Zhou L, Chen H, Xu P, Cong L N, Sciacchitano S, Li Y, Graham D, Jacobs A R, Taylor S I, Quon M J
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892-1754, USA.
Mol Endocrinol. 1999 Mar;13(3):505-14. doi: 10.1210/mend.13.3.0242.
The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.
胰岛素受体通过磷酸化多种细胞内底物来启动胰岛素作用。此前，我们已经证明胰岛素受体底物（IRS）-1和-2可介导胰岛素的作用，促进大鼠脂肪细胞中GLUT4葡萄糖转运体向细胞表面的转位。尽管IRS-1、-2和-4在整体结构上相似，但IRS-3大约短50%，并且在酪氨酸磷酸化位点方面有所不同。然而，如本研究所示，IRS-3和IRS-4也都能刺激GLUT4的转位。将大鼠脂肪细胞与血凝素（HA）表位标记的GLUT4（GLUT4-HA）以及人IRS-1、小鼠IRS-3或人IRS-4的表达载体共转染。在无胰岛素培养的细胞中，IRS-1的过表达导致细胞表面GLUT4-HA增加2倍；IRS-3或IRS-4的过表达引起细胞表面GLUT4-HA更大幅度的增加。实际上，在无胰岛素情况下IRS-3的作用比在未过表达IRS蛋白的细胞中最大刺激浓度胰岛素的作用大约大40%。由于磷脂酰肌醇（PI）3激酶对于胰岛素刺激的GLUT4转位至关重要，我们还研究了一种突变的IRS-3分子（IRS-3-F4），其中在所有四个YXXM基序（预测可结合并激活PI 3激酶的磷酸化位点）中苯丙氨酸取代了酪氨酸。有趣的是，IRS-3-F4的过表达并未促进GLUT4-HA的转位，反而实际上抑制了胰岛素刺激GLUT4-HA向细胞表面转位的能力。我们的数据表明，IRS-3和IRS-4能够介导脂肪细胞中胰岛素依赖PI 3激酶的代谢作用，并且IRS蛋白在介导GLUT4的转位中发挥生理作用。
