Shisheva A, Shechter Y
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1993 Mar 25;268(9):6463-9.
In previous studies we have identified a cytosolic protein tyrosine kinase (CytPTK) in rat adipocytes that is largely activated in vanadate-pretreated cells (Shisheva, A., and Shechter, Y. (1992) FEBS Lett. 300, 93-96). We report here that staurosporine and its analog K-252a are highly potent (ID50 = 3 and 100 nM, respectively) in inhibiting CytPTK activity of crude cell extract or partially purified enzyme preparations. Staurosporine and K-252a were less effective by more than 2 and 1 orders of magnitude, respectively, in inhibiting insulin receptor-catalyzed PolyGlu4Tyr phosphorylation in cell-free experiments. Preincubation of rat adipocytes with either staurosporine or K-252a selectively blocked the action of vanadate in activating glucose incorporation into lipids and its oxidation. Thus, staurosporine inhibited vanadate-stimulated lipogenesis and glucose oxidation (via glycolysis and the pentose phosphate pathway) in a concentration-dependent manner with ID50 of 75 and 300 nM, respectively. Insulin-stimulated bioeffects were not inhibited at this low range of staurosporine concentration. Staurosporine had no effect on vanadate-stimulated hexose uptake or on vanadate's antilipolytic action. Using staurosporine, we probed those insulinomimetic agents which facilitate their biological activity via the insulin receptor kinase (insulin, wheat germ agglutinin, concanavalin A, and pervanadate) or via CytPTK (vanadate and to a certain degree Mn2+ and Zn2+). These results suggest that (a) vanadate facilitates its insulin-like actions on glucose utilization via the cytosolic tyrosine kinase and (b) this enzyme does not participate in vanadate effects in stimulating hexose uptake and in inhibiting lipolysis. These findings explain further vanadate's post-insulin receptor actions and raise possible application in the management of glucose metabolism in insulin-independent fashion in pathological conditions.
在先前的研究中，我们在大鼠脂肪细胞中鉴定出一种胞质蛋白酪氨酸激酶（CytPTK），该激酶在经钒酸盐预处理的细胞中被大量激活（希舍娃，A.，和谢克特，Y.（1992年）《欧洲生物化学学会联合会快报》300，93 - 96）。我们在此报告，星形孢菌素及其类似物K - 252a在抑制粗细胞提取物或部分纯化的酶制剂的CytPTK活性方面具有高效性（ID50分别为3和100 nM）。在无细胞实验中，星形孢菌素和K - 252a在抑制胰岛素受体催化的聚谷氨酸4酪氨酸磷酸化方面的效果分别降低了超过2个和1个数量级。用星形孢菌素或K - 252a对大鼠脂肪细胞进行预孵育，可选择性地阻断钒酸盐在激活葡萄糖掺入脂质及其氧化方面的作用。因此，星形孢菌素以浓度依赖性方式抑制钒酸盐刺激的脂肪生成和葡萄糖氧化（通过糖酵解和磷酸戊糖途径），ID50分别为75和300 nM。在星形孢菌素的这一低浓度范围内，胰岛素刺激的生物效应未受到抑制。星形孢菌素对钒酸盐刺激的己糖摄取或钒酸盐的抗脂解作用没有影响。我们使用星形孢菌素探究了那些通过胰岛素受体激酶（胰岛素、麦胚凝集素、伴刀豆球蛋白A和过钒酸盐）或通过CytPTK（钒酸盐以及在一定程度上的Mn2 +和Zn2 +）来促进其生物活性的胰岛素模拟剂。这些结果表明：（a）钒酸盐通过胞质酪氨酸激酶促进其对葡萄糖利用的胰岛素样作用；（b）这种酶不参与钒酸盐在刺激己糖摄取和抑制脂解方面的作用。这些发现进一步解释了钒酸盐在胰岛素受体后的作用，并提出了在病理条件下以不依赖胰岛素的方式管理葡萄糖代谢的可能应用。
