【作者】 周军；

【导师】 黄开勋；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 糖尿病是一种常见的易发性代谢疾病,其中90%以上为2型糖尿病。胰岛素抵抗是2型糖尿病的主要特征,其本质是胰岛素信号转导通路中的某些环节出现故障。近年来越来越多的证据表明,活性氧和活性氮介导糖尿病及其并发症的病理过程。氧化应激状态下NO衍生得到的ONOO^-是一个强氧化剂和硝化剂,可与多种生物大分子反应,引起脂质过氧化、DNA损伤和蛋白质修饰。蛋白质酪氨酸硝化是一种重要的蛋白质翻译后修饰,它与炎症、心血管疾病和神经退行性疾病等多种病症相关,而ONOO^-路径是导致体内蛋白质酪氨酸硝化的重要途径。蛋白质酪氨酸硝化能影响蛋白质酪氨酸磷酸化,从而影响酪氨酸磷酸化介导的信号转导通路。研究表明ONOO^-与胰岛素抵抗及糖尿病的发生发展密切相关,但ONOO^-对胰岛素信号转导通路的调控作用及其机理尚不完全清楚。本论文从体外化学体系、细胞培养实验和体内动物实验三个方面入手,以纯化胰岛素受体、HepG2细胞和原代骨骼肌细胞、2型糖尿病小鼠模型为研究对象,探讨了ONOO^-对胰岛素信号转导的调控作用及其机理,所取得的主要结果有:（1）ONOO^-对骨骼肌细胞中胰岛素信号分子基因表达的影响以原代培养骨骼肌细胞为研究对象,采用MTT检测和实时定量PCR技术研究了化学合成ONOO^-和SIN-1对IR/IRSs/PI3-K/Akt通路和IR/CAP/Cb1通路中信号分子基因表达的影响。结果表明,50-1000μM的ONOO^-处理显著减少了骨骼肌细胞中IR、IRS-1、CAP、Cb1和Glut4的基因表达水平,并具有浓度依赖性。而且,SIN-1浓度依赖地减少了骨骼肌细胞中IR、Cb1和Glut4的基因表达水平,对IRS-1、CAP、p85α和SHIP2的基因表达水平则具有双向调节作用,即低浓度下表现为促进作用,高浓度下表现为抑制作用。以上结果表明较高浓度的ONOO^-可能下调IR/IRSs/PI3-K/Akt通路和IR/CAP/Cb1通路中信号分子的基因表达水平,从而抑制骨骼肌细胞中的胰岛素信号转导。（2）ONOO^-对HepG2细胞中胰岛素磷酸化信号的影响以HepG2细胞为研究对象,采用亚细胞分级和Western Blotting技术,研究了ONOO^-引起的蛋白质酪氨酸硝化对胰岛素刺激的酪氨酸磷酸化信号的影响,以及硝化蛋白和磷酸化蛋白的亚细胞定位。结果表明ONOO^-浓度依赖性地引起了HepG2细胞蛋白质的硝化,硝化蛋白主要位于细胞膜和细胞核上。ONOO^-在低浓度下（10-50μM）促进膜蛋白和胞质蛋白的酪氨酸磷酸化,可能起到信号分子的作用;在高浓度下（100-200μM）抑制膜蛋白和胞质蛋白的磷酸化,可能与胰岛素抵抗的发生有关,提示ONOO^-对胰岛素信号转导通路可能具有双向调节作用。（3） ONOO^-对胰岛素受体自磷酸化和激酶活性的影响采用免疫沉淀、免疫印迹、放射配体结合实验、小鼠血糖测定和实时定量PCR等方法研究了ONOO^-在体外和体内对胰岛素受体自磷酸化和激酶活性的影响。体外实验结果表明,无论在基础状态下还是在胰岛素刺激状态下,低浓度的ONOO^-（50μM）促进了胰岛素受体的自磷酸化和激酶活性,可能作为一个信号分子,而高浓度的ONOO^-（100-500μM）抑制了胰岛素受体的自磷酸化和激酶活性,可能介导胰岛素抵抗的发生。ONOO^-的这种双向调节作用可能与它对胰岛素受体的硝化和氧化修饰有关。此外,酪氨酸磷酸酯酶活性的降低以及胰岛素受体与¹²⁵I-胰岛素结合能力的降低也参与了这一调节过程。进一步的体内研究表明,腹腔注射0.1mg/kg和0.25mg/kgSIN-1显著降低了正常小鼠的血糖,其作用机理与IR/IRS-1酪氨酸磷酸化水平升高有关。腹腔注射0.25mg/kg和0.5mg/kg SIN-1显著升高了糖尿病小鼠的血糖,其作用机理与IR/IRS-1酪氨酸磷酸化水平降低有关。以上研究结果首次阐明了ONOO^-在体外和体内如何影响胰岛素受体功能,提示ONOO^-对胰岛素受体的自磷酸化和激酶活性具有双向调节作用。（4）ONOO^-在小鼠骨骼肌胰岛素抵抗发生中的作用采用胰岛素耐量测试、免疫沉淀和免疫印迹等方法研究了SIN-1对正常小鼠胰岛素敏感性的影响,以及骨骼肌中IR、IRS-1和Akt的硝化和磷酸化。采用高脂饮食诱导胰岛素抵抗小鼠模型,检测骨骼肌中IR和IRS-1的硝化和磷酸化,并观察了ONOO^-清除剂FeTPPS对上述效应的干预作用。研究结果表明,正常小鼠腹腔注射10mg/kg SIN-1后,与空白对照组相比胰岛素敏感性显著降低,骨骼肌中IR、IRS-1和Akt的硝化水平显著增加,而且在胰岛素刺激状态下IR、IRS-1和Akt的磷酸化水平显著降低,这可能是SIN-1导致胰岛素敏感性降低的重要原因。此外,SIN-1慢性注射使IRS-1蛋白质表达水平显著降低。在高脂饮食诱导的胰岛素抵抗小鼠中,与正常对照组相比骨骼肌中IR和IRS-1硝化水平显著升高,磷酸化水平显著降低,胰岛素敏感性也显著降低。使用FeTPPS治疗后,与高脂饮食组相比骨骼肌中IR和IRS-1硝化水平显著降低,磷酸化水平显著升高,胰岛素敏感性也显著升高。以上研究结果提示IR、IRS-1和Akt的硝化是iNOS介导的胰岛素抵抗的一种新的机理。（5）糖尿病小鼠肝脏和骨骼肌中硝化蛋白质组的初步研究采用免疫沉淀结合SDS-PAGE分离、免疫印迹、胶内酶切和LC-ESI-MS/MS等方法鉴定了体外硝化BSA的硝化位点,并探讨了2型糖尿病小鼠肝脏和骨骼肌中的蛋白质硝化。研究结果表明,BSA发生硝化的酪氨酸为Tyr161和Tyr424。与正常小鼠相比,糖尿病小鼠肝脏和骨骼肌中蛋白质硝化水平显著升高,肌浆/内质网Ca²⁺-ATP酶是骨骼肌中主要的硝化蛋白之一。更多还原

【Abstract】 Diabetes is a common kind of metabolic diseases. Type 2 diabetes, accounting for 90% of diabetes, is characterized by peripheral insulin resistance with an insulin-secretory defect that varies in severity. Insulin resistance results from impairment of one or more steps in insulin signal transduction pathway. In the past few years, there has been accumulating evidence that RNS/ROS make a significant contribution to the progression of diabetes and its complications. Peroxynitrite （ONOO^-）, derived from NO under oxidative stress, is a powerful oxidizing and nitrating species, causing DNA damage, lipid peroxidation, oxidation of protein-associated thiol groups, and nitration of protein tyrosine residues. Protein tyrosine nitration is an important posttranslational modification, and involved in a large variety of inflammatory, cardiovascular and neurodegenerative diseases. Nitration by peroxynitrite is a principal pathway, where nitrotyrosine is formed by free radical reaction. Protein tyrosine nitration can affect tyrosine phosphorylation, and thus affect signal transduction pathways involving tyrosine phosphorylation. Accumulating evidence suggests that peroxynitrite is involved in the pathogenesis of insulin resistance and diabetes, however, the regulation mechanism of insulin signal transduction pathway by peroxynitrite remains poorly understood.In this paper, the regulation mechanism of insulin signal transduction pathway by peroxynitrite was investigated in in vitro purified insulin receptor （IR）, ex vivo primary skeletal muscle cells and HepG2 cells, and in vivo experimental mouse model of type 2 diabetes. The main results are as follows:（1） Effects of peroxynitrite on mRNA expression of insulin signaling molecules in primary skeletal muscle cellsTo investigate the effects of peroxynitrite treatment on gene expression profiles of insulin signaling molecules, primary skeletal muscle cells were exposed to various concentrations of peroxynitrite or 3-morpholinosydnonimine hydrochloride （SIN-1）, followed by RNA isolation and real time RT-PCR analysis. The results showed that exposure of primary skeletal muscle cells to peroxynitrite （50-1000μM） led to a dose-dependent decrease in mRNA expression of IR, IRS-1, CAP, Cb1 and GLUT4. In contrast, under the same conditions, peroxynitrite had no effect on mRNA expression of PI3-K p85αand SHIP2. Moreover, exposure of primary skeletal muscle cells to SIN-1 also led to a dose-dependent decrease in mRNA expression of IR, Cbl and GLUT4. Of interest, SIN-1 treatment induced differential regulation of mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. At relatively low concentrations SIN-1 upregulated mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. However, at higher concentrations SIN-1 downregulated mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. Taken together, these data provide evidence that high concentrations of peroxynitrite may impair insulin signal transduction in skeletal muscle cells by reducing mRNA expression of insulin signaling molecules.（2） Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cellsHepG2 cells were exposed to various concentrations of peroxynitrite, follwed by 100 nM insulin stimulation. Subcellular fractionation was performed, and thereafter protein tyrosine nitration and tyrosine phosphorylation were detected by western blotting. The results showed that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations （10-50μM） peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations （100-200μM） peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.