叶酸补充对高脂饮食诱导的肥胖小鼠的影响及其机制研究

发布时间：2018-05-16 03:41

本文选题：肥胖 + DNA甲基化　；参考：《北京协和医学院》2017年博士论文

【摘要】：第一章叶酸补充对高脂饮食诱导的胰岛素抵抗的影响及其机制研究背景:肥胖症的流行是引起胰岛素抵抗、高血压、2型糖尿病和心血管疾病等慢性疾病发生率增加的主要原因。肥胖相关的胰岛素抵抗是遗传和环境因素共同作用的结果。这些因素在疾病发展期间通过表观遗传修饰发生相互作用,表观遗传学标志可能解释了生活方式和疾病风险之间的联系。白色脂肪组织(WAT)在肥胖相关的胰岛素抵抗中起着重要作用。然而,WAT的功能异常以及在促进肥胖相关胰岛素抵抗中的确切机制尚不清楚。叶酸(FA)通过提供一碳单位在S-腺苷甲硫氨酸的合成通路中发挥重要作用,FA补充能够改变全基因组DNA的甲基化特点,预防DNA损伤,促进DNA修复。已有研究发现,在伴有2型糖尿病的肥胖症患者,FA补充改善了胰岛素抵抗、降低了血浆同型半胱氨酸(Hcy)水平,同时改善了血糖控制。然而,对于FA补充在肥胖风险和胰岛素抵抗中的作用仍然缺乏系统性研究。我们因此拟采用全基因组甲基化测序的方法,直接探索FA补充对小鼠脂肪组织DNA甲基化特点的影响,并检测这些作用带来的功能改变。方法:雄性C57BL/6J小鼠分别给予高脂饮食(HFD),正常饮食(ND),或HFD补充FA饮食(FAD),喂养10周后,获取小鼠的附睾脂肪组织,利用甲基化免疫共沉淀测序(MeDIP-seq)技术,对小鼠脂肪组织进行全基因组甲基化测序。通过亚硫酸氢盐测序的方法验证高通量的测序结果,随后,采用荧光定量PCR(RT-qPCR)方法检测相关甲基化改变基因的相应表达量变化。结果:小鼠经HFD喂养10周后,相比对照组,体重、附睾脂肪重量均显著增加,同时HFD组小鼠空腹血糖和血清胰岛素水平也明显升高。FA补充显著减少了 HFD诱导的脂肪累积,降低了空腹血糖水平,改善了胰岛素抵抗。MeDIP-seq结果显示,差异甲基化区(DMRs)分布在整个小鼠基因组上,在HFD组小鼠,出现明显的DNA高甲基化。在FAD组与HFD组之间,共鉴定出3787个差异甲基化注释基因,通路分析这些差异甲基化基因(DMGs)发现,与insulinsecretion,pancreatic secretion,type2 diabetes信号通路密切相关。亚硫酸氢盐测序验证了这些通路中的基因(Adcy3和Rapgef4)甲基化改变,基因表达量改变同样证实,相比HFD组,FA补充引起Adcy3和Rapgef4基因的表达显著增加。结论:FA补充饮食改善了 HFD诱导的胰岛素抵抗,减少了脂肪块累积,诱导了与能量代谢和胰岛素分泌相关基因的甲基化和表达量改变。第二章叶酸补充对肥胖相关心功能异常和心肌纤维化的作用及其机制研究背景:肥胖被认为是不合理的脂肪组织累积和功能异常引起的能量平衡异常,导致了2型糖尿病和心血管疾病的盛行。大量研究证实,肥胖引起了心肌结构重塑和心功能异常。然而,这些改变的潜在机制尚不完全清楚。氧化应激和炎症被认为参与了多种类型的心血管疾病的发病机制和疾病进展。有研究发现,氧化应激在肥胖相关的心血管异常中起着关键作用。在糖尿病心脏病患者,使用抗氧化剂能够明显缓解心肌纤维化和炎症反应。叶酸作为一种抗氧化剂,近来发现能够减少心肌缺血期间的活性氧产物的产生,缓解心功能异常。一项对随机对照实验的meta分析也证实,叶酸补充能够明显降低心血管疾病的风险。FA的抗氧化作用被认为是预防心血管疾病的关键因素。此外,高Hcy血症也被发现与高风险心血管疾病相关,饮食中补充FA则可以将Hcy转换为蛋氨酸降低血浆Hcy水平。因此,FA被认为具有预防ROS产生和缓解高Hcy血症的作用,而ROS和Hcy与心功能异常密切相关。然而,FA在肥胖相关性心肌病中的作用尚未有研究报道。方法:雄性6周龄C57BL/6J小鼠分别给予ND,HFD和FAD,喂养14周后,在麻醉条件下,经胸壁超声心动图检测各组小鼠的心脏功能。同时测量小鼠体重变化、心脏重量、血糖水平、心脏组织中FA和Hcy含量,以及心肌氧化应激状态,组织病理观察各组小鼠心肌的病理变化。结果:HFD引起了小鼠空腹血糖升高,体重和心脏重量增加。FA补充增加了心肌组织中的FA含量,改善了 HFD诱导的空腹血糖水平,降低了小鼠心脏重量和HFD诱导的心脏Hcy含量升高。此外,相比ND组,HFD组小鼠的心脏射血分数和短轴缩短率均明显下降,而FA补充显著改善了 HFD诱导的小鼠心功能异常。同时,FA补充预防了 HFD引起的心肌纤维化。相比对照组,HFD明显增加了心脏组织MDA含量,降低了抗氧化酶GSH和CAT活性,而FA补充显著改善了 HFD诱导的心肌氧化应激状态。结论:FA补充显著降低了肥胖小鼠的空腹血糖水平,减少了心脏Hcy含量,逆转了肥胖相关的心功能异常。这些FA补充对心脏功能的改善可能通过缓解氧化应激和减少心肌纤维化介导。
[Abstract]:Chapter 1 the effect of folic acid supplementation on insulin resistance induced by high fat diet and its mechanism research background: obesity is the main cause of the increase in the incidence of chronic diseases such as insulin resistance, hypertension, type 2 diabetes and cardiovascular disease. Obesity related insulin resistance is the result of the combination of genetic and environmental factors. These factors interact through epigenetic modification during the development of the disease. Epigenetic markers may explain the link between lifestyle and the risk of disease. White fat tissue (WAT) plays an important role in obesity related insulin resistance. However, the dysfunction of WAT and the promotion of obesity related insulin can be used. The exact mechanism of resistance is not clear. Folic acid (FA) plays an important role in the synthesis pathway of S- adenosine methionine by providing a single carbon unit, FA supplementation can change the methylation of whole genome DNA, prevent DNA damage and promote DNA repair. The study found that FA supplementation improved the islets in obese patients with type 2 diabetes. Hormone resistance reduces plasma homocysteine (Hcy) levels and improves blood glucose control. However, there is still a lack of systematic study on the role of FA supplementation in obesity and insulin resistance. We should therefore use a complete genome sequencing method to explore the DNA methylation characteristics of FA supplementation in mouse adipose tissue directly. Method: male C57BL/6J mice were given high fat diet (HFD), normal diet (ND), or HFD supplemented FA diet (FAD). After feeding 10 weeks, the epididymal adipose tissue of mice was obtained, and the methylation immunoprecipitation sequencing (MeDIP-seq) technique was used to carry out the whole genome methylation of the mouse adipose tissue. The results of high throughput sequencing were verified by the method of hydrogen sulfite sequencing. Subsequently, the corresponding expression changes of related methylation altered genes were detected by fluorescence quantitative PCR (RT-qPCR). Results: after 10 weeks of feeding in HFD, the weight and epididymal fat weight of mice increased significantly compared with the control group, while the fasting blood glucose in the group HFD mice was at the same time. The level of.FA supplementation significantly reduced the accumulation of fat induced by HFD, reduced the level of fasting blood glucose, and improved the results of insulin resistance.MeDIP-seq, and the difference methylation area (DMRs) was distributed throughout the genome of the mice, and in the HFD group, the obvious hypermethylation of DNA was found. Between the FAD group and the HFD group, a total of DNA hypermethylation was found. 