Sandu O A, Ragolia L, Begum N
Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA.
Diabetes. 2000 Dec;49(12):2178-89. doi: 10.2337/diabetes.49.12.2178.
Our laboratory has demonstrated that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity in vascular smooth muscle cells (VSMCs). In this study, we examined whether diabetes is accompanied by alterations in MBP activation and elucidated the components of the signaling pathway that mediate the effects of diabetes. VSMCs isolated from Goto-Kakizaki (GK) diabetic rats (a model for type 2 diabetes) exhibited marked impairment in MBP activation by insulin that was accompanied by failure of insulin to decrease the phosphorylation of a regulatory myosin-bound subunit (MBS) of MBP and inhibit Rho kinase activity resulting in increased myosin light-chain (MLC)20 phosphorylation and VSMC contraction. In VSMCs isolated from control rats, insulin inactivates Rho kinase and decreases MBS phosphorylation, leading to MBP activation. In addition to this pathway, insulin also appears to activate MBP by stimulating the phosphatidylinositol (PI) 3-kinase/nitric oxide (NO)/cGMP signaling pathway because treatment with the synthetic inhibitors of PI 3-kinase, NO synthase (NOS), and cGMP all blocked insulin's effect on MBP activation, whereas cGMP agonists and sodium nitroprusside (SNP) mimicked insulin's effect on MBP activation. VSMCs from diabetic GK rats exhibit reductions in insulin-mediated induction of inducible NOS protein expression and cGMP generation but normal MBP activation in response to SNP and cGMP agonist. This observation led us to examine the effect of diabetes on the activation status of the upstream insulin-signaling components. Although GK diabetes did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor or its content, insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was severely impaired. This was accompanied by marked reductions in IRS-1-associated PI 3-kinase activity. We conclude that insulin stimulates MBP via its regulatory subunit, MBS, by inactivating Rho kinase and stimulating NO/cGMP signaling via PI 3-kinase as part of a complex signaling network that controls MLC20 phosphorylation and VSMC contraction. Defective signaling via Rho kinase and the IRS-1/PI 3-kinase/NOS/cGMP pathway may mediate the inhibitory effects of hyperglycemia and diabetes on MBP activation in this experimental model.
我们实验室已证明，胰岛素可迅速刺激血管平滑肌细胞（VSMC）中肌球蛋白结合磷酸酶（MBP）的活性。在本研究中，我们检测了糖尿病是否伴有MBP激活的改变，并阐明了介导糖尿病作用的信号通路的组成成分。从Goto-Kakizaki（GK）糖尿病大鼠（2型糖尿病模型）分离的VSMC表现出胰岛素对MBP激活的显著损伤，同时胰岛素无法降低MBP调节性肌球蛋白结合亚基（MBS）的磷酸化水平，也无法抑制Rho激酶活性，导致肌球蛋白轻链（MLC）20磷酸化增加和VSMC收缩。在从对照大鼠分离的VSMC中，胰岛素使Rho激酶失活并降低MBS磷酸化，从而导致MBP激活。除了这条通路外，胰岛素似乎还通过刺激磷脂酰肌醇（PI）3激酶/一氧化氮（NO）/环鸟苷酸（cGMP）信号通路来激活MBP，因为用PI 3激酶、NO合酶（NOS）和cGMP的合成抑制剂处理均阻断了胰岛素对MBP激活的作用，而cGMP激动剂和硝普钠（SNP）模拟了胰岛素对MBP激活的作用。来自糖尿病GK大鼠的VSMC表现出胰岛素介导的诱导型NOS蛋白表达和cGMP生成的减少，但对SNP和cGMP激动剂的反应中MBP激活正常。这一观察结果促使我们研究糖尿病对上游胰岛素信号成分激活状态的影响。尽管GK糖尿病不影响胰岛素刺激的胰岛素受体酪氨酸磷酸化或其含量，但胰岛素刺激的胰岛素受体底物（IRS）-1酪氨酸磷酸化严重受损。这伴随着IRS-1相关PI 3激酶活性的显著降低。我们得出结论，胰岛素通过使其调节亚基MBS失活并通过PI 3激酶刺激NO/cGMP信号通路，经由其调节亚基MBS刺激MBP，这是控制MLC20磷酸化和VSMC收缩的复杂信号网络的一部分。在这个实验模型中，经由Rho激酶和IRS-1/PI 3激酶/NOS/cGMP通路的信号缺陷可能介导了高血糖和糖尿病对MBP激活的抑制作用。