（3） Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitriteThis report describes the effect of peroxynitrite on insulin receptor （IR） autophosphorylation and kinase activity. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, IR preparations exhibited dramatically impaired ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity with increasing peroxynitrite concentrations. Moreover, the in vivo study showed that SIN-1 administration （0.1 mg/kg and 0.25 mg/kg） decreased blood glucose levels in normal mice via upregulation of IR and IRS-1 tyrosine phosphorylation. In contrast, SIN-1 （0.25 mg/kg and 0.5 mg/kg） increased blood glucose levels in diabetic mice via downregulation of IR and IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity.（4） Peroxynitrite mediates muscle insulin resistance in mice via nitration of IRβ/IRS-1 and AktIn the current study, we investigated whether insulin resistance in vivo could be mediated by nitration of proteins involved in the early steps of the insulin signal transduction pathway. Exogenous peroxynitrite donated by 10 mg/kg SIN-1 induced in vivo nitration of the insulin receptorβsubunit （IRβ）, IRS-1 and Akt, and dramatically reduced insulin signaling in skeletal muscle of mice. In high-fat diet （HFD）-fed insulin resistant mice, we observed enhanced nitration of IRβand IRS-1 and reduced insulin signaling in skeletal muscle. Reversal of nitration of these proteins by treatment with the peroxynitrite decomposition catalyst FeTPPS yielded an improvement in insulin action in skeletal muscle, in parallel with decreased nitration of IRβand IRS-1. Taken together, these findings provide new mechanistic insights for the involvement of peroxynitrite in the development of insulin resistance, and suggest that nitration of proteins involved in the early steps of insulin signal transduction is a novel molecular mechanism of HFD-induced muscle insulin resistance.（5） Preliminary study on nitroproteome in liver and skeletal muscle of diabetic miceThe nitration sites on bovine serum albumin （BSA） exposed to SIN-1 were identifiedby capillary high-performance liquid chromatography-coupled electrospray ionization tandem mass Spectrometry （LC-ESI-MS/MS）. Moreover, the nitrotyrosine-containing proteins in liver and skeletal muscle from diabetic mice were investigated, and anti-nitrotyrosine immunoprecipitation products from diabetic mouse liver and skeletal muscle were analyzed by LC-ESI-MS/MS. The results showed that the nitration sites on BSA were Tyr161 and Tyr424. Moreover, protein nitration was greater in diabetic mouse liver and skeletal muscle than normal mouse liver and skeletal muscle. Sarcoplasmic reticulum Ca²⁺-ATPase was identified as one of the nitrated proteins in diabetic mouse skeletal muscle.更多还原