【作者】 唐晶；

【导师】 高翔；

【作者基本信息】 南京大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 MAGED1,也被称作NRAGE或者Dlxin-1.作为一个连结蛋白,它可以和很多蛋白结合,从而行使多种功能。目前已知的有关MAGED1的功能研究大多建立在体外模型上,对于它的生理功能,知识依然很缺乏。2008年有一例Maged1敲除小鼠被报道,文章显示敲除Maged1会影响发育中的凋亡过程,而在成体中没有明显的可辨异常。我们实验室也构建了一个Maged1敲除小鼠模型。敲除的位置稍有不同,也同样没有从外观上观察到异常。敲除小鼠可以育成并且繁殖。在纯化背景的过程中,我们发现敲除小鼠在晚期发生肥胖。肥胖的时间随小鼠批次的不同而有变化。12月龄已发生肥胖的小鼠表现出体重增加、脂肪增加以及脂肪百分比增加,但是肌肉的质量和同窝的对照相比没有显著差异。正常摄食状态下,血糖和对照持平,禁食后,略有增加,但远没有到达糖尿病的程度。在敲除小鼠体重和对照相差不大的时候,尽管前者体内分泌的胰岛素表现为不足,但是对葡萄糖的耐受能力正常或有所提高；对胰岛素的响应正常或更敏感。利用CLAMS (Comprehensive Lab Animal Monitoring System)对体重相当的小鼠进行监测,发现敲除小鼠的摄食减少,并且活动也减少。细胞水平上,敲除小鼠来源的永生化胚胎成纤维细胞分化成脂肪细胞的能力有所提高,分析表明该细胞对胰岛素更加敏感,可能与增加的IRS-1表达有关。此外胰岛素诱导产生的JNK的活化降低,这也可能导致了细胞对胰岛素的敏感性增加。UV和TNF-a对JNK的活化不受基因敲除的影响,提示JNK对胰岛素的反应降低是特异的。进一步研究发现年轻时的敲除小鼠有体温下降和leptin含量升高的表现。在正常小鼠体内注入leptin,会导致食欲降低、体重减轻。高水平的leptin和缓慢的体重增加提示体内可能存在leptin耐受的情况。在今后的工作中,需要增加敲除小鼠体内的生理指标的测定,用来帮助研究其中的机制。除了晚期肥胖的表型,在筛选新的生物节律相关蛋白的过程中,我们所建立的Maged1敲除小鼠还被发现有周期缩短的现象。生物钟的存在,使机体可以对外界的变化进行适应和预测。Maged1敲除后,可能对中心的分子调控网络产生影响,从而影响了节律。研究表明,MAGED1可以与RORa结合,通过作用于RORE,增加Bmal1和E4bp4的表达,抑制Rev-erbα的表达。在敲除小鼠内就表现为Bmal1和E4bp4的表达降低,Rev-erbα表达增加。尽管MAGED1本身并不表现出节律,与RORa的结合也没有周期性,但是它增加了体内时钟的稳定性。MAGED1被认为是一个新的节律调控因子,对体内时钟有不可或缺的作用。更多还原

【Abstract】 MAGED1, also named NRAGE or Dlxin-1, is regarded as an adaptor protein. MAGED1can interact with many proteins and play important roles in several intracellular signaling pathways. However, most conclusions are drawn on the basis of in vitro functional analysis. And the knowledge of the physiological roles of Magedl is limited.Up till now, only one Magedl knockout mouse has been reported. The deletion just appeared to cause defect in developmental apoptosis. A Magedl knockout mouse strain was also generated in our lab. Similar to those of the reported, the mice are viable, fertile, and normal in gross physical observation. However, the knockout mice were weightier and showed slightly higher levels of blood glucose in fasted status than wild-type mice although these phenotypes were variable among individuals. Compared with the wild-type littermates, the knockout mice exhibited normal or improved glucose tolerance and insulin sensitivity despite of low levels of insulin secretion, when their body weight was comparable. The data from Comprehensive Lab Animal Monitoring System showed that there was almost no difference in oxygen consumption and carbon dioxide production between the knockout and the wild-type mice with undistinguishable body weight. But the knockout mice had less food intake and were less active than wild-type mice. Cellular experiments demonstrated that immortalized Mefs of Magedl knockout mice had a stronger potential to differentiate into adipocytes than those of wild-type mice. In addition, the cells of knockout mice had higher protein levels of IRS-1and continuing phosphorylation of AKT under the stimulation of insulin, indicating that these cells had increased insulin sensitivity. Therefore, the increased adipogenesis observed in knockout Mefs was probably attributed to the improved insulin sensitivity. JNK has been demonstrated to regulate insulin pathway by negative feedback. Interestingly, insulin induced activation of JNK was downregulated in Magedl null cells, which indicated that low activity of JNK was probably responsible for the improved insulin sensitivity. The attenuated activation of JNK by insulin may be unique, because the phosphorylation of JNK caused by UV and TNF-a was comparable in the Maged1knockout and wild-type Mefs. During the further investigation, hypothermia and hyperleptinemia were found in the knockout mice when they were young. In random fed lean mice, administration of exogenous leptin results in anorexia and weight loss. The coexistence of hyperleptinemia and slow weight gain/obesity suggested that there was some kind of leptin resistance in the knockout mice. And more detailed mechanisms are needed to elucidate such late-onset obesity resulting from Maged1knockout.Besides the late-onset obesity, the most penetrated phenotype of Maged1was shortened circadian period. As we know, the circadian clock plays a central role in physiological adaption and anticipation of day to night alternations. In a genetic screen for novel regulators of circadian rhythms, the Magedl knockout mice were noted for shortened period and altered rest-activity bouts. These circadian phenotypes were proposed to result from a direct change in the core molecular clock network that reduced the robustness of the circadian clock. In vitro and in vivo evidences indicated that MAGED1bound to RORa and then affected the expression of core clock genes, Bmall, Rev-erba and E4bp4, through the Rev-Erba/RORA responsive elements (RORE). Although Magedl was a non rhythmic gene, it enhanced rhythmic input and buffered the irrelevant, perturbing stimuli or noise by binding RORa in a non-circadian way. Magedl was thus identified and defined as a novel circadian regulator, which was indispensable for the robustness of the circadian clock to better serve the organism.更多还原