【作者】 姜明红；

【导师】 卢大儒；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 人口老龄化是全球日趋面临的挑战,其中涉及到健康和疾病等问题。机体随年龄增长常伴随肥胖,而衰老和肥胖又能够引发2型糖尿病(Type 2 diabetesmellitus,T2DM)等许多代谢性疾病。随着近几年代谢综合征(Metabolicsyndrome,MS)研究的不断深入,研究者发现MS共同的病理生理基础是胰岛素抵抗(insulin resistance,IR),2007年国际糖尿病联盟(DF)在第二届“糖尿病前期和代谢综合症”国际会议上已经把糖尿病前期的发病机制阐述为胰岛素抵抗,可见胰岛素抵抗在糖尿病的发病过程中具有重要的意义。衰老(年龄增长)和肥胖是增加胰岛素抵抗发生的两个独立危险因素。随着人类寿命的延长,阐明增龄和肥胖产生胰岛素抵抗的发生机理,对不同年龄人群,特别是老年人的健康及疾病的预防具有重要的指导价值。但到目前为止,胰岛素抵抗的分子生物学机制还不是十分清楚,而表观遗传改变有助于将环境、年龄与疾病发生风险联系起来,目前已有证据显示表观遗传在细胞和器官的衰老中扮演着重要的角色。在人类随年龄增长逐渐衰老的过程中,表观遗传标记比DNA序列更易受到环境因素的影响而发生变化,它的轻微变化就可能打开或关闭基因的表达从而产生一系列的影响,对胰岛素抵抗及糖尿病的发病可能具有重要的意义。肝脏是胰岛素作用的关键器官,肝脏对胰岛素的敏感性降低将导致肝脏的葡萄糖利用降低以及葡萄糖输出增加,与糖尿病的发病密切相关。研究肝脏在胰岛素抵抗以及糖尿病发病机理中的作用对于探讨糖尿病的病因以及治疗糖尿病具有重要意义。本课题拟从衰老和肥胖两方面探讨肝脏糖代谢相关基因的表观遗传学调控机制,为阐述胰岛素抵抗和糖尿病的分子生物学发生机制提供依据。方法和结果:一、不同鼠龄大鼠肝脏糖代谢相关基因的表观遗传学研究:1.大鼠的基本指标:14周龄(年轻鼠)、40周龄(中年鼠)和80周龄(老年鼠)的Wistar大鼠空腹血糖均正常,但空腹胰岛素水平随鼠龄增高而增高,表明中年及老年鼠处于胰岛素抵抗阶段。2.肝脏糖代谢相关基因的表达水平:肝脏糖代谢基因Glut2,Gck和L-PK基因的表达水平均随大鼠的鼠龄增加而降低,肝脏的糖原水平也随鼠龄的增加而逐渐降低。3.不同鼠龄大鼠肝脏糖代谢相关基因的表观遗传学研究:(1)建立了一种定量DNA甲基化的简单可行的方法,即DNA经亚硫酸氢盐处理后PCR扩增并直接测序,根据测序峰值图中“T”和“C”碱基的峰高计算CpG位点的甲基化程度。(2)利用该甲基化定量的方法,检测了Glut2、Gck和L-PK基因启动子的DNA甲基化程度,发现Glut2基因的启动子区甲基化程度在不同鼠龄大鼠肝脏中不存在差异,而Gck和L-PK基因启动子的DNA甲基化程度随大鼠鼠龄增加而逐渐增高,呈现明显的年龄依赖性特点,提示肝脏Gck和L-PK基因启动子区的甲基化程度与其基因的转录水平可能存在负相关关系。二、肥胖大鼠肝脏糖代谢相关基因与表观遗传学的研究肥胖是机体产生胰岛素抵抗的重要因素之一,我们探讨了表观遗传学机制在肥胖所致的胰岛素抵抗中的作用:1.肥胖大鼠模型的建立:大鼠4周龄开始给予高脂饲料喂养,大鼠体重随喂养时间延长而显著增加,喂养8周(鼠龄为12周)后成功地建立了肥胖合并胰岛素抵抗的大鼠模型。2.肥胖大鼠肝脏糖酵解的两个关键基因Gck和L-PK的表达水平显著低于正常大鼠;肥胖大鼠的肝糖原显著低于正常体重大鼠。3.肥胖大鼠Gck和L-PK基因启动子的甲基化程度显著高于正常体重大鼠,说明这两个基因启动子区的甲基化程度与基因的转录水平可能是一种负相关关系,提示肥胖可能是影响肝脏Gck和L-PK启动子甲基化程度的重要因素之一。三、游离脂肪酸诱导的胰岛素抵抗肝细胞的表观遗传学研究通过建立胰岛素抵抗肝细胞模型,在细胞水平上进一步研究表观遗传学与胰岛素抵抗之间的关系:1.胰岛素抵抗肝细胞模型的基因表达变化:浓度为0.1mM的PA诱导大鼠正常肝细胞株BRL细胞24h后即成胰岛素抵抗细胞模型(BRL/IR)。该细胞的Gck基因转录、翻译及酶活均显著降低,肝糖原含量显著降低。2.胰岛素抵抗肝细胞模型的Gck启动子区甲基化程度的变化:Gck启动子区的甲基化程度几乎在所有的CpG位点中都稍有增加,提示Gck基因表达与启动子区的甲基化之间可能存在负相关关系;去甲基化试剂5-Aza-CdR处理过的BRL/IR细胞Gck基因表达水平显著提高。3.药物盐酸小檗碱(BBR)对BRL/IR细胞的疗效观察:BBR能够显著提高BRL/IR细胞的葡萄糖消耗量及肝糖原含量;显著增加Gck基因在转录及翻译水平上的表达量以及Gck的活性;显著降低BRL/IR细胞内聚集的TG含量。BBR处理过的BRL/IR细胞Gck启动子区的甲基化程度稍降低,提示去甲基化机制可能参与了BBR药物对Gck基因转录水平的调控。4.表达Gck siRNA和Gck基因的腺病毒制备及对胰岛素抵抗细胞作用的初步研究:成功制备了表达Gck RNA干扰的腺病毒Ad-Gck-siRNA和表达Gck基因的腺病毒Ad-Gck。实验证实Ad-Gck-siRNA腺病毒能够显著降低肝细胞的Gck基因表达量,显著降低肝细胞的葡萄糖消耗量以及肝糖原含量;Ad-Gck腺病毒能够在肝细胞内过量表达Gck基因,显著提高BRL/IR细胞的葡萄糖消耗量以及肝糖原含量。通过在肝细胞中过量表达或抑制表达Gck基因,初步证实了Gck基因在肝细胞的胰岛素抵抗状态中具有重要的作用,为今后在动物体内进行Gck基因的功能研究打下了基础。结论:胰岛素抵抗是逐步发展形成的,而衰老和肥胖是产生胰岛素抵抗的两个重要因素,并与葡萄糖代谢密切相关。本课题即从表观遗传学的角度分析衰老及肥胖大鼠发生胰岛素抵抗的分子生物学机制,发现了肝脏糖代谢关键基因Gck和L-PK在衰老和肥胖两种胰岛素抵抗大鼠肝脏中均存在启动子区的甲基化程度与基因表达的负相关关系,影响了肝脏的胰岛素利用及葡萄糖代谢,是参与胰岛素抵抗的重要机制。药物BBR能够上调胰岛素抵抗肝细胞的Gck基因表达量,同时也能够降低Gck基因启动子的甲基化程度,因此我们推测BBR激活Gck基因表达的机制可能包括Gck基因启动子的去甲基化机制。综上所述,本课题对胰岛素抵抗动物和细胞模型的甲基化机制进行了初步探索,对今后胰岛素抵抗及相关的代谢性疾病的表观遗传学研究提供了新的思路,同时也为探讨通过调控基因组DNA甲基化状态来防治临床的代谢性疾病提供了新的依据。更多还原

【Abstract】 Background and AimsAging has been posing a global challenge to mankind, which is concomitant with many health issues. When bodies grow old, obesity accompanies, and these two factors make bodies susceptible to Type 2 diabetes mellitus(T2DM) and a number of other metabolic diseases. With the progress in the research of Metabolic Syndrome (MS), researchers have pinned the common pathophysiological basis on insulin resistance(IR). During The Second International Congress on Prediabetes and the Metabolic Syndrome" hosted by International Diabetes Federation in 2007, the etiology of prediabetes was attributed to insulin resistance, which reflected the central importance of insulin resistance