【作者】 赵红叶；

【导师】 黎健；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 目的：蛋白磷酸酶4(Protein phosphatase4, PP4)是PP2A家族的重要成员,参与调节许多重要的细胞进程,如中心体的成熟、微管的组织和生长、DNA损伤修复,并与胰岛素受体底物4(Insulin receptor4, IRS4)的降解和肝糖代谢有关。本研究通过建立TNF-a诱导的胰岛素抵抗的细胞和动物模型,分析细胞或肝脏组织中PP4的表达及活性的变化,探讨PP4在TNF-a诱导的肝脏胰岛素抵抗中的作用及其机制。方法：TNF-α(10ng/ml)刺激人肝癌细胞系HepG2和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗的细胞模型；C57BL/6小鼠背部皮下埋植TNF-a(8μg/kg-day)缓释泵处理7天,建立TNF-a诱导的胰岛素抵抗动物模型。用G418筛选得到稳定转染PP4高表达质粒HA-PP4和转染PP4活性抑制突变体质粒FLAG-PP4RL的HepG2细胞克隆；应用PP4-siRNA及PP4-shRNA干扰HepG2细胞与原代肝细胞中PP4的表达,获得PP4低表达的HepG2细胞和原代肝细胞；构建PP4-shRNA腺病毒载体,通过尾静脉注射C57BL/6、鼠,建立肝脏PP4低表达的动物模型。测定小鼠血糖、血脂、胰岛素和TNF-a水平。用蒽酮法检测细胞和肝脏组织中的糖原水平,Real-time PCR测定糖异生相关基因(G6Pase、PEPCK、PGCl-α)的表达,Western blot分析细胞或肝脏组织胰岛素信号通路中JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、GSK、P-GSK和PP4的水平,用免疫沉淀结合丝／苏氨酸磷酸酶活性分析法测定PP4的活性。免疫共沉淀结合Western blot检测PP4与IRS1的相互作用。结果：1.我们首先以2型糖尿病小鼠-db/db小鼠(C57BKS/J背景)为胰岛素抵抗模型,检测到血糖、胰岛素和TNF-a水平显著升高,而胰岛素敏感指数(ISI)降低。蒽酮法检测结果显示肝组织糖原含量显著降低,Western blot结果表明糖原合成和糖异生相关信号通路受损：JNK的磷酸化和IRS1的307-Ser磷酸化水平增强,而GSK、AKT的磷酸化水平下降,同时伴有IRS1表达下降及PEPCK表达增加。这些结果表明db/db小鼠的肝脏发生了胰岛素抵抗。我们还观察到db/db小鼠肝组织中PP4的表达和活性均显著增强,提示PP4可能参与了肝脏胰岛素抵抗的发生。在体外试验中,我们采用10ng/mlTNF-a处理HepG2细胞和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗细胞模型。在这两种细胞的胰岛素抵抗模型中,糖原含量显著下降,糖异生相关基因表达增加,细胞的糖原合成信号通路受损： JNK的磷酸化和IRS1的307-Ser磷酸化水平升高,而GSK和AKT的磷酸化水平下降。Western blot和磷酸酶活性分析结果显示PP4的表达和活性均增强。在TNF-a诱导的C57BL/6小鼠胰岛素抵抗动物模型中,血清胰岛素水平升高,ISI降低,肝脏组织中糖原含量显著下降,糖异生相关基因(G6Pase、PEPCK、PGC1-a)的mRNA水平升高。肝脏组织的糖原合成信号通路受损：IRS1和JNK的磷酸化水平显著增加,而GSK和AKT的磷酸化水平及IRS1表达水平显著降低。同时肝脏组织PP4的表达和活性增强。细胞和动物实验结果均提示PP4参与了TNF-a诱导的肝脏胰岛素抵抗的发生。2.为了进一步探讨PP4在TNF-a诱导的肝脏胰岛素抵抗中的作用及机制,我们针对PP4的表达和活性,分别建立了稳定高表达PP4以及PP4活性被抑制的HepG2细胞克隆,应用PP4-siRNA及PP4-shRNA获得PP4低表达的HepG2细胞和原代肝细胞以及肝脏PP4低表达的C57BL/6小鼠。这些细胞和动物的分析结果表明①PP4高表达的HepG2细胞中糖原含量降低,糖原合成信号通路受损；②PP4表达降低可以使HepG2细胞、原代肝细胞以及C57BL/6小鼠中糖原含量增加,糖异生相关基因表达降低,糖原合成信号通路恢复；③抑制PP4活性,HepG2细胞的糖原合成增加,糖原合成信号通路得以恢复。此外,免疫共沉淀及Westernblot的结果显示,在HepG2细胞和原代肝细胞中PP4与IRS1存在着相互调节作用。这种相互调节可能在TNF-a诱导的肝脏胰岛素抵抗中起关键作用。结论：1.在肝胰岛素抵抗的细胞和动物模型中,PP4的表达和活性均显著增强；2.PP4是TNF-α信号通路的正调控因子,促进TNF-α诱导的胰岛素抵；3.PP4通过两种方式参与TNF-α诱导的胰岛素抵抗。一方面,通过激活JNK调控胰岛素信号通路；另一方面,可能通过与IRS1直接作用而调节TNF-α诱导的胰岛素抵抗。更多还原

【Abstract】 Objectives:Protein phosphatase4(PP4) is a member of PP2A-family and plays important role in many cellular processes, such as centrosome maturation, microtubule growth and organization, and DNA repair. PP4also involves in the down-regulation of IRS4and hepatic glucose metabolism. In the present study, we established the TNF-a-induced insulin resistance models in vivo and in vitro and analyzed the expression and phosphatase activity of PP4in hepatocytes and liver. Moreover, we explored the role of PP4in TNF-a-induced hepatic insulin resistance.Methods:We established cellular insulin resistance model by treatment of human HepG2cells and C57BL/6mouse primary hepatocytes with lOng/ml TNF-a for24h. Implanting the TNF-a osmotic pumps to the back subcuticle of mice was used to establish TNF-a-induced insulin resistance animal model. The HepG2cell clones expressing HA-PP4and FLAG-PP4RL were obtained by G418selection. The expression of PP4in HepG2cells and primary hepatocytes was knock-downed by transfection of PP4-siRNA and