Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: Role of oxidative stress
Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: Role of oxidative stress
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DOI：
10.1124/jpet.104.066522
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发表时间：
2004-08-01
影响因子：
3.5
通讯作者：
Novak, RF
中科院分区：
文献类型：
--
作者：
Abdelmegeed, MA;Kim, SK;Novak, RF
糖尿病的特征是酮体乙酰乙酸酯(AA)和3-羟基丁酸酯(3HB)水平升高。高水平的酮体与细胞氧化应激的产生有关。然而，与氧化应激相关的细胞信号通路的酮体激活还没有建立起来。因此，我们研究了酮体对原代培养的大鼠肝细胞中激酶活性的影响。AA处理增加了细胞外信号调节蛋白1/2(ERK1/2)和p38丝裂原活化蛋白激酶(MAPK)的磷酸化，最大分别增加了2.5倍和4倍。AA不能激活c-jun氨基末端激酶。AA介导的ERK1/2和p38MAPK在处理后3h即可被激活，12h达到最大激活，而3HB则未能激活其中的任何一种。AA也使Raf和MKK3/6的磷酸化水平升高。蛋白激酶C(PKC)抑制剂双吲哚马来酰亚胺和RAS法尼化抑制剂B581可抑制AA介导的ERK1/2和p38MAPK的激活，提示PKC和RAS在介导ERK1/2和p38MAPK激活中起作用。有趣的是，酪氨酸激酶抑制剂Genistein阻止了AA介导的ERK1/2的磷酸化，但不能阻止p38MAPK的磷酸化。AA处理导致细胞内活性氧(ROS)的产生和细胞内谷胱甘肽水平的降低，而抗氧化剂N-乙酰-L-半胱氨酸(N-Acetyl-L-cysteine，NAc)和特罗克斯(6-hydroxy-2，5，7，8-tetramethylchroman-2-carboxylic酸(Trolox)可以缓解这一现象。NAC和Trolox还改善了AA介导的ERK1/2和p38MAPK的激活，表明这种激活与ROS和氧化应激有关。
Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by similar to2.5- and 4-fold, respectively. AA failed to activate c-Jun NH2-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-L-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.