衰老及肥胖大鼠肝脏糖代谢相关基因的表观遗传学研究
|
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。
Background and Aims
Aging 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..
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。
Background and Aims
Aging 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..
引文
1. Reaven GM, Reaven ER Age, glucose intolerance, and non-insulin-dependent diabetes mellitus. J Am Geriatr Soc 1985;33:286-290.
2. Narimiya M, Azhar S, Dolkas CB, Mondon CE, Sims C, Wright DW, Reaven GM. Insulin resistance in older rats. Am J Physiol 1984;246:E397-404.
3. Stewart KJ. Physical activity and aging. Ann N Y Acad Sci 2005;1055:193-206.
4. Fontana L, Klein S. Aging, adiposity, and calorie restriction. Jama 2007;297:986-994.
5. da Silva RC, Miranda WL, Chacra AR, Dib SA. Insulin resistance, beta-cell function, and glucose tolerance in Brazilian adolescents with obesity or risk factors for type 2 diabetes mellitus. J Diabetes Complications 2007;21:84-92.
6. Fontana L. Excessive adiposity, calorie restriction, and aging. Jama 2006;295:1577-1578.
7. Moore G, Knoblaugh S, Gollahon K, Rabinovitch P, Ladiges W. Hyperinsulinemia and insulin resistance in Wrn null mice fed a diabetogenic diet. Mech Ageing Dev 2008; 129:201-206.
8. Huang X, Charbeneau RA, Fu Y, Kaur K, Gerin I, MacDougald OA, Neubig RR. Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele. Diabetes 2008;57:77-85.
9. Raz I, Eldor R, Cernea S, Shafrir E. Diabetes: insulin resistance and derangements in lipid metabolism. Cure through intervention in fat transport and storage. Diabetes Metab Res Rev 2005;21:3-14.
10. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 2001 ;86:3574-3578.
11. Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, Sharpless NE. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 2006;443:453-457.
12. Li LC, Shiina H, Deguchi M, Zhao H, Okino ST, Kane CJ, Carroll PR, et al. Age-dependent methylation of ESR1 gene in prostate cancer. Biochem Biophys Res Commun 2004;321:455-461.
13. Martens JW, Sieuwerts AM, Bolt-deVries J, Bosma PT, Swiggers SJ, Klijn JG, Foekens JA. Aging of stromal-derived human breast fibroblasts might contribute to breast cancer progression. Thromb Haemost 2003;89:393-404.
14. Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ. Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene 1999;18:1313-1324.
15. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res 2001 ;61:3573-3577.
16. Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 1994;7:536-540.
17. Tohgi H, Utsugisawa K, Nagane Y, Yoshimura M, Genda Y, Ukitsu M. Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex. Brain Res Mol Brain Res 1999;70:288-292.
18. Ling C, Poulsen P, Simonsson S, Ronn T, Holmkvist J, Almgren P, Hagert P, et al. Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle. J Clin Invest 2007;117:3427-3435.
19. Bontemps F, Hue L, Hers HG. Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone. Biochem J 1978; 174:603-611.
20. Davidson AL, Arion WJ. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. Arch Biochem Biophys 1987;253:156-167.
21. Turner N, Li JY, Gosby A, To SW, Cheng Z, Miyoshi H, Taketo MM, et al. Berberine, and its more Biologically Available Derivative Dihydroberberine, Inhibit Mitochondrial Respiratory Complex I: A Mechanism for the Action of Berberine to Activate AMPK and Improve Insulin Action. Diabetes 2008.
22. Yin J, Gao Z, Liu D, Liu Z, Ye J. Berberine improves glucose metabolism through induction of glycolysis. Am J Physiol Endocrinol Metab 2008;294:E148-156.
23. Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, Ye JM, et al. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 2006;55:2256-2264.
24. Van Der Vies J. Two methods for the determination of glycogen in liver. Biochem J 1954;57:410-416.
25. Templeton M. Microdetermination of glycogen with anthrone reagent. J Histochem Cytochem 1961;9:670-672.
26. Chen J, Rider DA, Ruan R. Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. J Gerontol A Biol Sci Med Sci 2006;61:20-27.
27. Ginzinger DG Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002;30:503-512.
28. Grunau C, Clark SJ, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001;29:E65-65.
29. Bogdarina I, Murphy HC, Burns SP, Clark AJ. Investigation of the role of epigenetic modification of the rat glucokinase gene in fetal programming. Life Sci 2004;74:1407-1415.
30. Gallou-Kabani C, Junien C. Nutritional epigenomics of metabolic syndrome: new perspective against the epidemic. Diabetes 2005;54:1899-906.
31. Li LC, Chui R, Nakajima K, Oh BR, Au HC, Dahiya R. Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression. Cancer Res 2000;60:702-706.
32. Paul CL, Clark SJ. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. Biotechniques 1996;21:126-133.
33. Stirzaker C, Millar DS, Paul CL, Warnecke PM, Harrison J, Vincent PC, Frommer M, et al. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res 1997;57:2229-2237.
34. Lewin J, Schmitt AO, Adorjan P, Hildmann T, Piepenbrock C. Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates. Bioinformatics 2004;20:3005-3012.
35. Ngo V, Gourdji D, Laverriere JN. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression. Mol Cell Biol 1996;16:3245-3254.
36. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF. Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue. Blood 1998;91:1373-1381.
37. Murumagi A, Vahamurto P, Peterson P. Characterization of regulatory elements and methylation pattern of the autoimmune regulator (AIRE) promoter. J Biol Chem 2003 ;278:19784-19790.
38. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 2000;6:87-97.
39. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999;274:305-315.
40. Magnuson MA, Shelton KD. An alternate promoter in the glucokinase gene is active in the pancreatic beta cell. J Biol Chem 1989;264:15936-15942.
41. Frese T, Bazwinsky I, Muhlbauer E, Peschke E. Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats. Horm Metab Res 2007;39:567-574.
1.张家庆.新的HOMA2IR—从空腹血糖、空腹胰岛素测胰岛素抵抗.中华糖尿病杂志,2005,13:245-246.
2.Moore G,Knoblaugh S,Gollahon K,Rabinovitch P,Ladiges W.Hyperinsulinemia and insulin resistance in Wrn null mice fed a diabetogenic diet.Mech Ageing Dev 2008;129:201-206.
3.Huang X,Charbeneau RA,Fu Y,Kaur K,Gerin I,MacDougald OA,Neubig RR.Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele.Diabetes 2008;57:77-85.
4.Raz I,Eldor R,Cemea S,Shafrir E.Diabetes:insulin resistance and derangements in lipid metabolism.Cure through intervention in fat transport and storage.Diabetes Metab Res Rev 2005;21:3-14.
5.Nair S,Lee YH,Lindsay RS,Walker BR,Tataranni PA,Bogardus C,Baier LJ,et al.11beta-Hydroxysteroid dehydrogenase Type 1:genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.Diabetologia 2004;47:1088-1095.
6.Dominguez LJ,Barbagallo M.The cardiometabolic syndrome and sarcopenic obesity in older persons.J Cardiometab Syndr 2007;2:183-9.
7.Slawik M,Vidal-Puig AJ.Lipotoxicity,ovemutrition and energy metabolism in aging.Ageing Res Rev 2006;5:144-64.
8.Morgan HD,Sutherland HG,Martin DI,Whitelaw E.Epigenetic inheritance at the agouti locus in the mouse.Nat Genet 1999;23:314-318.
9.Dolinoy DC,Huang D,Jirtle RL.Matemal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development.Proc Natl Acad Sci U S A 2007;104:13056-13061.
10.Wolff GL,Roberts DW,Galbraith DB.Prenatal determination of obesity,tumor susceptibility,and coat color pattern in viable yellow(Avy/a) mice.The yellow mouse syndrome.J Hered 1986;77:151-158.
11.Zemel MB,Kim JH,Woychik RP,Michaud EJ,Kadwell SH,Patel IR,Wilkison WO.Agouti regulation of intracellular calcium:role in the insulin resistance of viable yellow mice.Proc Natl Acad Sci U S A 1995;92:4733-4737.
12.Kuklin AI,Mynatt RL,Klebig ML,Kiefer LL,Wilkison WO,Woychik RP,Michaud EJ.Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes.Mol Cancer 2004;3:17
13.Kaul S,Ritschel WA.Total body water as an index for predicting body fat in rats.Arzneimittelforschung 1986;36:112-6.
14.Peralta S,Carrascosa JM,Gallardo N,Ros M,Arribas C.Ageing increases SOCS-3 expression in rat hypothalamus:effects of food restriction.Biochem Biophys Res Commun.2002;16;296(2):425-8.
15.Bemardis LL,Patterson BD.Correlation between 'Lee index' and carcass fat content in weanling and adult female rats with hypothalamic lesions.J Endocrinol.1968;40:527-8.
16.Harder T,Rake A,Rohde W,Doerner G,Plagemann A.Overweight and increased diabetes susceptibility in neonatally insulin-treated adult rats.Endocr Regul 1999;33:25-31.
