Elucidation of the molecular mechanisms underlying metabolic disorders using protemomics analysis

IRS蛋白被激活的胰岛素受体在多个酪氨酸残基上磷酸化。我们用32P-ATP标记的IRS-1探针筛选了一个与IRS-1相关的cDNA文库，并用小鼠肝脏的myc和FLAG标记鉴定了过表达的IRS-1免疫沉淀物中的Pin1。据报道，Pin1与含有基序的pSer/Pro或pThr/Pro结合，转化Praline残基的顺式-反式构象变化，从而导致靶蛋白的构象变化。不仅在利用SF-9和HepG2细胞进行的过表达实验中观察到Pin1与IRS-1的关联，而且在小鼠肝脏中也观察到了这种关联。冈田酸处理后，细胞中Pin1和IRS-1的相关性显著增强。GST-Pin1可下调SF-9细胞产生的IRS-1，但不能单独作用于GST，而碱性磷酸酶处理的IRS-1则不能。使用缺失和点突变的PIN1和IRS-1构建物的分析表明，WW d…在IRS-1的SAIN结构域中，Pin1和Ser434的N端有更多的缺失参与了这两种信号的相互作用。在HepG2中，Pin1的过表达显著增强了胰岛素诱导的IRS-1的磷酸化及其下游事件、PI-3-K与IRS-1的结合、Akt的磷酸化和GSK3β的磷酸化。相比之下，用Pin1抑制剂胡桃仁处理的HepG2细胞抑制了它们的表达。此外，Pin1在ob/ob小鼠肝脏中的过度表达可促进IRS-1介导的胰岛素信号转导，而Pin1 ko小鼠则表现出胰岛素抵抗和糖耐量异常。高脂饲料喂养2周后，肝脏和肌肉中Pin1蛋白的表达和与Pin1相关的IRS-1的表达均增加了数倍。结果还表明，在禁食条件下，Pin1的表达水平较低，重新投喂后，Pin1的表达水平有所提高。综上所述，我们得出结论，Pin1的表达受营养条件的调节，并且根据营养条件在增强IRS-1介导的胰岛素作用方面起着重要作用。较少

IRS-proteins are phosphorylated on multiple tyrosine residues by the activated insulin receptor. We identified Pin1 to be associated with IRS-1 by screening a cDNA library using 32P-ATP labeled IRS-1 probe and identifying Pin1 in the immunoprecipitates of overexpressed IRS-1.with myc and FLAG tags in mouse livers. Pin1 reportedly binds to pSer/Pro or pThr/Pro containing motif and converts the cis-trans conformational change of praline residue, which induce the conformational change of target protein. The association of Pin1 with IRS-1 was observed in not only overexpression experiments using Sf-9 and HepG2 cells, but also endogenously in the mouse liver. The association between Pin1 and IRS-1 in HepG2 cells was significantly increased by the treatment of okadaic acid. IRS-1 produced in Sf-9 cells was pulldowned by GST-pin1 but not GST alone, while IRS-1 treated with alkaline phosphatase was not. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed that WW d … More omain located in the N-terminus of Pin1 and Ser 434 in the SAIN domain of IRS-1 is involved in their association.Next, we investigated the role of Pin1 on IRS-1 mediating insulin signaling. The overexpression of Pin1 in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events; PI 3-kinase binding with IRS-1, Akt phosphorylation and GSK3beta phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 inhibitor Juglone suppressed them. In addition, overxpression of Pin1 in ob/ob mouse liver elevated IRS-1 mediating insulin signaling, while Pin1 KO mice exhibited insulin resistance and glucose intolerance.Finally, the regulation of Pin1 expression in the mouse liver and muscle was investigated. After 2 weeks high-fat diet feeding, in both liver and muscle, Pin1 protein expression and IRS-1 associated with Pin1 was increased by several folds. It was also shown that Pin1 expression is low in the fasted condition, and increased by re-feeding. Taken all results together, we conclude that Pin1 expression is regulated by the nutrient conditions, and play an important role on enhancing IRS-1 mediating insulin actions in accordance with the nutrient conditions. Less