in the development of diabetes.Aging and obesity are two independent risk factors in insulin resistance. With the increase of human lifespan, the elucidation of the effect of aging and obesity on insulin resistance will provide valuable information for the health and prevention of diseases in older population. The molecular mechanism of insulin resistance remains unclear up to now. Epigenetic changes link environmental factors, age and disease risk, and has been indicated to play an important role in cellular and individual aging. Epigenetic marks are easier to change than DNA sequence, under the influence of environmental factors. A slight change of epigenetic marks may activate or silence gene expression and produce a series of effects on the development of insulin resistance and diabetes. In this project, we sought to dissect the epigenetic mechanism of insulin resistance from two aspects—aging and obesity, using glucose-metabolic genes.Methods and results:1. The epigenetic study of aging rat hepatic glucose-metabolism-related genes.1) Determination of expression of hepatic glucose-metabolism-related genes.(1) Physiological indices of rats used in the experiments. Age: 14 wks (young), 40 wks (adult), 80 wks (aged). While fasting blood glucose levels in the three groups are normal, blood insulin increase with age, and the increase was significant in adult and aged rats, compared to the young group, indicating that rats were insulin resistant in the former two groups.(2) Transcription level of hepatic glucose-metabolism-related genes: hepatic glycogen decrease with increasing age, and the expression of hepatic glucose-metabolism-related genes Glut2, Gck and L-PK decrease with increasing age. What’s more, the degree of reduction of the activity and transcription of Gck and L-PK were similar.2). Epigenetic study of hepatic glucose-metabolism-related genes in aging rats: the methylation levels of Glut2、Gck and L-PK promoters were measured(1) The methylation degree of each of the 12 CpG sites in Glut2 promoter is showed no difference among the three groups. This result indicated that the methylation degree of Glut2 promoter methylation is not related to aging, ruling out the regulatory effect of DNA methylation on transcription.(2) The methylation levels of Gck and L-PK promoter increased with age in the three groups of rat livers, showing an age-dependent manner, which indicated a negative correlation between Gck and L-PK promoter methylation and their transcriptions.2. Epigenetic study of hepatic glucose-metabolism-related genes in obesity rats.(1) Establishment of obese rat model: the rats were fed high-fat food beginning at the age of 4 wks. Body weights of these rats increase quickly with time, and after 8 wks of high-fat feeding we confirmed the successful establishment of obese rat model.(2) Detection of transcription of glucose-metabolism-related genes in obese rat livers: glycogen contents of obese rats were higher than normal rats; Real time PCR detection of glycolysis-Gck and L-PK-is significantly lower than normal rats.(3) The epigenetic study of obese rat hepatic Gck and L-PK genes: the methylation level of both Gck and L-PK promoters are significantly higher than normal-weighted rats, indicating a negative correlation between the methylation levels of the these promoters and transcription, which hinted that obesity is probably a primary factor in the hypermethylation of hepatic Gck and L-PK promoter.3. Epigenetic study of insulin resistant hepatocytes induced by free fatty acids. 