3787 differential methylation annotation genes were identified. Pathway analysis of these differentially methylated genes (DMGs) was found to be closely related to insulinsecretion, pancreatic secretion, and type2 diabetes signaling pathways. Compared with the HFD group, FA supplementation caused a significant increase in the expression of Adcy3 and Rapgef4 genes. Conclusion: FA supplementation improves the insulin resistance induced by HFD, reduces the accumulation of fat blocks, induces the methylation and expression of genes related to energy metabolism and insulin secretion. The second chapter folic acid supplementation to obesity is concerned with abnormal function and myocardium. The role of fibrosis and its mechanism research background: obesity is considered to be an abnormal energy balance caused by the accumulation and dysfunction of adipose tissue, which leads to the prevalence of type 2 diabetes and cardiovascular disease. A large number of studies have confirmed that obesity has caused myocardial remodeling and cardiac dysfunction. However, the potential mechanisms of these changes are not yet completed. It is fully clear that oxidative stress and inflammation are considered to be involved in the pathogenesis and disease progression of various types of cardiovascular diseases. Studies have found that oxidative stress plays a key role in obesity related cardiovascular abnormalities. In patients with diabetes, the use of antioxidants can significantly alleviate myocardial fibrosis and inflammatory reactions. As an antioxidant, it has recently been found to reduce the production of reactive oxygen products during myocardial ischemia and relieve cardiac dysfunction. A meta analysis of randomized controlled trials also confirmed that folic acid supplementation can significantly reduce the risk of cardiovascular disease. The antioxidant effect of.FA is considered to be a key factor in preventing cardiovascular disease. In addition, high H CY has also been found to be associated with high risk cardiovascular disease. Dietary supplementation of FA can convert Hcy to methionine to reduce plasma Hcy levels. Therefore, FA is considered to have the effect of preventing ROS production and alleviating hyperlipidemia, and ROS and Hcy are closely related to cardiac dysfunction. However, the role of FA in obesity related cardiomyopathy has not yet been studied. Methods: the male C57BL/6J mice of 6 weeks of age were given ND, HFD and FAD respectively. After 14 weeks of feeding, the cardiac function of each group was detected by the chest wall echocardiography. The body weight change, heart weight, blood glucose level, the content of FA and Hcy in the heart tissue, and the oxidative stress state of the heart were measured, and the pathological observation of the myocardium was observed. Pathological changes in myocardium of mice in each group. Results: HFD caused the increase of fasting blood glucose in mice. The increase of weight and heart weight.FA supplemented the FA content in the myocardium, improved the HFD induced fasting blood glucose level, reduced the weight of the heart of the mice and the Hcy content in the heart induced by HFD. In addition, compared to the ND group, the cardiac ejection of the HFD mice. FA supplementation significantly improved cardiac dysfunction induced by HFD, and FA supplementation prevented myocardial fibrosis induced by HFD. HFD significantly increased the MDA content of cardiac tissue, reduced the activity of antioxidant enzyme GSH and CAT, and FA supplementation significantly improved HFD induced myocardial oxygen. Conclusion: FA supplementation significantly reduces fasting blood glucose levels in obese mice, reduces cardiac Hcy content and reverses obesity related cardiac dysfunction. These FA supplements may be mediated by alleviating oxidative stress and reducing myocardial fibrosis.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2

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