17.Rector RS,Warner SO,Liu Y,Hinton PS,Sun GY,Cox RH,Stump CS,Laughlin MH,Dellsperger KC,Thomas TR.Exercise and diet induced weight loss improves measures of oxidative stress and insulin sensitivity in adults with characteristics of the metabolic syndrome.Am J Physiol Endocrinol Metab 2007;293:E500-6.
18.Clark JM.Weight loss as a treatment for nonalcoholic fatty liver disease.J Clin Gastroenterol 2006;40:S39-43.
1.王镜岩,朱圣庚,徐长法.生物化学下册[M].北京:高等教育出版社,2002:232.
2.Bain JR,Schisler JC,Takeuchi K,Newgard CB,Becket TC.An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.Diabetes 2004;53:2190-2194.
3.Salavert A,lynedjian PBRegulation of phosphoenolpyruvate carboxykinase(GTP)synthesis in rat liver cells.Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone.J Biol Chem.1982;257:13404-13412.
4.Butte NF,Puyau MR,Vohra FA,Adolph AL,Mehta NR,Zakeri I.Body size,body composition,and metabolic profile explain higher energy expenditure in overweight children.J Nutr 2007;137:2660-7.
5.Miles JM,Nelson RH.Contribution of triglyceride-rich lipoproteins to plasma free fatty acids.Horm Metab Res 2007;39(10):726-9.
6.Jensen MD.Adipose tissue metabolism-an aspect we should not neglect? Horm Metab Res 2007;39:722-5.
7.Parekh S,Anania FA.Abnormal lipid and glucose metabolism in obesity:implications for nonalcoholic fatty liver disease.Gastroenterology 2007;132:2191-207.
8.Delarue J,Magnan C.Free fatty acids and insulin resistance.Curr Opin Clin Nutr Metab Care 2007;10(2):142-8.
9.Mlinar B,Marc J,Janez A,Pfeifer M.Molecular mechanisms of insulin resistance and associated diseases.Clin Chim Acta 2007;375(1-2):20-35.
10.Schinner S,Scherbaum WA,Bornstein SR,Barthel A.Molecular mechanisms of insulin resistance.Diabet Med 2005;22:674-82.
11.Reid BN,Ables GP,Otlivanchik OA,Schoiswohl G,Zechner R,Blaner WS,Goldberg IJ,Schwabe R,Chua SC Jr,Huang LS.Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation,stimulates direct release of free fatty acids and ameliorates steatosis.J Biol Chem.2008 Mar 12
12.Anderwald C,Brunmair B,Stadlbauer K,Krebs M,Furnsinn C,Roden M.Effects of free fatty acids on carbohydrate metabolism and insulin signalling in perfused rat liver.Eur J Clin Invest 2007;37:774-82.
13.Holland WL,Knotts TA,Chavez JA,Wang LP,Hoehn KL,Summers SA.Lipid mediators of insulin resistance.Nutr Rev 2007;65:S39-46.
14.Lall CG,Aisen AM,Bansal N,Sandrasegaran K.Nonalcoholic Fatty liver disease.AJR Am J Roentgenol 2008;190:993-1002.
15.Turkish AR.Nonalcoholic fatty liver disease:emerging mechanisms and consequences.Curr Opin Clin Nutr Metab Care 2008;11:128-33.
16.Streba LA,Carstea D,Mitru,t P,Vere CC,Dragomir N,Streba CT.Nonalcoholic fatty liver disease and metabolic syndrome:a concise review.Rom J Morphol Embryol 2008;49:13-20.
17.Turner N,Li JY,Gosby A,To SW,Cheng Z,Miyoshi H,Taketo MM,Cooney GJ,Kraegen EW,James DE,Hu LH,Li J,Ye JM.Berberine,and its more Biologically Available Derivative Dihydroberberine,Inhibit Mitochondrial Respiratory Complex Ⅰ:A Mechanism for the Action of Berberine to Activate AMPK and Improve Insulin Action.Diabetes 2008.
18.Yin J,Gao Z,Liu D,Liu Z,Ye J.Berberine improves glucose metabolism through induction of glycolysis.Am J Physiol Endocrinol Metab 2008;294:E148-56.
19.Lee YS,Kim WS,Kim KH,Yoon MJ,Cho HJ,Shen Y,Ye JM,Lee CH,Oh WK,Kim CT,Hohnen-Behrens C,Gosby A,Kraegen EW,James DE,Kim JB.Berberine,a natural plant product,activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states.Diabetes 2006;55:2256-64.
20.黄天福.黄连素加小剂量优降糖治疗老年Ⅱ型糖尿病30例疗效观察[J].临床医学,1999,19(10):21-22.
21.刘欣荣.黄连素在糖尿病中的应用[J].山西医药杂志,2007,36(7):628-629.
22.张洁,王立琴.黄连素治疗糖尿病的研究进展[J].中西医结合心脑血管病杂志,2003,1(3):160-161.
23.冷三华,陆付耳.黄连素治疗2型糖尿病临床及实验研究进展中国中西医结合杂志,2002,22(10):794-795.
24.Yin J,Gao Z,Liu D,Liu Z,Ye J.Berberine improves glucose metabolism through induction of glycolysis.Am J Physiol Endocrinol Metab.2008;294:E148-56.
25.Walker DG,Rao S.The role of glucokinase in the phosphorylation of glucose by rat liver.Biochem J 1964;90:360-368.
26.Magnuson MA,Andreone TL,Printz RL,Koch S,Granner DK.Rat glucokinase gene:structure and regulation by insulin.Proc Natl Acad Sci U S A 1989;86:4838-4842.
27.Salas M,Vinuela E,Sols A.Insulin-Dependent Synthesis of Liver Glucokinase in the Rat.J Biol Chem 1963;238:3535-3538
28.Iynedjian PB,Gjinovci A,Renold AE.Stimulation by insulin of glucokinase gene transcription in liver of diabetic rats.J Biol Chem 1988;263:740-744.
29.Iynedjian PB,Pilot PR,Nouspikel T,et al.Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.Proc Natl Acad Sci U S A 1989;86:7838-7842.
30.Caro JF,Triester S,Patel VK,Tapscott EB,Frazier NL,Dohm GL.Liver glucokinase:decreased activity in patients with type Ⅱ diabetes.Horm Metab Res 1995;27:19-22.
31.Stoffel M,Patel P,Lo YM,et al.Missense glucokinase mutation in maturity-onset diabetes of the young and mutation screening in late-onset diabetes.Nat Genet 1992;2:153-156.
32.Vionnet N,Stoffel M,Takeda J,et al.Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus.Nature 1992;356:721-722.
1.Wilson JE.Hexokinases.Rev Physiol Biochem Pharmacol 1995;126:65-198.
2.Weinhouse S.Regulation of glucokinase in liver.Curr Top Cell Regul 1976;11:1-50.
3.Meglasson MD,Matschinsky FM.Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma.J Biol Chem 1983;258:6705-6708.
4. Matschinsky FM, Glaser B, Magnuson MA. Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes 1998;47:307-315.
5. Meglasson MD. Matschinsky FM. New perspectives on pancreatic islet glucokinase. Am J Physiol 1984;246:E1-13.
6. Liang Y, Najafi H, Smith RM, Zimmerman EC, Magnuson MA, Tal M, Matschinsky FM. Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture. Diabetes 1992;41:792-806.
7. Wang H, Iynedjian PB. Modulation of glucose responsiveness of insulinoma beta-cells by graded overexpression of glucokinase. Proc Natl Acad Sci U S A 1997;94:4372-4377.
8. Grossbard L, Schimke RT. Multiple hexokinases of rat tissues. Purification and comparison of soluble forms. J Biol Chem 1966;241:3546-3560.
9. Ferre T, Riu E, Bosch F, Valera A. Evidence from transgenic mice that glucokinase is rate limiting for glucose utilization in the liver. FASEB J 1996;10:1213-1218.
10. Hariharan N, Farrelly D, Hagan D, Hillyer D, Arbeeny C, Sabrah T, Treloar A, et al. Expression of human hepatic glucokinase in transgenic mice liver results in decreased glucose levels and reduced body weight. Diabetes 1997;46:11-16.
11. Niswender KD, Shiota M, Postic C, Cherrington AD, Magnuson MA. Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism. J Biol Chem 1997;272:22570-22575.
12. Seoane J, Gomez-Foix AM, O'Doherty RM, Gomez-Ara C, Newgard CB, Guinovart JJ. Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase. J Biol Chem 1996;271:23756-23760.
13. Aiston S, Trinh KY, Lange AJ, Newgard CB, Agius L. Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase. J Biol Chem 1999;274:24559-24566.
14. Vaulont S, Kahn A. Transcriptional control of metabolic regulation genes by carbohydrates. FASEB J 1994;8:28-35.
15. Shih H, Towle HC. Definition of the carbohydrate response element of the rat S14 gene. Context of the CACGTG motif determines the specificity of carbohydrate regulation. J Biol Chem 1994;269:9380-9387.
16. Foufelle F, Girard J, Ferre P. Regulation of lipogenic enzyme expression by glucose in liver and adipose tissue: a review of the potential cellular and molecular mechanisms. Adv Enzyme Regul 1996;36:199-226.
