【作者】 葛伊莉；

【导师】 黄开勋；

【作者基本信息】 华中科技大学 ， 无机化学， 2007， 博士

【摘要】 由于糖尿病对人类健康的严重威胁,相关科技工作者对糖尿病的病因学、病理学研究以及治疗糖尿病新药的开发给于了高度的关注。四氧嘧啶常常用来诱导I型糖尿病的动物模型来研究其病理并评价候选药物降血糖效果。在这些研究中,四氧嘧啶对动物胰腺损伤的研究已经有许多报道,但其对肝脏和肌肉组织糖代谢过程中胰岛素信号传导分子mRNA表达的影响尚不多见。蛋白质磷酸化在细胞信号传导中起着关键的作用。在胰岛素信号系统中,胰岛素受体在胰岛素作用下首先自磷酸化而激活酪氨酸激酶活性,从而向下游传导信号。蛋白质硝化是一种重要的蛋白质翻译后修饰,它与多种疾病有关。硝基酪氨酸的形成可能干扰胰岛素信号传导分子的磷酸化,因此进行酪氨酸硝化对酪氨酸磷酸化影响的研究对于了解蛋白质硝化在胰岛素抵抗型糖尿病中的作用具有重要的意义。本文以胰岛素信号传导系统中PI（3）K通路为对象,建立了体外测定胰岛素受体自磷酸化的³¹P-NMR波谱法,研究了体外胰岛素受体和受体底物的酪氨酸磷酸化反应,探讨了酪氨酸硝化对酪氨酸磷酸化的影响,并研究了四氧嘧啶对大鼠肝和肌肉组织该通路信号传导分子mRNA表达的影响,取得了以下主要结果:（1）通过探索胰岛素受体自磷酸化的适宜反应条件和适宜测试条件,建立了体外测定胰岛素受体自磷酸化的³¹P-NMR波谱法,并将此方法用于胰岛素受体自磷酸化的体外研究。结果表明:体外胰岛素受体自磷酸化反应进程能够用此方法实时检测并且可以作相对定量检测,所得到的胰岛素受体自磷酸化的结果与免疫印迹分析方法的结果相吻合,说明所建立的³¹P-NMR波谱方法是可行的。此方法的最大优点在于避免了放射性。（2）用四氧嘧啶腹腔注射法对Wistar大鼠进行糖尿病造模,运用³¹P-NMR波谱法和免疫印迹法发现四氧嘧啶诱导的糖尿病大鼠肝脏中胰岛素受体（IR）体外自磷酸化水平下降,研究还揭示酪氨酸残基的硝化对IR自磷酸的影响在体外是依赖于SIN-1浓度的,即在较低的SIN-1浓度下IR自磷酸水平上调,在较高的SIN-1浓度下IR自磷酸水平下调,这说明IR酪氨酸激酶在较低的过氧亚硝酸根离子浓度下活性增强,而在较高的过氧亚硝酸根离子浓度下IR酪氨酸激酶的活性受到抑制。并且运用所建立的³¹P-NMR波谱法检测了模拟胰岛素受体底物1（IRS1）磷酸化部位的基序所合成多肽的酪氨酸磷酸化信号,实验数据表明模拟IRS1的硝化多肽中硝基酪氨酸的磷酸化完全被抑制,说明3-硝基酪氨酸的形成干扰了酪氨酸酚羟基上的磷酸化反应。上述磷酸化水平的削弱可能会干扰胰岛素信号的传导,进而导致糖尿病大鼠肝脏的胰岛素抵抗。（3）采用巯基测试法测定了正常及糖尿病模型大鼠和家兔肝脏的巯基含量,结果显示糖尿病大鼠和家兔肝脏存在氧化损伤。用¹²⁵I-胰岛素标记法测定了大鼠和家兔肝细胞膜中胰岛素受体与胰岛素的结合能力,实验结果表明胰岛素与胰岛素受体专一结合与pH明显相关,最适宜结合的pH值大约是7.5,与血液pH相当,而且发现糖尿病家兔肝细胞膜不论高亲和位点还是低亲和位点的IR,与胰岛素的解离都显著减弱。（4）采用RT-PCR和免疫印迹法揭示了四氧嘧啶对其诱导的糖尿病大鼠肝脏的胰岛素信号传导PI（3）K通路主要分子的表达有显著的影响,四氧嘧啶诱导的糖尿病大鼠肝脏的IR、IRS1、葡萄糖转运体2（GLUT2）和葡萄糖激酶（GK）的mRNA表达和IR蛋白质表达下调,而IRS2和胰岛素磷脂酰肌醇3激酶（PI（3）K）的mRNA表达上调。蒽酮比色测定发现胰岛素治疗没有使四氧嘧啶诱导的糖尿病大鼠恢复到正常大鼠的肝糖原水平,说明四氧嘧啶诱导的糖尿病大鼠肝脏存在一定的胰岛素抵抗。（5）运用荧光定量PCR研究了大鼠肌肉组织胰岛素信号传导PI（3）K通路中重要的信号传导分子的基因表达,结果表明四氧嘧啶诱导的糖尿病大鼠肌肉组织的IR、IRS1和GLUT4的mRNA的表达下调,PI（3）K的mRNA的表达上调。糖尿病大鼠骨骼肌GLUT4的mRNA含量下降可能会引起糖摄取能力下降,说明四氧嘧啶诱导的糖尿病大鼠肌肉组织也存在一定的胰岛素抵抗。更多还原

