Heller-Harrison R A, Morin M, Guilherme A, Czech M P
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA.
J Biol Chem. 1996 Apr 26;271(17):10200-4. doi: 10.1074/jbc.271.17.10200.
Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.
磷脂酰肌醇（PI）3激酶被认为是细胞内GLUT4葡萄糖转运体在胰岛素作用下急性重新分布至质膜过程中的一个信号元件。然而，一些受体激活PI 3激酶却不引起GLUT4易位，这表明特定的细胞定位可能对该PI 3激酶功能至关重要。与这一观点一致，在3T3-L1脂肪细胞中，含有与胰岛素受体底物1（IRS-1）结合的PI 3激酶的复合物与细胞内膜相关（赫勒-哈里森，R.，莫林，M.和捷克，M.（1995年）《生物化学杂志》270，24442 - 24450）。我们在此报告，在胰岛素作用下，可从用免疫吸附法从3T3-L1脂肪细胞制备的含GLUT4囊泡的去污剂提取物中，用抗IRS-1抗体免疫沉淀激活的IRS-1.PI 3激酶复合物。还证实了使用蔗糖速度梯度超速离心制备的囊泡将PI 3激酶靶向大鼠脂肪细胞含GLUT4的囊泡。胰岛素处理使这些含GLUT4的囊泡中免疫反应性p85蛋白增加了2.3倍，而含GLUT4囊泡提取物中PI 3激酶活性的抗p85免疫沉淀物也增加到类似程度。当以PI、PI 4-P和PI 4,5-P2为底物时，对含GLUT4囊泡相关的PI 3激酶活性进行HPLC分析表明，胰岛素介导PI 3-P、PI 3,4-P2和PI 3,4,5-P3增加。我们的数据表明，胰岛素促使PI 3激酶与3T3-L1和大鼠脂肪细胞中含GLUT4的囊泡结合，这与PI 3激酶参与胰岛素调节的GLUT4向质膜移动的假说一致。
