Draznin B, Lewis D, Houlder N, Sherman N, Adamo M, Garvey W T, LeRoith D, Sussman K
Research Service, Veterans Administration Medical Center, Denver, Colorado 80220.
Endocrinology. 1989 Nov;125(5):2341-9. doi: 10.1210/endo-125-5-2341.
We have recently provided evidence that elevated levels of cytosolic free Ca2+ ([Ca2+]i) decreased insulin-stimulated glucose uptake in isolated rat adipocytes. To investigate the mechanism of Ca2+ action, we examined the effects of elevated levels of [Ca2+]i on insulin binding, autophosphorylation, and tyrosine kinase activity (TKA) of insulin receptors as well as basal and insulin-stimulated cellular distribution of glucose transporters. The latter was assessed by cytochalasin-B binding to plasma membrane and cytosolic fractions. Elevated concentrations of [Ca2+]i were maintained by incubating adipocytes with a depolarizing concentration of K+ (40 mM). Basal nonstimulated glucose uptake was not altered by increased levels of [Ca2+]i. Adipocytes with higher [Ca2+]i (220 +/- 15 nM) showed 30% reduction in insulin-stimulated 2-deoxyglucose uptake compared with control cells ([Ca2+]i, 140 +/- 18 nM). Moreover, adipocytes with higher levels of [Ca2+]i demonstrated an approximately 10% reduction in autophosphorylation and TKA of insulin receptors without a change in insulin binding. Both basal and insulin-stimulated distributions of glucose transporters were unaffected by sustained levels of [Ca2+]i. The effects of elevated [Ca2+]i were not mimicked by protein kinase-C activation. These observations suggest that 1) elevated or sustained levels of [Ca2+]i impair insulin-stimulated glucose uptake; and 2) Ca2+-induced impairment appears to reside at the postbinding steps of insulin action and probably interferes with the TKA of insulin receptors and the intrinsic activity of glucose transporters.
我们最近提供的证据表明，在分离的大鼠脂肪细胞中，胞质游离Ca2+（[Ca2+]i）水平升高会降低胰岛素刺激的葡萄糖摄取。为了研究Ca2+作用的机制，我们检测了[Ca2+]i水平升高对胰岛素受体的胰岛素结合、自身磷酸化和酪氨酸激酶活性（TKA）以及葡萄糖转运蛋白的基础和胰岛素刺激的细胞分布的影响。后者通过细胞松弛素B与质膜和胞质部分的结合来评估。通过用去极化浓度的K+（40 mM）孵育脂肪细胞来维持[Ca2+]i的升高浓度。[Ca2+]i水平升高并未改变基础非刺激状态下的葡萄糖摄取。与对照细胞（[Ca2+]i，140±18 nM）相比，[Ca2+]i较高（220±15 nM）的脂肪细胞胰岛素刺激的2-脱氧葡萄糖摄取降低了30%。此外，[Ca2+]i水平较高的脂肪细胞胰岛素受体的自身磷酸化和TKA降低了约10%，而胰岛素结合没有变化。葡萄糖转运蛋白的基础和胰岛素刺激分布均不受[Ca2+]i持续水平的影响。蛋白激酶-C激活未模拟[Ca2+]i升高的作用。这些观察结果表明：1）[Ca2+]i升高或持续水平会损害胰岛素刺激的葡萄糖摄取；2）Ca2+诱导的损害似乎存在于胰岛素作用的结合后步骤，可能会干扰胰岛素受体的TKA和葡萄糖转运蛋白的内在活性。