【Abstract】 Because of the serious threat of diabetes mellitus to human health, some scientists pay attention to the aetiology and pathematology as well as the development of new medicine for diabetes. Animal models of Type 1 diabetes mellitus was induced by alloxan to research its pathematology and the effects of selected drugs on cutting down blood sugar. Although the investigation of the damage of alloxan to pancreaticβcells has been well reported, the effects of alloxan-induced diabetes on the mRNA expression levels of insulin signal transmission molecules in carbohydrate metabolism of rat liver and muscle have been little reported.Protein phosphorylation plays a crucial role in cellular signal transduction. In insulin signaling system, insulin transmits the signal downstream by means of autophosphorylation on tyrosine residues of insulin receptor （IR） and tyrosine phosphorylation of a family of insulin receptor substrate （IRS） proteins. Protein tyrosine nitration is an important posttranslational modification, and involves in a variety of diseases. The formation of nitrotyrosine may interfere with normal signal transduction pathways and the study of its effects on tyrosine phosphorylation is important to comprehend the function of tyrosine nitration in diabetes mellitus of insulin resistance type.In this paper, A new method with ³¹P-NMR spectroscopy has been set up to investigate in vitro phosphorylation of insulin receptor and its substrate in the PI（3）K pathway in insulin signaling transmission system.The tyrosine phosphorylation of insulin receptor and insulin receptor substrate and the effects of nitration on tyrosine phosphorylation were investigated in vitro. The effects of alloxan-induced diabetes on the mRNA expression levels of insulin signal transmission molecules in carbohydrate metabolism of rat liver and muscle were also studied. The main results obtained were as follows: （1） A new protocol with ³¹P-NMR spectroscopy has been applied to investigate in vitro autophosphorylation of insulin receptor. The results of the present study demonstrated that the time-courses or quantitative reaction of in vitro autophosphorylation of insulin receptor could be monitored by the new method. Our data illustrated that the results determined by our developed ³¹P-NMR protocol coincided with those in western-blotting analysis, which confirmed the feasibility of the established ³¹P-NMR method, and the outstanding advantage of this method is non-radioactive.（2） Three consecutive intraperitoneal injections of alloxan in Wistar rats induced diabetic model. The results of the present study demonstrated that autophosphorylation level of IR in alloxan-induced diabetic rat livers was attenuated in vitro. This alteration in the early steps of insulin signaling might be one of many insulin resistance states. Our findings confirmed that the effect of the nitration of tyrosine residue on autophosphorylation of IR was concentration-dependent of SIN-1 in vitro, and autophosphorylation level of IR was up-regulated at relatively low concentration of SIN-1 and down-regulated at relatively high concentration of SIN-1, which suggested that IR tyrosine kinase was activated at lower concentration of peroxynitrite and inactivated at higher one. Moreover, our data proved that the nitrotyrosine in the nitrated peptides mimicking the nitration of IRS1 could inhibit their insulin-stimulated tyrosine phosphorylation in vitro. Because of the electronic and steric effects of nitrotyrosine on the tyrosine residue phosphorylation, the formation of 3-nitrotyrosine might interfere with the phosphorylation reaction on phenolic hydroxyl group of tyrosine. Then, the attenuation of phosphorylation level might lead to insulin resistance in insulin signal system.（3） The thiol group amounts in liver of rats and rabbits were determined by thiol group assay. The results revealed that oxidative damage existed in liver tissue of diabetic rats and rabbits. The properties of insulin receptor in liver membrane of rats and rabbits were assayed by ¹²⁵I-insulin marking method. The optimal pH was 7.5, and it corresponded to that of blood. The experiment showed that the dissociation of IR with insulin was obviously decreased at high and low affinity sites in the liver membrane of the diabetic rat and rabbit.（4） The present study revealed that alloxan-induced diabetes had significant effects on the expression of insulin signal transmission molecules in PI（3）K pathway in rat livers by RT-PCR. Alloxan-induced diabetes attenuated the mRNA expression levels of IR, IRS1, GK and GLUT2 and the protein expression of IR in rat liver tissue, and increased the mRNA expression levels of IRS2 and PI（3）K. The hepatic glycogen content of alloxan-diabetic rats treated with insulin did not restore to the levels of normal rats under above experimental conditions. These results implied that when alloxan was used to induce diabetes rats as model animals of Type 1 diabetes mellitus, the factor of insulin resistance should be considered.（5） The effects of alloxan-induced diabetes on the mRNA expression levels of important insulin signal molecules in PI（3）K pathway in rat muscle were also studied by Real-time Quantitative Polymerase Chain Reaction. The results demonstrated that alloxan-induced diabetes attenuated the mRNA expression levels of IR, IRS1 and GLUT4 in rat muscle tissues, and increased the mRNA expression levels of PI（3）K. The decrease in mRNA expression level of GLUT4 in rat muscle tissue implied that there might be insulin resistance in insulin signal system due to the decrease in its ability of uptake glucose.更多还原