17. Rencurel F, Waeber G, Antoine B, Rocchiccioli F, Maulard P, Girard J, Leturque A. Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver. Biochem J 1996;314 (Pt 3):903-909.
18. Girard J, Ferre P, Foufelle F. Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes. Annu Rev Nutr 1997;17:325-352.
19. Magnuson MA. Glucokinase gene structure. Functional implications of molecular genetic studies. Diabetes 1990;39:523-527.
20. Van Schaftingen E. A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem 1989; 179:179-184.
21. Postic C, Niswender KD, Decaux JF, Parsa R, Shelton KD, Gouhot B, Pettepher CC, et al. Cloning and characterization of the mouse glucokinase gene locus and identification of distal liver-specific DNase I hypersensitive sites. Genomics 1995;29:740-750.
22. Hughes SD, Quaade C, Milburn JL, Cassidy L, Newgard CB. Expression of normal and novel glucokinase mRNAs in anterior pituitary and islet cells. J Biol Chem 1991;266:4521-4530.
23. Jetton TL, Liang Y, Pettepher CC, Zimmerman EC, Cox FG, Horvath K, Matschinsky FM, et al. Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut. J Biol Chem 1994;269:3641-3654.
24. Jetton TL, Moates JM, Lindner J, Wright CV, Magnuson MA. Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells. Proc Natl Acad Sci U S A 1998;95:8654-8659.
25. Shelton KD, Franklin AJ, Khoor A, Beechem J, Magnuson MA. Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells. Mol Cell Biol 1992;12:4578-4589.
26. Moates JM, Shelton KD, Magnuson MA. Characterization of the Pal motifs in the upstream glucokinase promoter: binding of a cell type-specific protein complex correlates with transcriptional activation. Mol Endocrinol 1996; 10:723-731.
27. Iynedjian PB, Jotterand D, Nouspikel T, Asfari M, Pilot PR. Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system. J Biol Chem 1989;264:21824-21829.
28. Magnuson MA, Andreone TL, Printz RL, Koch S, Granner DK. Rat glucokinase gene: structure and regulation by insulin. Proc Natl Acad Sci U S A 1989;86:4838-4842.
29. Niswender KD, Postic C, Jetton TL, Bennett BD, Piston DW, Efrat S, Magnuson MA. Cell-specific expression and regulation of a glucokinase gene locus transgene. J Biol Chem 1997;272:22564-22569.
30. Iynedjian PB. Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene. Biochem J 1998;333 ( Pt 3):705-712.
31. Moates JM, Postic C, Decaux JF, Girard J, Magnuson MA. Variable expression of hepatic glucokinase in mice is due to a regulational locus that cosegregates with the glucokinase gene. Genomics 1997;45:185-193.
32. Foretz M, Guichard C, Ferre P, Foufelle F. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc Natl Acad Sci U S A 1999;96:12737-12742.
33. rown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331-340.
34. Sebastian S, Horton JD, Wilson JE. Anabolic function of the type II isozyme of hexokinase in hepatic lipid synthesis. Biochem Biophys Res Commun 2000;270:886-891.
35. Vandercammen A, Van Schaftingen E. The mechanism by which rat liver glucokinase is inhibited by the regulatory protein. Eur J Biochem 1990;191:483-489.
36. de la Iglesia N, Veiga-da-Cunha M, Van Schaftingen E, Guinovart JJ, Ferrer JC. Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase. FEBS Lett 1999;456:332-338.
37. Shiota C, Coffey J, Grimsby J, Grippo JF, Magnuson MA. Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase. J Biol Chem 1999;274:37125-37130.
38. de la Iglesia N, Mukhtar M, Seoane J, Guinovart JJ, Agius L. The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J Biol Chem 2000;275:10597-10603.
39. Farrelly D, Brown KS, Tieman A, Ren J, Lira SA, Hagan D, Gregg R, et al. Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: a sequestration mechanism in metabolic regulation. Proc Natl Acad Sci U S A 1999;96:14511-14516.
40. Grimsby J, Coffey JW, Dvorozniak MT, Magram J, Li G, Matschinsky FM, Shiota C, et al. Characterization of glucokinase regulatory protein-deficient mice. J Biol Chem 2000;275:7826-7831.
41. Shiota M, Postic C, Fujimoto Y, Jetton TL, Dixon K, Pan D, Grimsby J, et al. Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes. Diabetes 2001;50:622-629.
42. Bosco D, Meda P, Iynedjian PB. Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization. Biochem J 2000;348 Pt 1:215-222.
43. Froguel P, Vaxillaire M, Sun F, Velho G, Zouali H, Butel MO, Lesage S, et al. Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus. Nature 1992;356:162-164.
44. Vionnet N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, et al. Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 1992;356:721-722.
45. Velho G, Blanche H, Vaxillaire M, Bellanne-Chantelot C, Pardini VC, Timsit J, Passa P, et al. Identification of 14 new glucokinase mutations and description of the clinical profile of 42 MODY-2 families. Diabetologia 1997;40:217-224.
46. Pilkis SJ, Weber IT, Harrison RW, Bell GI. Glucokinase: structural analysis of a protein involved in susceptibility to diabetes. J Biol Chem 1994;269:21925-21928.
47. Liang Y, Kesavan P, Wang LQ, Niswender K, Tanizawa Y, Permutt MA, Magnuson MA, et al. Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. Biochem J 1995;309 ( Pt 1): 167-173.
48. Guazzini B, Gaffi D, Mainieri D, Multari G, Cordera R, Bertolini S, Pozza G, et al. Three novel missense mutations in the glucokinase gene (G80S; E221K; G227C) in Italian subjects with maturity-onset diabetes of the young (MODY). Mutations in brief no. 162. Online. Hum Mutat 1998;12:136.
49. Miller SP, Anand GR, Karschnia EJ, Bell GI, LaPorte DC, Lange AJ. Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype. Diabetes 1999;48:1645-1651.
50. Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, et al. Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 1998;338:226-230.
51. Davis EA, Cuesta-Munoz A, Raoul M, Buettger C, Sweet I, Moates M, Magnuson MA, et al. Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis. Diabetologia 1999;42:1175-1186.
52. Agius L, Peak M, Newgard CB, Gomez-Foix AM, Guinovart JJ. Evidence for a role of glucose-induced translocation of glucokinase in the control of hepatic glycogen synthesis. J Biol Chem 1996;271:30479-30486.
53. O'Doherty RM, Lehman DL, Seoane J, Gomez-Foix AM, Guinovart JJ, Newgard CB. Differential metabolic effects of adenovirus-mediated glucokinase and hexokinase I overexpression in rat primary hepatocytes. J Biol Chem 1996;271:20524-20530.
54. O'Doherty RM, Lehman DL, Telemaque-Potts S, Newgard CB. Metabolic impact of glucokinase overexpression in liver: lowering of blood glucose in fed rats is accompanied by hyperlipidemia. Diabetes 1999;48:2022-2027.
55. Araki K, Araki M, Miyazaki J, Vassalli P. Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A 1995;92:160-164.
56. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999;274:305-315.
57. Duvillie B, Cordonnier N, Deltour L, Dandoy-Dron F, Itier JM, Monthioux E, Jami J, et al. Phenotypic alterations in insulin-deficient mutant mice. Proc Natl Acad Sci U S A 1997;94:5137-5140.
58. Accili D, Drago J, Lee EJ, Johnson MD, Cool MH, Salvatore P, Asico LD, et al. Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet 1996; 12:106-109.
59. Anton M, Graham FL. Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression. J Virol 1995;69:4600-4606.
60. Bali D, Svetlanov A, Lee HW, Fusco-DeMane D, Leiser M, Li B, Barzilai N, et al. Animal model for maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene. J Biol Chem 1995;270:21464-21467.
61. Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D, Goodyear LJ, et al. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 1998;2:559-569.
62. Burcelin R, del Carmen Munoz M, Guillam MT, Thorens B. Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway. J Biol Chem 2000;275:10930-10936.
63. Cabrera-Valladares G, German MS, Matschinsky FM, Wang J, Fernandez-Mejia C. Effect of retinoic acid on glucokinase activity and gene expression and on insulin secretion in primary cultures of pancreatic islets. Endocrinology 1999;140:3091-3096.
64. DeFronzo RA. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidaemia and atherosclerosis. Neth J Med 1997;50:191-197.
65. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol 1993;225:890-900.
66. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 1988;85:5166-5170.
67. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21:70-71.
68. Gannon M, Shiota C, Postic C, Wright CV, Magnuson M. Analysis of the Cre-mediated recombination driven by rat insulin promoter in embryonic and adult mouse pancreas. Genesis 2000;26:139-142.
69. Postic C, Magnuson MA. DNA excision in liver by an albumin-Cre transgene occurs progressively with age. Genesis 2000;26:149-150.
70. Meglasson MD, Matschinsky FM. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 1986;2:163-214.
71. Sweet IR, Li G, Najafi H, Berner D, Matschinsky FM. Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets. Am J Physiol 1996;271:E606-625.
72. Grupe A, Hultgren B, Ryan A, Ma YH, Bauer M, Stewart TA. Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis. Cell 1995;83:69-78.
