Begum N, Ragolia L, Srinivasan M
Diabetes Research Laboratory, Winthrop University Hospital, Mineola, NY 11501, USA.
Eur J Biochem. 1996 May 15;238(1):214-20. doi: 10.1111/j.1432-1033.1996.0214q.x.
Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
肿瘤坏死因子-α（TNF-α）被认为是肥胖/糖尿病动物胰岛素抵抗的介质，它通过影响胰岛素受体及其底物胰岛素受体底物-1的酪氨酸磷酸化发挥作用。在本研究中，我们检测了TNF-α对培养的大鼠骨骼肌细胞系L6中丝裂原活化蛋白激酶（MAPK）信号级联反应的急性影响。用胰岛素处理L6细胞导致MAPK活性迅速增加（10 nM胰岛素处理5分钟后增加两倍以上）。预先用TNF-α处理60分钟会以剂量和时间依赖的方式阻断随后胰岛素诱导的MAPK活化。用无机[32P]磷酸盐进行代谢标记研究，随后对MAPK及其上游激活剂丝裂原活化蛋白激酶激酶进行免疫沉淀，结果表明在暴露于TNF-α的细胞中，胰岛素刺激后MAPK及其激酶的磷酸化水平降低。TNF-α的这种作用并非由于抑制胰岛素刺激的p21ras-GTP负载或Raf-1磷酸化。低浓度（2 nM）的冈田酸（一种丝氨酸/苏氨酸磷酸酶抑制剂）可防止TNF-α诱导的MAPK抑制，并恢复胰岛素对MAPK活性的影响，而原钒酸盐（一种酪氨酸磷酸酶抑制剂）、抑制剂2（磷酸酶-1抑制剂）和FK506（磷酸酶-2B抑制剂）则无效。这些结果表明，一种对冈田酸敏感的丝氨酸/苏氨酸磷酸酶参与了TNF-α诱导的胰岛素对MAPK及其激酶作用的阻断。因此，我们检测了TNF-α对蛋白磷酸酶-1（PP-1）和蛋白磷酸酶-2A（PP-2A）活性的影响。正如我们之前报道的，胰岛素在对照细胞中迅速刺激PP-1并同时抑制PP-2A活性。TNF-α处理阻断了胰岛素诱导的PP-1活化。与PP-1相反，TNF-α使PP-2A活性增加60%，胰岛素无法阻止TNF-α的这种作用。TNF-α激活PP-2A的时间进程先于MAPK抑制的动力学过程。细胞可渗透的神经酰胺类似物模拟了TNF-α对MAPK抑制和PP-2A激活的作用。我们得出结论，TNF-α通过活化的磷酸酶增加MAPK激酶的去磷酸化，从而消除胰岛素对MAPK活化的作用。