1). Establishment of insulin resistant hepatic cell line. Glucose utilization the "gold standard" glucose uptake were significantly altered after 24 hrs induction of normal rat hepatocytes with 0.1 mM PA, confirmed that the model was insulin resistant cell model (BRL/IR for short).3). Changes in the expression of glucose-metabolism-related genes in BRL/IR cells: hepatic glycogen content of BRL/IR cells ware significantly reduced; transcription, translation and enzymatic activity of Gck was significantly reduced.4). Effect of BBR on the treatment of BRL/IR: 20μM BBR signifieantly increase glucose uptake and hepatic glycogen content in BRL/IR cells; significantly increase Gck transcription and translation and enzymatic activity. What’s more, BBR significantly reduced hepatic TG content in BRL/IR, and reversed hepatic steatosis.5). Epigenetic study of rat hepatocytes-BRL cell line Gck promoter(1) Determination of BRL cells Gck promoter methylation revealed a minor and variable increase in almost every CpG site after 24 hrs PA induction and showed a negative correlation.(2) Transcription of Gck in BRL/IR was elevated by demethylating agent 5-Aza-CdR. The decrease of methylation in hypomethylated CpG sites is significant, but not significant in hypermethylated CpG sites.(3) The methylation level of hypomethylated CpG sites in BRL/IR cells Gck promoter were significantly reduced by BBR treatment, but the reduction is not significant in hypermethylated CpG sites. However, the demethylating effect of BBR is higher than 5-Aza-CdR, indicating that BBR might activate Gck expression through demethylation of Gck promoter.(4) Construction of Gck-expressing and Gck-siRNA-expressing adenovirus and their effects on insulin resistant cells: Adenoviruses expressing Gck (Ad-Gck) and Gck siRNA (Ad-Gck-siRNA) were contructed. Our experiments showed that overexpression of Ad-Gck can significantly improve glucose uptake and hepatic glycogen in BRL/IR cells; Ad-Gck-siRNA can signifieantly repress hepatic Gck expression and decrease hepatic glucose uptake and glycogen. The role of Gck in hepatic insulin resistance was evidenced by overexpression or repression of Gck, paving the way for functional study of Gck in vivo. Conclusions:Insulin resistance develops gradually, and aging and obesity are two primary factors inducing insulin resistance, which was intimately related with glucose tolerance. In this project, we analyzed the molecular mechanism of aging and obese rats’ susceptibility to diabetes from the epigenetic point of view, and discovered the reverse relationship between gene transcription and the promoter methylation status of two hepatic glucose metabolizing genes: Gck and L-PK. The hypermethylation of these promoters impaired insulin’s effect and glucose metabolism, and contributes to the susceptibility to diabetes. In the cell model of insulin resistance, the reverse relationship between Gck transcription and promoter methylation is confirmed, and we found that BBR increase Gck transcription through promoter demethylation. This study is a primary exploration of DNA methylation in insulin resistant cell and animal models, and will shed a new light on further study of the epigenetics of diabetes, and provide foundation for epigenetic therapy of metabolic diseases by regulating DNA methylation..更多还原