73. Terauchi Y, Sakura H, Yasuda K, Iwamoto K, Takahashi N, Ito K, Kasai H, et al. Pancreatic beta-cell-specific targeted disruption of glucokinase gene. Diabetes mellitus due to defective insulin secretion to glucose. J Biol Chem 1995;270:30253-30256.
74. Piston DW, Knobel SM, Postic C, Shelton KD, Magnuson MA. Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion. J Biol Chem 1999;274:1000-1004.
75. Velho G, Petersen KF, Perseghin G, Hwang JH, Rothman DL, Pueyo ME, Cline GW, et al. Impaired hepatic glycogen synthesis in glucokinase-deficient (MODY-2) subjects. J Clin Invest 1996;98:1755-1761.
76. Tappy L, Dussoix P, Iynedjian P, Henry S, Schneiter P, Zahnd G, Jequier E, et al. Abnormal regulation of hepatic glucose output in maturity-onset diabetes of the young caused by a specific mutation of the glucokinase gene. Diabetes 1997;46:204-208.
77. Rossetti L, Chen W, Hu M, Hawkins M, Barzilai N, Efrat S. Abnormal regulation of HGP by hyperglycemia in mice with a disrupted glucokinase allele. Am J Physiol 1997;273:E743-750.
78. Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, et al. Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1). Nature 1996;384:458-460.
79. Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, Southam L, et al. Mutations in the hepatocyte nuclear factor-1 alpha gene in maturity-onset diabetes of the young (MODY3). Nature 1996;384:455-458.
80. Stoffers DA, Ferrer J, Clarke WL, Habener JF. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 1997;17:138-139.
81. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 1997; 15:106-110.
82. Horikawa Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, Lindner T, et al. Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY. Nat Genet 1997;17:384-385.
83. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 2000;6:87-97.
84. Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR. Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 1999;96:329-339.
85. Hardouin N, Nagy A. Gene-trap-based target site for cre-mediated transgenic insertion. Genesis 2000;26:245-252.
86. Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, Jami J, Bucchini D. Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 1996;15:1542-1547.
87. Pontoglio M, Sreenan S, Roe M, Pugh W, Ostrega D, Doyen A, Pick AJ, et al. Defective insulin secretion in hepatocyte nuclear factor 1 alpha-deficient mice. J Clin Invest 1998;101:2215-2222.
88. Postic, C, and Magnuson, M.A.. In "Molecular Pathogenesis of MODYs" (F.M. Matschinsky and M.A. Magnuson, eds.), Frontiers in Diabetes, 2000b; vol. 15, pp 293-298. Karger, Basel, Switzerland.
89. Postic, C, and Magnuson, M.A. In "Molecular Basis of Endocrine Pancreas Development and Function" (M.A. Hussain and J.F. Habener, eds.). Kluwer Academic Publishers, Norwell. 2001.
90. Roncero I, Alvarez E, Vazquez P, Blazquez E. Functional glucokinase isoforms are expressed in rat brain. J Neurochem 2000;74:1848-1857.
91. Sreenan SK, Cockburn BN, Baldwin AC, Ostrega DM, Levisetti M, Grape A, Bell GI, et al. Adaptation to hyperglycemia enhances insulin secretion in glucokinase mutant mice. Diabetes 1998;47:1881-1888.
92. Sturis J, Kurland IJ, Byrne MM, Mosekilde E, Froguel P, Pilkis SJ, Bell GI, et al. Compensation in pancreatic beta-cell function in subjects with glucokinase mutations. Diabetes 1994;43:718-723.
93. Thomas PM, Cote GJ, Wohllk N, Haddad B, Mathew PM, Rabl W, Aguilar-Bryan L, et al. Mutations in the sulfonylurea receptor gene in familial persistent hypennsulinemic hypoglycemia of infancy. Science 1995;268:426-429.
1 Asao K,Kao WH,Baptiste-Roberts K,et al.Short stature and the risk of adiposity,insulin resistance,and type 2 diabetes in middle age:the Third National Health and Nutrition Examination Survey(NI-tANES Ⅲ),1988-1994.Diabetes Care,2006,29:1632-1637.
2 Forest C,Beale E.New developments in nutrition and diabetes:glyceroneogenesis comes of age.Biochimie,2003,85:1195-1197.
3 Colombo M,Kruhoeffer M,Gregersen S,et al.Energy restriction prevents the development of type 2 diabetes in Zucker diabetic fatty rats:coordinated patterns of gene expression for energy metabolism in insulin-sensitive tissues and pancreatic islets determined by oligonucleotide microarray analysis.Metabolism,2006,55:43-52.
4 Nair S,Lee YH,Lindsay RS,et al.11beta-Hydroxysteroid dehydrogenase Type 1:genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.Diabetologia,2004,47:1088-1095.
5 Ishihara H,Sasaoka T,Kagawa S,et al.Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population.FEBS Lett,2003,554:450-454.
6 Wang H,Chu WS,Lu T,et al.Uncoupling protein-2 polymorphisms in type 2diabetes,obesity,and insulin secretion.Am J Physiol Endocrinol Metab,2004,286:E1-7.
7 D'Alfonso R,Marini MA,Frittitta L,et al.Polymorphisms of the insulin receptor substrate-2 in patients with type 2 diabetes.J Clin Endocrinol Metab,2003,88:317-322.
8 Patti ME,Butte A J,Crunkhorn S,et al.Coordinated reduction of genes oxidative metabolism in humans with insulin resistance and diabetes:Potential role of PGC1 and NRF1.Proc Natl Acad Sci U S A,2003,100:8466-8471.
9 Guilleret I,Benhattar J.Demethylation of the human telomerase catalytic subunit (hTERT) gene promoter reduced hTERT expression and telomerase activity and shortened telomeres.Exp Cell Res,2003,289:326-334.
10 Guilleret I,Yan P,Grange F,et al.Hypermethylation of the human telomerase catalytic subunit(hTERT) gene correlates with telomerase activity.Int J Cancer,2002,101:335-341.
11 Liu L,Wylie RC,Andrews LG,et al.Aging,cancer and nutrition:the DNA methylation connection.Mech Ageing Dev,2003,124:989-998.
12 Richardson B.Impact of aging on DNA methylation.Ageing Res Rev,2003,2:245-261.
13 Ahuja N, Li Q, Mohan AL, et al. Aging and DNA methylation in colorectal mucosa and cancer. Cancer Res, 1998, 58: 5489-5494.
14 Maier S, Olek A. Diabetes: a candidate disease for efficient DNA methylation profiling. J Nutr, 2002, 132: 2440S-2443S.
15 Longnecker DS. Abnormal methyl metabolism in pancreatic toxicity and diabetes. J Nutr, 2002, 132: 2373S-2376S.
16 Issa JP, Ahuja N, Toyota M, et al. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res, 2001, 61: 3573-3577.
17 Post WS, Goldschmidt-Clermont PJ, Wilhide CC, et al. Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res, 1999, 43: 985-991.
18 Tohgi H, Utsugisawa K, Nagane Y, et al. Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex. Brain Res Mol Brain Res, 1999, 70: 288-292.
19 Poirier LA, Brown AT, Fink LM, et al. Blood S-adenosylmethionine concentrations and lymphocyte methylenetetrahydrofolate reductase activity in diabetes mellitus and diabetic nephropathy. Metabolism, 2001, 50: 1014-1018.
20 Yokomori N, Tawata M, Onaya T DNA demethylation during the differentiation of 3T3-L1 cells affects the expression of the mouse GLUT4 gene. Diabetes, 1999, 48: 685-690.
21 Yokomori N, Tawata M, Onaya T. DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells. Diabetologia, 2002, 45: 140-148.
22 Jin B, Seong JK, Ryu DY. Tissue-specific and de novo promoter methylation of the mouse glucose transporter 2. Biol Pharm Bull, 2005, 28: 2054-2057.
23 Carretero MV, Torres L, Latasa U, et al. Transformed but not normal hepatocytes express UCP2. FEBS Lett, 1998, 439: 55-58.
24 Faber S, Ip T, Granner D, et al. The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression. Nucleic Acids Res, 1991, 19: 4681-4688.
25 Lillycrop KA, Phillips ES, Jackson AA, et al. Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring. J Nutr, 2005, 135: 1382-1386.
26 Temple IK, Gardner RJ, Mackay DJ, et al. Transient neonatal diabetes: widening the understanding of the etiopathogenesis of diabetes. Diabetes, 2000, 49: 1359-1366.
27 Ling C, Poulsen P, Simonsson S, et al. Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle. J Clin Invest, 2007, 117: 3427-3435.
28 Lyko F, Brown R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst, 2005, 97: 1498-1506.
29 Strathdee G,Brown R.Epigenetic cancer therapies:DNA methyltransferase inhibitors.Expert Opin Investig Drugs,2002,11:747-754.
30 Wang CY,Ma Q,He ZX.Activity of DNAmethylase and DNase Ⅰ sensitivity assay of genome DNA of liver from rat of diabetic nephropathy.Journal of Beijing Normal University(Natural Science),2002,6:810-814.[江琛颖,何忠效,马晴.糖尿病肾病大鼠肝DNA甲基化酶活性及基因组DNA 对DNase Ⅰ敏感性的分析.北京师范大学学报(自然科学版),2002,6:810-814.]
