Akt)與Angplt8之間的關係。此外，觀察GNMT-KO與wild type小鼠肝臟組織中Angplt8與p-Akt的濃度是否亦有差別，以及GNMT-KO肝細胞在加入LY294002 、MK2206後，Akt (p-Akt)與Angplt8兩者之間的交互作用。 結果： 在第一群組實驗裡，在相同的食物攝取量下，HFD組無論在體重增加、餵食效益比（FER）、BMI、肝臟重量、肝體重比例、總膽固醇、血糖及胰島素上都顯著高於ND組，此外在肝細胞及血漿中，Angptl8在HDF組亦高於ND組，而在肝細胞及脂肪細胞中，GNMT在HDF組則較低。在第二組實驗裡，肝細胞在經過胰島素的刺激後，Angptl8蛋白會明顯上升，且mRNA表現量會隨著胰島素劑量及暴露時間上升，而以LY294002 、MK2206及dn-Akt處理過後的肝細胞發現抑制掉Akt會連帶使Angptl8表現不活化，但以myrAkt處理後結果則完全相反。第三組實驗發現GNMT-KO組較wild type組的確有較明顯因Akt磷酸化導致Angptl8活化的現象，但以LY294002 、MK2206干擾Akt磷酸化後，亦導致Angptl8不活化。 結論： 藉由小鼠高脂飲食模擬現代人過分攝食的狀況下，研究肝細胞及脂肪細胞在胰島素過分刺激後促進Akt活化，導致Angptl8亦被活化，進而造成脂肪代謝受阻而升高血液中的三酸甘油脂，且在此肥胖狀況下亦致使GNMT被抑制，其抑制Akt磷酸化的過程受阻，最終結果也活化Angptl8的作用。藉此實驗了解高脂飲食下GNMT及Angptl8在脂質代謝中反應的變化，讓肥胖疾病造成的脂質代謝障礙交互作用的藍圖有更進一步了解。
並列摘要
Background: By the achievement of human culture, people's life has changed from hunting, grazing, cultivating to industry. Under the high pressure environment and the colleague's competition, it’s no easy to go to bed early and get up early. Living for overwork, lost of sleep quality and abandoning exercise are as usual as breathing. Lots of people can only get compensations from eating, resulting in producing many potential civilization illnesses in the busy 20th century. In the older ages, rich people with plump body shape were thought as an expression of wealth and people-desired lifestyle. But now, obesity is regarded as a hidden killer of health. Not only being era trend, but many evident issues have proved the strong relationship between obesity and metabolic syndromes, cardiovascular disease, renal and liver insufficiency. Recent years, researches in cholesterol and carbohydrates metabolism are hot spots. Proteins in muscle, liver and fat tissues have showed the connections to lots of metabolic diseases. When facing the fact that overweight becomes an inexorable trend, exploring the biological mechanism of how high fat diet affects metabolic abnormalities in tissues and what the protein's role is in lipid metabolism are urgent. Recent literatures point out that Angiopoietin-like protein 8 (Angptl8) produced from liver tissue and adipose cell regulates triglyceride function. By animal experiments, the blockage of plasma Angptl8 promotes triglyceride clearance and increases the weight loss. However, the molecular regulation of Angptl8 is still unknown. As we know, the expression of Angptl8 can be regulated through insulin-mediated PI3K/Akt pathway. Also Akt could be activated by inhibition of Glycine N-Methyltransferase (GNMT). But the connection between Angptl8 and GNMT is still unknown. Methods: Twenty age-matched C57BL/6J mice (6 weeks old; male; Charles River. Technology, BioLASCO Taiwan Co, Ltd., Taipei, Taiwan) were randomly divided into ND-fed and HFD-fed groups with 10 mice each. The mice were fed with a control diet or a high-fat diet for 12 weeks. Body weight gain and feed consumption were monitored weekly to determine the feed efficiency ratio (FER). The end points of the experiment were designed in three comparisons. The first one is the difference between the normal diet group and the high fat diet group. After experiment, the blood samples, the liver and adipose cells were collected to identify the expression between Angptl8, GNMT and mRNA in two groups. The second point is the change of protein in the normal diet feeding mice's liver cell under different biochemical intervention. The relationship between insulin-induced p-Akt and Angplt8 in liver cells were studied under the intervention with PI3K inhibitor (LY294002), Akt inhibitor (MK2206), kinase-dead Akt plasmid (dn-Akt) and myrAkt. The last one is the level comparison of Angplt8 and p-Akt in the liver tissue between the wide type and GNMT-knock-out mice group. And the interaction after the intervention of PI3K inhibitor and Akt inhibitor in the GNMT-KO mice liver cell. Results: In the first experiment under the same food consumption, the body wight gain, food efficiency ratio (FER), body mass index (BMI), liver weight, hepatosomatic index (HSI), cholesterol, blood glucose and insulin level were significantly higher (p < 0.05) in a HFD group. In liver and serum, Angptl8 level is also higher in HFD group. But in liver and adipose tissue, GNMT is lower in HFD group. In the second experiment in ND group, after the stimulation via insulin, Angptl8 elevated obviously and the expression of mRNA increased by dose and by time. After inhibition of Akt phosphorylation by LYT294002, MK2206 and dn-Akt, Angptl8 was down-regulated. But treated by myrAkt, the result is exactly the opposite. In the last experiment, Angptl8 is activated more by Akt phosphorylation in GNMT-KO than in wide type group. But after the interference of Akt phosphorylation by LYT294002, MK2206, Angptl8 expression is inhibited. Conclusions: Through the high fat diet mice experiment simulating the over-feeding situation in modern habit, excessive release of insulin changes the physiochemical reaction in liver and adipose cells. Exposure in high insulin level from high fat diet-related obesity results in activation of PI3K-Akt pathway. It promotes the expression of Angptl8 making lipoprotein lipase dysfunction. Therefore, poor clearance of lipid metabolism makes triglyceride elevated. In the other hand, GNMT was down-regulated in HFD group. Therefore, GNMT can’t play the role to inhibit Akt phosphorylation. In the end, Angptl8 was activated.