Sentinelli F, Filippi E, Cavallo M G, Romeo S, Fanelli M, Baroni M G
Department of Clinical Sciences, Division of Endocrinology, Policlinico Umberto I, University of Rome 'La Sapienza', Viale del Policlinico 155, 00161 Rome, Italy.
J Endocrinol. 2006 Feb;188(2):271-85. doi: 10.1677/joe.1.06290.
The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6-8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARgamma expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARgamma agonist, restored differentiation with higher level of PPARgamma expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARgamma agonist restores the normal phenotype of 3T3L1 cells.
胰岛素受体底物1（IRS-1）在胰岛素敏感性方面发挥着核心作用，关联研究表明IRS-1 G972R变异体是胰岛素抵抗的一个危险因素。然而，这种突变如何导致胰岛素敏感性受损仍有待确定。我们的研究旨在评估在3T3L1脂肪细胞中转染IRS-1 G972R变异体后，该突变对胰岛素信号传导和细胞分化的影响。用含有人类野生型IRS-1或G972R变异体的pcDNA3表达载体转染3T3L1细胞。诱导分化后，转染野生型IRS-1的3T3L1细胞在6-8天内分化，而转染G972R变异体的细胞未分化。为了确定IRS-1的缺陷是否是造成这种情况的原因，我们分析了胰岛素信号通路中几个相关基因的表达。结果显示，转染突变型IRS-1的细胞中PPARγ表达显著降低，同时磷脂酰肌醇-3激酶（PI 3-激酶）与IRS-1 G972R的结合以及PI 3-激酶活性显著下降。此外，我们观察到胰岛素受体（IR）与IRS-1 G972R蛋白之间的相互作用增强，并且与3T3L1-WT相比，3T3L1-G972R细胞中IR的自磷酸化显著受到抑制。用PPARγ激动剂吡格列酮（PIO）处理3T3L1-G972R细胞，可恢复分化，PPARγ表达水平升高，PI 3-激酶与IRS-1 G972R的结合以及PI 3-激酶活性恢复。IR自磷酸化也增加。在完全分化的3T3L1-G972R细胞中撤去PIO，可导致胰岛素信号缺陷再次出现。最后，我们观察到IRS-2表达水平较高，这表明IRS-2可能在脂肪细胞胰岛素信号传导中发挥更重要的作用。总之，IRS-1 G972R变异体损害胰岛素信号传导，用PPARγ激动剂治疗可恢复3T3L1细胞的正常表型。