2. Narimiya M, Azhar S, Dolkas CB, Mondon CE, Sims C, Wright DW, Reaven GM. Insulin resistance in older rats. Am J Physiol 1984;246:E397-404.
3. Stewart KJ. Physical activity and aging. Ann N Y Acad Sci 2005;1055:193-206.
4. Fontana L, Klein S. Aging, adiposity, and calorie restriction. Jama 2007;297:986-994.
5. da Silva RC, Miranda WL, Chacra AR, Dib SA. Insulin resistance, beta-cell function, and glucose tolerance in Brazilian adolescents with obesity or risk factors for type 2 diabetes mellitus. J Diabetes Complications 2007;21:84-92.
6. Fontana L. Excessive adiposity, calorie restriction, and aging. Jama 2006;295:1577-1578.
7. Moore G, Knoblaugh S, Gollahon K, Rabinovitch P, Ladiges W. Hyperinsulinemia and insulin resistance in Wrn null mice fed a diabetogenic diet. Mech Ageing Dev 2008; 129:201-206.
8. Huang X, Charbeneau RA, Fu Y, Kaur K, Gerin I, MacDougald OA, Neubig RR. Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele. Diabetes 2008;57:77-85.
9. Raz I, Eldor R, Cernea S, Shafrir E. Diabetes: insulin resistance and derangements in lipid metabolism. Cure through intervention in fat transport and storage. Diabetes Metab Res Rev 2005;21:3-14.
10. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 2001 ;86:3574-3578.
11. Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, Sharpless NE. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 2006;443:453-457.
12. Li LC, Shiina H, Deguchi M, Zhao H, Okino ST, Kane CJ, Carroll PR, et al. Age-dependent methylation of ESR1 gene in prostate cancer. Biochem Biophys Res Commun 2004;321:455-461.
13. Martens JW, Sieuwerts AM, Bolt-deVries J, Bosma PT, Swiggers SJ, Klijn JG, Foekens JA. Aging of stromal-derived human breast fibroblasts might contribute to breast cancer progression. Thromb Haemost 2003;89:393-404.
14. Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ. Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene 1999;18:1313-1324.
15. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res 2001 ;61:3573-3577.
16. Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 1994;7:536-540.
17. Tohgi H, Utsugisawa K, Nagane Y, Yoshimura M, Genda Y, Ukitsu M. Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex. Brain Res Mol Brain Res 1999;70:288-292.
18. Ling C, Poulsen P, Simonsson S, Ronn T, Holmkvist J, Almgren P, Hagert P, et al. Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle. J Clin Invest 2007;117:3427-3435.
19. Bontemps F, Hue L, Hers HG. Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone. Biochem J 1978; 174:603-611.
20. Davidson AL, Arion WJ. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. Arch Biochem Biophys 1987;253:156-167.
21. Turner N, Li JY, Gosby A, To SW, Cheng Z, Miyoshi H, Taketo MM, et al. Berberine, and its more Biologically Available Derivative Dihydroberberine, Inhibit Mitochondrial Respiratory Complex I: A Mechanism for the Action of Berberine to Activate AMPK and Improve Insulin Action. Diabetes 2008.
22. Yin J, Gao Z, Liu D, Liu Z, Ye J. Berberine improves glucose metabolism through induction of glycolysis. Am J Physiol Endocrinol Metab 2008;294:E148-156.
23. Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, Ye JM, et al. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 2006;55:2256-2264.
24. Van Der Vies J. Two methods for the determination of glycogen in liver. Biochem J 1954;57:410-416.
25. Templeton M. Microdetermination of glycogen with anthrone reagent. J Histochem Cytochem 1961;9:670-672.
26. Chen J, Rider DA, Ruan R. Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. J Gerontol A Biol Sci Med Sci 2006;61:20-27.
27. Ginzinger DG Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002;30:503-512.
28. Grunau C, Clark SJ, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001;29:E65-65.
29. Bogdarina I, Murphy HC, Burns SP, Clark AJ. Investigation of the role of epigenetic modification of the rat glucokinase gene in fetal programming. Life Sci 2004;74:1407-1415.
30. Gallou-Kabani C, Junien C. Nutritional epigenomics of metabolic syndrome: new perspective against the epidemic. Diabetes 2005;54:1899-906.
31. Li LC, Chui R, Nakajima K, Oh BR, Au HC, Dahiya R. Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression. Cancer Res 2000;60:702-706.
32. Paul CL, Clark SJ. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. Biotechniques 1996;21:126-133.
33. Stirzaker C, Millar DS, Paul CL, Warnecke PM, Harrison J, Vincent PC, Frommer M, et al. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res 1997;57:2229-2237.
34. Lewin J, Schmitt AO, Adorjan P, Hildmann T, Piepenbrock C. Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates. Bioinformatics 2004;20:3005-3012.
35. Ngo V, Gourdji D, Laverriere JN. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression. Mol Cell Biol 1996;16:3245-3254.
36. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF. Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue. Blood 1998;91:1373-1381.
37. Murumagi A, Vahamurto P, Peterson P. Characterization of regulatory elements and methylation pattern of the autoimmune regulator (AIRE) promoter. J Biol Chem 2003 ;278:19784-19790.
38. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 2000;6:87-97.
39. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999;274:305-315.
40. Magnuson MA, Shelton KD. An alternate promoter in the glucokinase gene is active in the pancreatic beta cell. J Biol Chem 1989;264:15936-15942.
41. Frese T, Bazwinsky I, Muhlbauer E, Peschke E. Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats. Horm Metab Res 2007;39:567-574.
1.张家庆.新的HOMA2IR—从空腹血糖、空腹胰岛素测胰岛素抵抗.中华糖尿病杂志,2005,13:245-246.
2.Moore G,Knoblaugh S,Gollahon K,Rabinovitch P,Ladiges W.Hyperinsulinemia and insulin resistance in Wrn null mice fed a diabetogenic diet.Mech Ageing Dev 2008;129:201-206.
3.Huang X,Charbeneau RA,Fu Y,Kaur K,Gerin I,MacDougald OA,Neubig RR.Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele.Diabetes 2008;57:77-85.
4.Raz I,Eldor R,Cemea S,Shafrir E.Diabetes:insulin resistance and derangements in lipid metabolism.Cure through intervention in fat transport and storage.Diabetes Metab Res Rev 2005;21:3-14.
5.Nair S,Lee YH,Lindsay RS,Walker BR,Tataranni PA,Bogardus C,Baier LJ,et al.11beta-Hydroxysteroid dehydrogenase Type 1:genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.Diabetologia 2004;47:1088-1095.
6.Dominguez LJ,Barbagallo M.The cardiometabolic syndrome and sarcopenic obesity in older persons.J Cardiometab Syndr 2007;2:183-9.
7.Slawik M,Vidal-Puig AJ.Lipotoxicity,ovemutrition and energy metabolism in aging.Ageing Res Rev 2006;5:144-64.
8.Morgan HD,Sutherland HG,Martin DI,Whitelaw E.Epigenetic inheritance at the agouti locus in the mouse.Nat Genet 1999;23:314-318.
9.Dolinoy DC,Huang D,Jirtle RL.Matemal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development.Proc Natl Acad Sci U S A 2007;104:13056-13061.
10.Wolff GL,Roberts DW,Galbraith DB.Prenatal determination of obesity,tumor susceptibility,and coat color pattern in viable yellow(Avy/a) mice.The yellow mouse syndrome.J Hered 1986;77:151-158.
11.Zemel MB,Kim JH,Woychik RP,Michaud EJ,Kadwell SH,Patel IR,Wilkison WO.Agouti regulation of intracellular calcium:role in the insulin resistance of viable yellow mice.Proc Natl Acad Sci U S A 1995;92:4733-4737.
12.Kuklin AI,Mynatt RL,Klebig ML,Kiefer LL,Wilkison WO,Woychik RP,Michaud EJ.Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes.Mol Cancer 2004;3:17
13.Kaul S,Ritschel WA.Total body water as an index for predicting body fat in rats.Arzneimittelforschung 1986;36:112-6.
14.Peralta S,Carrascosa JM,Gallardo N,Ros M,Arribas C.Ageing increases SOCS-3 expression in rat hypothalamus:effects of food restriction.Biochem Biophys Res Commun.2002;16;296(2):425-8.
15.Bemardis LL,Patterson BD.Correlation between 'Lee index' and carcass fat content in weanling and adult female rats with hypothalamic lesions.J Endocrinol.1968;40:527-8.
16.Harder T,Rake A,Rohde W,Doerner G,Plagemann A.Overweight and increased diabetes susceptibility in neonatally insulin-treated adult rats.Endocr Regul 1999;33:25-31.
17.Rector RS,Warner SO,Liu Y,Hinton PS,Sun GY,Cox RH,Stump CS,Laughlin MH,Dellsperger KC,Thomas TR.Exercise and diet induced weight loss improves measures of oxidative stress and insulin sensitivity in adults with characteristics of the metabolic syndrome.Am J Physiol Endocrinol Metab 2007;293:E500-6.