PP4-shRNA. Moreover, Ad-PP4-shRNA was delivered to the livers in C57BL/6mice via tail veil injection before implanting the TNF-a osmotic pumps to the back subcuticle of mice. Serum insulin and TNF-a levels were detected by ELISA, and glycogen content was measured by Anthrone method. The expression of the G6Pase, PEPCK and PGCl-α was analyzed by Real-time PCR, and the levels of JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、 GSK、P-GSK、and PP4were detected by Western blot. Phosphatase activity of PP4was also analyzed with immunoprecipitation and phosphatase activity assay. In addition, the interaction between PP4and IRS1was measured by co-immunoprecipitation and Western blot. Results:The db/db mice displayed increased levels of serum GLU, insulin and TNF-a, and decreased ISI. We also found that glycogen levels in the liver of db/db mice, suggesting a state of insulin resistance. Importantly, levels of phosphorylated JNK and IRS1, and PEPCK expression were elevated in liver of db/db mice, companied by decreased IRS1expression and phosphorylated levels of GSK and AKT. It is noteworthy that elevated PP4activity and expression were detected in liver of db/db mice, suggesting that PP4may be involved in TNF-a-induced insulin resistance. To extend these observations from db/db mice to the cellular models of insulin resistance, human HepG2cells and C57BL/6mouse primary hepatocytes were treated with TNF-a to induce insulin resistance. These two cellular models displayed a state of insulin resistance, as assessed by declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling. The expression and activity of PP4were also increased. Moreover, C57BL/6mice implanted the TNF-a osmotic pumps to the back subcuticle displayed declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling, companied by reduction of PP4expression and activity. Taken together, these data suggest that PP4may play a key role in TNF-a-induced insulin resistance.In order to further assess whether PP4is involved in TNF-a-induced insulin resistance and explore the possible mechanism, HA-PP4, PP4-siRNA, PP4-shRNA and PP4RL were used to investigate the effect of PP4expression and activity on TNF-a-induced hepatic insulin resistance. The results suggested that up-regulation of PP4expression resulted in decreased glycogen content in HepG2cells and impaired glycogen synthesis and gluconeogenesis signaling. In contrast, down-regulation of PP4expression led to increase of glycogen content in HepG2cells and C57BL/6mouse primary hepatocytes and liver of C57BL/6mice, companied by restoration of insulin signaling. Similarly, inhibition of PP4activity in HepG2cells also led to elevated glycogen content. Moreover, the interaction between PP4and IRS1was assessed by co-immunoprecipitation and Western blot.Conclusions: 1. In cellular or animal insulin resistance models, the expression and activity of PP4were increased in hepatocytes and liver.2. PP4functions as a positive regulator of TNF-a-signaling and a linker between inflammation and hepatic insulin resistance.3. PP4plays a key role in TNF-a-induced hepatic insulin resistance via regulating IRS1by activation of JNK or interaction with IRS1directly.更多还原