18.Clark JM.Weight loss as a treatment for nonalcoholic fatty liver disease.J Clin Gastroenterol 2006;40:S39-43.
1.王镜岩,朱圣庚,徐长法.生物化学下册[M].北京:高等教育出版社,2002:232.
2.Bain JR,Schisler JC,Takeuchi K,Newgard CB,Becket TC.An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.Diabetes 2004;53:2190-2194.
3.Salavert A,lynedjian PBRegulation of phosphoenolpyruvate carboxykinase(GTP)synthesis in rat liver cells.Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone.J Biol Chem.1982;257:13404-13412.
4.Butte NF,Puyau MR,Vohra FA,Adolph AL,Mehta NR,Zakeri I.Body size,body composition,and metabolic profile explain higher energy expenditure in overweight children.J Nutr 2007;137:2660-7.
5.Miles JM,Nelson RH.Contribution of triglyceride-rich lipoproteins to plasma free fatty acids.Horm Metab Res 2007;39(10):726-9.
6.Jensen MD.Adipose tissue metabolism-an aspect we should not neglect? Horm Metab Res 2007;39:722-5.
7.Parekh S,Anania FA.Abnormal lipid and glucose metabolism in obesity:implications for nonalcoholic fatty liver disease.Gastroenterology 2007;132:2191-207.
8.Delarue J,Magnan C.Free fatty acids and insulin resistance.Curr Opin Clin Nutr Metab Care 2007;10(2):142-8.
9.Mlinar B,Marc J,Janez A,Pfeifer M.Molecular mechanisms of insulin resistance and associated diseases.Clin Chim Acta 2007;375(1-2):20-35.
10.Schinner S,Scherbaum WA,Bornstein SR,Barthel A.Molecular mechanisms of insulin resistance.Diabet Med 2005;22:674-82.
11.Reid BN,Ables GP,Otlivanchik OA,Schoiswohl G,Zechner R,Blaner WS,Goldberg IJ,Schwabe R,Chua SC Jr,Huang LS.Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation,stimulates direct release of free fatty acids and ameliorates steatosis.J Biol Chem.2008 Mar 12
12.Anderwald C,Brunmair B,Stadlbauer K,Krebs M,Furnsinn C,Roden M.Effects of free fatty acids on carbohydrate metabolism and insulin signalling in perfused rat liver.Eur J Clin Invest 2007;37:774-82.
13.Holland WL,Knotts TA,Chavez JA,Wang LP,Hoehn KL,Summers SA.Lipid mediators of insulin resistance.Nutr Rev 2007;65:S39-46.
14.Lall CG,Aisen AM,Bansal N,Sandrasegaran K.Nonalcoholic Fatty liver disease.AJR Am J Roentgenol 2008;190:993-1002.
15.Turkish AR.Nonalcoholic fatty liver disease:emerging mechanisms and consequences.Curr Opin Clin Nutr Metab Care 2008;11:128-33.
16.Streba LA,Carstea D,Mitru,t P,Vere CC,Dragomir N,Streba CT.Nonalcoholic fatty liver disease and metabolic syndrome:a concise review.Rom J Morphol Embryol 2008;49:13-20.
17.Turner N,Li JY,Gosby A,To SW,Cheng Z,Miyoshi H,Taketo MM,Cooney GJ,Kraegen EW,James DE,Hu LH,Li J,Ye JM.Berberine,and its more Biologically Available Derivative Dihydroberberine,Inhibit Mitochondrial Respiratory Complex Ⅰ:A Mechanism for the Action of Berberine to Activate AMPK and Improve Insulin Action.Diabetes 2008.
18.Yin J,Gao Z,Liu D,Liu Z,Ye J.Berberine improves glucose metabolism through induction of glycolysis.Am J Physiol Endocrinol Metab 2008;294:E148-56.
19.Lee YS,Kim WS,Kim KH,Yoon MJ,Cho HJ,Shen Y,Ye JM,Lee CH,Oh WK,Kim CT,Hohnen-Behrens C,Gosby A,Kraegen EW,James DE,Kim JB.Berberine,a natural plant product,activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states.Diabetes 2006;55:2256-64.
20.黄天福.黄连素加小剂量优降糖治疗老年Ⅱ型糖尿病30例疗效观察[J].临床医学,1999,19(10):21-22.
21.刘欣荣.黄连素在糖尿病中的应用[J].山西医药杂志,2007,36(7):628-629.
22.张洁,王立琴.黄连素治疗糖尿病的研究进展[J].中西医结合心脑血管病杂志,2003,1(3):160-161.
23.冷三华,陆付耳.黄连素治疗2型糖尿病临床及实验研究进展中国中西医结合杂志,2002,22(10):794-795.
24.Yin J,Gao Z,Liu D,Liu Z,Ye J.Berberine improves glucose metabolism through induction of glycolysis.Am J Physiol Endocrinol Metab.2008;294:E148-56.
25.Walker DG,Rao S.The role of glucokinase in the phosphorylation of glucose by rat liver.Biochem J 1964;90:360-368.
26.Magnuson MA,Andreone TL,Printz RL,Koch S,Granner DK.Rat glucokinase gene:structure and regulation by insulin.Proc Natl Acad Sci U S A 1989;86:4838-4842.
27.Salas M,Vinuela E,Sols A.Insulin-Dependent Synthesis of Liver Glucokinase in the Rat.J Biol Chem 1963;238:3535-3538
28.Iynedjian PB,Gjinovci A,Renold AE.Stimulation by insulin of glucokinase gene transcription in liver of diabetic rats.J Biol Chem 1988;263:740-744.
29.Iynedjian PB,Pilot PR,Nouspikel T,et al.Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.Proc Natl Acad Sci U S A 1989;86:7838-7842.
30.Caro JF,Triester S,Patel VK,Tapscott EB,Frazier NL,Dohm GL.Liver glucokinase:decreased activity in patients with type Ⅱ diabetes.Horm Metab Res 1995;27:19-22.
31.Stoffel M,Patel P,Lo YM,et al.Missense glucokinase mutation in maturity-onset diabetes of the young and mutation screening in late-onset diabetes.Nat Genet 1992;2:153-156.
32.Vionnet N,Stoffel M,Takeda J,et al.Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus.Nature 1992;356:721-722.
1.Wilson JE.Hexokinases.Rev Physiol Biochem Pharmacol 1995;126:65-198.
2.Weinhouse S.Regulation of glucokinase in liver.Curr Top Cell Regul 1976;11:1-50.
3.Meglasson MD,Matschinsky FM.Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma.J Biol Chem 1983;258:6705-6708.
4. Matschinsky FM, Glaser B, Magnuson MA. Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes 1998;47:307-315.
5. Meglasson MD. Matschinsky FM. New perspectives on pancreatic islet glucokinase. Am J Physiol 1984;246:E1-13.
6. Liang Y, Najafi H, Smith RM, Zimmerman EC, Magnuson MA, Tal M, Matschinsky FM. Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture. Diabetes 1992;41:792-806.
7. Wang H, Iynedjian PB. Modulation of glucose responsiveness of insulinoma beta-cells by graded overexpression of glucokinase. Proc Natl Acad Sci U S A 1997;94:4372-4377.
8. Grossbard L, Schimke RT. Multiple hexokinases of rat tissues. Purification and comparison of soluble forms. J Biol Chem 1966;241:3546-3560.
9. Ferre T, Riu E, Bosch F, Valera A. Evidence from transgenic mice that glucokinase is rate limiting for glucose utilization in the liver. FASEB J 1996;10:1213-1218.
10. Hariharan N, Farrelly D, Hagan D, Hillyer D, Arbeeny C, Sabrah T, Treloar A, et al. Expression of human hepatic glucokinase in transgenic mice liver results in decreased glucose levels and reduced body weight. Diabetes 1997;46:11-16.
11. Niswender KD, Shiota M, Postic C, Cherrington AD, Magnuson MA. Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism. J Biol Chem 1997;272:22570-22575.
12. Seoane J, Gomez-Foix AM, O'Doherty RM, Gomez-Ara C, Newgard CB, Guinovart JJ. Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase. J Biol Chem 1996;271:23756-23760.
13. Aiston S, Trinh KY, Lange AJ, Newgard CB, Agius L. Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase. J Biol Chem 1999;274:24559-24566.
14. Vaulont S, Kahn A. Transcriptional control of metabolic regulation genes by carbohydrates. FASEB J 1994;8:28-35.
15. Shih H, Towle HC. Definition of the carbohydrate response element of the rat S14 gene. Context of the CACGTG motif determines the specificity of carbohydrate regulation. J Biol Chem 1994;269:9380-9387.
16. Foufelle F, Girard J, Ferre P. Regulation of lipogenic enzyme expression by glucose in liver and adipose tissue: a review of the potential cellular and molecular mechanisms. Adv Enzyme Regul 1996;36:199-226.
17. Rencurel F, Waeber G, Antoine B, Rocchiccioli F, Maulard P, Girard J, Leturque A. Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver. Biochem J 1996;314 (Pt 3):903-909.
18. Girard J, Ferre P, Foufelle F. Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes. Annu Rev Nutr 1997;17:325-352.
19. Magnuson MA. Glucokinase gene structure. Functional implications of molecular genetic studies. Diabetes 1990;39:523-527.
20. Van Schaftingen E. A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem 1989; 179:179-184.
21. Postic C, Niswender KD, Decaux JF, Parsa R, Shelton KD, Gouhot B, Pettepher CC, et al. Cloning and characterization of the mouse glucokinase gene locus and identification of distal liver-specific DNase I hypersensitive sites. Genomics 1995;29:740-750.
22. Hughes SD, Quaade C, Milburn JL, Cassidy L, Newgard CB. Expression of normal and novel glucokinase mRNAs in anterior pituitary and islet cells. J Biol Chem 1991;266:4521-4530.
23. Jetton TL, Liang Y, Pettepher CC, Zimmerman EC, Cox FG, Horvath K, Matschinsky FM, et al. Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut. J Biol Chem 1994;269:3641-3654.
24. Jetton TL, Moates JM, Lindner J, Wright CV, Magnuson MA. Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells. Proc Natl Acad Sci U S A 1998;95:8654-8659.
25. Shelton KD, Franklin AJ, Khoor A, Beechem J, Magnuson MA. Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells. Mol Cell Biol 1992;12:4578-4589.
26. Moates JM, Shelton KD, Magnuson MA. Characterization of the Pal motifs in the upstream glucokinase promoter: binding of a cell type-specific protein complex correlates with transcriptional activation. Mol Endocrinol 1996; 10:723-731.
27. Iynedjian PB, Jotterand D, Nouspikel T, Asfari M, Pilot PR. Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system. J Biol Chem 1989;264:21824-21829.
28. Magnuson MA, Andreone TL, Printz RL, Koch S, Granner DK. Rat glucokinase gene: structure and regulation by insulin. Proc Natl Acad Sci U S A 1989;86:4838-4842.
29. Niswender KD, Postic C, Jetton TL, Bennett BD, Piston DW, Efrat S, Magnuson MA. Cell-specific expression and regulation of a glucokinase gene locus transgene. J Biol Chem 1997;272:22564-22569.
30. Iynedjian PB. Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene. Biochem J 1998;333 ( Pt 3):705-712.
31. Moates JM, Postic C, Decaux JF, Girard J, Magnuson MA. Variable expression of hepatic glucokinase in mice is due to a regulational locus that cosegregates with the glucokinase gene. Genomics 1997;45:185-193.
32. Foretz M, Guichard C, Ferre P, Foufelle F. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc Natl Acad Sci U S A 1999;96:12737-12742.
33. rown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331-340.
34. Sebastian S, Horton JD, Wilson JE. Anabolic function of the type II isozyme of hexokinase in hepatic lipid synthesis. Biochem Biophys Res Commun 2000;270:886-891.
35. Vandercammen A, Van Schaftingen E. The mechanism by which rat liver glucokinase is inhibited by the regulatory protein. Eur J Biochem 1990;191:483-489.
36. de la Iglesia N, Veiga-da-Cunha M, Van Schaftingen E, Guinovart JJ, Ferrer JC. Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase. FEBS Lett 1999;456:332-338.
37. Shiota C, Coffey J, Grimsby J, Grippo JF, Magnuson MA. Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase. J Biol Chem 1999;274:37125-37130.
38. de la Iglesia N, Mukhtar M, Seoane J, Guinovart JJ, Agius L. The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J Biol Chem 2000;275:10597-10603.
39. Farrelly D, Brown KS, Tieman A, Ren J, Lira SA, Hagan D, Gregg R, et al. Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: a sequestration mechanism in metabolic regulation. Proc Natl Acad Sci U S A 1999;96:14511-14516.
40. Grimsby J, Coffey JW, Dvorozniak MT, Magram J, Li G, Matschinsky FM, Shiota C, et al. Characterization of glucokinase regulatory protein-deficient mice. J Biol Chem 2000;275:7826-7831.
41. Shiota M, Postic C, Fujimoto Y, Jetton TL, Dixon K, Pan D, Grimsby J, et al. Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes. Diabetes 2001;50:622-629.
42. Bosco D, Meda P, Iynedjian PB. Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization. Biochem J 2000;348 Pt 1:215-222.
43. Froguel P, Vaxillaire M, Sun F, Velho G, Zouali H, Butel MO, Lesage S, et al. Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus. Nature 1992;356:162-164.
44. Vionnet N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, et al. Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 1992;356:721-722.
45. Velho G, Blanche H, Vaxillaire M, Bellanne-Chantelot C, Pardini VC, Timsit J, Passa P, et al. Identification of 14 new glucokinase mutations and description of the clinical profile of 42 MODY-2 families. Diabetologia 1997;40:217-224.
46. Pilkis SJ, Weber IT, Harrison RW, Bell GI. Glucokinase: structural analysis of a protein involved in susceptibility to diabetes. J Biol Chem 1994;269:21925-21928.
47. Liang Y, Kesavan P, Wang LQ, Niswender K, Tanizawa Y, Permutt MA, Magnuson MA, et al. Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme. Biochem J 1995;309 ( Pt 1): 167-173.
48. Guazzini B, Gaffi D, Mainieri D, Multari G, Cordera R, Bertolini S, Pozza G, et al. Three novel missense mutations in the glucokinase gene (G80S; E221K; G227C) in Italian subjects with maturity-onset diabetes of the young (MODY). Mutations in brief no. 162. Online. Hum Mutat 1998;12:136.
49. Miller SP, Anand GR, Karschnia EJ, Bell GI, LaPorte DC, Lange AJ. Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype. Diabetes 1999;48:1645-1651.
50. Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, et al. Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 1998;338:226-230.
51. Davis EA, Cuesta-Munoz A, Raoul M, Buettger C, Sweet I, Moates M, Magnuson MA, et al. Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis. Diabetologia 1999;42:1175-1186.
52. Agius L, Peak M, Newgard CB, Gomez-Foix AM, Guinovart JJ. Evidence for a role of glucose-induced translocation of glucokinase in the control of hepatic glycogen synthesis. J Biol Chem 1996;271:30479-30486.
53. O'Doherty RM, Lehman DL, Seoane J, Gomez-Foix AM, Guinovart JJ, Newgard CB. Differential metabolic effects of adenovirus-mediated glucokinase and hexokinase I overexpression in rat primary hepatocytes. J Biol Chem 1996;271:20524-20530.
54. O'Doherty RM, Lehman DL, Telemaque-Potts S, Newgard CB. Metabolic impact of glucokinase overexpression in liver: lowering of blood glucose in fed rats is accompanied by hyperlipidemia. Diabetes 1999;48:2022-2027.
55. Araki K, Araki M, Miyazaki J, Vassalli P. Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A 1995;92:160-164.
56. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999;274:305-315.
57. Duvillie B, Cordonnier N, Deltour L, Dandoy-Dron F, Itier JM, Monthioux E, Jami J, et al. Phenotypic alterations in insulin-deficient mutant mice. Proc Natl Acad Sci U S A 1997;94:5137-5140.
58. Accili D, Drago J, Lee EJ, Johnson MD, Cool MH, Salvatore P, Asico LD, et al. Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet 1996; 12:106-109.
59. Anton M, Graham FL. Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression. J Virol 1995;69:4600-4606.
60. Bali D, Svetlanov A, Lee HW, Fusco-DeMane D, Leiser M, Li B, Barzilai N, et al. Animal model for maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene. J Biol Chem 1995;270:21464-21467.
61. Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D, Goodyear LJ, et al. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 1998;2:559-569.
62. Burcelin R, del Carmen Munoz M, Guillam MT, Thorens B. Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway. J Biol Chem 2000;275:10930-10936.
63. Cabrera-Valladares G, German MS, Matschinsky FM, Wang J, Fernandez-Mejia C. Effect of retinoic acid on glucokinase activity and gene expression and on insulin secretion in primary cultures of pancreatic islets. Endocrinology 1999;140:3091-3096.
64. DeFronzo RA. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidaemia and atherosclerosis. Neth J Med 1997;50:191-197.
65. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol 1993;225:890-900.
66. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 1988;85:5166-5170.
67. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21:70-71.
68. Gannon M, Shiota C, Postic C, Wright CV, Magnuson M. Analysis of the Cre-mediated recombination driven by rat insulin promoter in embryonic and adult mouse pancreas. Genesis 2000;26:139-142.
69. Postic C, Magnuson MA. DNA excision in liver by an albumin-Cre transgene occurs progressively with age. Genesis 2000;26:149-150.
70. Meglasson MD, Matschinsky FM. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 1986;2:163-214.
71. Sweet IR, Li G, Najafi H, Berner D, Matschinsky FM. Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets. Am J Physiol 1996;271:E606-625.
72. Grupe A, Hultgren B, Ryan A, Ma YH, Bauer M, Stewart TA. Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis. Cell 1995;83:69-78.
73. Terauchi Y, Sakura H, Yasuda K, Iwamoto K, Takahashi N, Ito K, Kasai H, et al. Pancreatic beta-cell-specific targeted disruption of glucokinase gene. Diabetes mellitus due to defective insulin secretion to glucose. J Biol Chem 1995;270:30253-30256.
74. Piston DW, Knobel SM, Postic C, Shelton KD, Magnuson MA. Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion. J Biol Chem 1999;274:1000-1004.
75. Velho G, Petersen KF, Perseghin G, Hwang JH, Rothman DL, Pueyo ME, Cline GW, et al. Impaired hepatic glycogen synthesis in glucokinase-deficient (MODY-2) subjects. J Clin Invest 1996;98:1755-1761.
76. Tappy L, Dussoix P, Iynedjian P, Henry S, Schneiter P, Zahnd G, Jequier E, et al. Abnormal regulation of hepatic glucose output in maturity-onset diabetes of the young caused by a specific mutation of the glucokinase gene. Diabetes 1997;46:204-208.
77. Rossetti L, Chen W, Hu M, Hawkins M, Barzilai N, Efrat S. Abnormal regulation of HGP by hyperglycemia in mice with a disrupted glucokinase allele. Am J Physiol 1997;273:E743-750.
78. Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, et al. Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1). Nature 1996;384:458-460.
79. Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, Southam L, et al. Mutations in the hepatocyte nuclear factor-1 alpha gene in maturity-onset diabetes of the young (MODY3). Nature 1996;384:455-458.
80. Stoffers DA, Ferrer J, Clarke WL, Habener JF. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 1997;17:138-139.
81. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 1997; 15:106-110.
82. Horikawa Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, Lindner T, et al. Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY. Nat Genet 1997;17:384-385.
83. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 2000;6:87-97.
84. Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR. Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 1999;96:329-339.
85. Hardouin N, Nagy A. Gene-trap-based target site for cre-mediated transgenic insertion. Genesis 2000;26:245-252.
86. Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, Jami J, Bucchini D. Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 1996;15:1542-1547.
87. Pontoglio M, Sreenan S, Roe M, Pugh W, Ostrega D, Doyen A, Pick AJ, et al. Defective insulin secretion in hepatocyte nuclear factor 1 alpha-deficient mice. J Clin Invest 1998;101:2215-2222.
88. Postic, C, and Magnuson, M.A.. In "Molecular Pathogenesis of MODYs" (F.M. Matschinsky and M.A. Magnuson, eds.), Frontiers in Diabetes, 2000b; vol. 15, pp 293-298. Karger, Basel, Switzerland.
89. Postic, C, and Magnuson, M.A. In "Molecular Basis of Endocrine Pancreas Development and Function" (M.A. Hussain and J.F. Habener, eds.). Kluwer Academic Publishers, Norwell. 2001.
90. Roncero I, Alvarez E, Vazquez P, Blazquez E. Functional glucokinase isoforms are expressed in rat brain. J Neurochem 2000;74:1848-1857.
91. Sreenan SK, Cockburn BN, Baldwin AC, Ostrega DM, Levisetti M, Grape A, Bell GI, et al. Adaptation to hyperglycemia enhances insulin secretion in glucokinase mutant mice. Diabetes 1998;47:1881-1888.
92. Sturis J, Kurland IJ, Byrne MM, Mosekilde E, Froguel P, Pilkis SJ, Bell GI, et al. Compensation in pancreatic beta-cell function in subjects with glucokinase mutations. Diabetes 1994;43:718-723.
93. Thomas PM, Cote GJ, Wohllk N, Haddad B, Mathew PM, Rabl W, Aguilar-Bryan L, et al. Mutations in the sulfonylurea receptor gene in familial persistent hypennsulinemic hypoglycemia of infancy. Science 1995;268:426-429.
1 Asao K,Kao WH,Baptiste-Roberts K,et al.Short stature and the risk of adiposity,insulin resistance,and type 2 diabetes in middle age:the Third National Health and Nutrition Examination Survey(NI-tANES Ⅲ),1988-1994.Diabetes Care,2006,29:1632-1637.
2 Forest C,Beale E.New developments in nutrition and diabetes:glyceroneogenesis comes of age.Biochimie,2003,85:1195-1197.
3 Colombo M,Kruhoeffer M,Gregersen S,et al.Energy restriction prevents the development of type 2 diabetes in Zucker diabetic fatty rats:coordinated patterns of gene expression for energy metabolism in insulin-sensitive tissues and pancreatic islets determined by oligonucleotide microarray analysis.Metabolism,2006,55:43-52.
4 Nair S,Lee YH,Lindsay RS,et al.11beta-Hydroxysteroid dehydrogenase Type 1:genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.Diabetologia,2004,47:1088-1095.
5 Ishihara H,Sasaoka T,Kagawa S,et al.Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population.FEBS Lett,2003,554:450-454.
6 Wang H,Chu WS,Lu T,et al.Uncoupling protein-2 polymorphisms in type 2diabetes,obesity,and insulin secretion.Am J Physiol Endocrinol Metab,2004,286:E1-7.
7 D'Alfonso R,Marini MA,Frittitta L,et al.Polymorphisms of the insulin receptor substrate-2 in patients with type 2 diabetes.J Clin Endocrinol Metab,2003,88:317-322.
8 Patti ME,Butte A J,Crunkhorn S,et al.Coordinated reduction of genes oxidative metabolism in humans with insulin resistance and diabetes:Potential role of PGC1 and NRF1.Proc Natl Acad Sci U S A,2003,100:8466-8471.
9 Guilleret I,Benhattar J.Demethylation of the human telomerase catalytic subunit (hTERT) gene promoter reduced hTERT expression and telomerase activity and shortened telomeres.Exp Cell Res,2003,289:326-334.
10 Guilleret I,Yan P,Grange F,et al.Hypermethylation of the human telomerase catalytic subunit(hTERT) gene correlates with telomerase activity.Int J Cancer,2002,101:335-341.
11 Liu L,Wylie RC,Andrews LG,et al.Aging,cancer and nutrition:the DNA methylation connection.Mech Ageing Dev,2003,124:989-998.
12 Richardson B.Impact of aging on DNA methylation.Ageing Res Rev,2003,2:245-261.
13 Ahuja N, Li Q, Mohan AL, et al. Aging and DNA methylation in colorectal mucosa and cancer. Cancer Res, 1998, 58: 5489-5494.
14 Maier S, Olek A. Diabetes: a candidate disease for efficient DNA methylation profiling. J Nutr, 2002, 132: 2440S-2443S.
15 Longnecker DS. Abnormal methyl metabolism in pancreatic toxicity and diabetes. J Nutr, 2002, 132: 2373S-2376S.
16 Issa JP, Ahuja N, Toyota M, et al. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res, 2001, 61: 3573-3577.
17 Post WS, Goldschmidt-Clermont PJ, Wilhide CC, et al. Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res, 1999, 43: 985-991.
18 Tohgi H, Utsugisawa K, Nagane Y, et al. Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex. Brain Res Mol Brain Res, 1999, 70: 288-292.
19 Poirier LA, Brown AT, Fink LM, et al. Blood S-adenosylmethionine concentrations and lymphocyte methylenetetrahydrofolate reductase activity in diabetes mellitus and diabetic nephropathy. Metabolism, 2001, 50: 1014-1018.
20 Yokomori N, Tawata M, Onaya T DNA demethylation during the differentiation of 3T3-L1 cells affects the expression of the mouse GLUT4 gene. Diabetes, 1999, 48: 685-690.
21 Yokomori N, Tawata M, Onaya T. DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells. Diabetologia, 2002, 45: 140-148.
22 Jin B, Seong JK, Ryu DY. Tissue-specific and de novo promoter methylation of the mouse glucose transporter 2. Biol Pharm Bull, 2005, 28: 2054-2057.
23 Carretero MV, Torres L, Latasa U, et al. Transformed but not normal hepatocytes express UCP2. FEBS Lett, 1998, 439: 55-58.
24 Faber S, Ip T, Granner D, et al. The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression. Nucleic Acids Res, 1991, 19: 4681-4688.
25 Lillycrop KA, Phillips ES, Jackson AA, et al. Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring. J Nutr, 2005, 135: 1382-1386.
26 Temple IK, Gardner RJ, Mackay DJ, et al. Transient neonatal diabetes: widening the understanding of the etiopathogenesis of diabetes. Diabetes, 2000, 49: 1359-1366.
27 Ling C, Poulsen P, Simonsson S, et al. Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle. J Clin Invest, 2007, 117: 3427-3435.
28 Lyko F, Brown R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst, 2005, 97: 1498-1506.
29 Strathdee G,Brown R.Epigenetic cancer therapies:DNA methyltransferase inhibitors.Expert Opin Investig Drugs,2002,11:747-754.
30 Wang CY,Ma Q,He ZX.Activity of DNAmethylase and DNase Ⅰ sensitivity assay of genome DNA of liver from rat of diabetic nephropathy.Journal of Beijing Normal University(Natural Science),2002,6:810-814.[江琛颖,何忠效,马晴.糖尿病肾病大鼠肝DNA甲基化酶活性及基因组DNA 对DNase Ⅰ敏感性的分析.北京师范大学学报(自然科学版),2002,6:810-814.]