Imoto Koujiro, Kukidome Daisuke, Nishikawa Takeshi, Matsuhisa Takako, Sonoda Kazuhiro, Fujisawa Kazuo, Yano Miyuki, Motoshima Hiroyuki, Taguchi Tetsuya, Tsuruzoe Kaku, Matsumura Takeshi, Ichijo Hidenori, Araki Eiichi
Department of Metabolic Medicine, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
Diabetes. 2006 May;55(5):1197-204. doi: 10.2337/db05-1187.
Tumor necrosis factor (TNF)-alpha inhibits insulin action; however, the precise mechanisms are unknown. It was reported that TNF-alpha could increase mitochondrial reactive oxygen species (ROS) production, and apoptosis signal-regulating kinase 1 (ASK1) was reported to be required for TNF-alpha-induced apoptosis. Here, we examined roles of mitochondrial ROS and ASK1 in TNF-alpha-induced impaired insulin signaling in cultured human hepatoma (Huh7) cells. Using reduced MitoTracker Red probe, we confirmed that TNF-alpha increased mitochondrial ROS production, which was suppressed by overexpression of either uncoupling protein-1 (UCP)-1 or manganese superoxide dismutase (MnSOD). TNF-alpha significantly activated ASK1, increased serine phosphorylation of insulin receptor substrate (IRS)-1, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, and all of these effects were inhibited by overexpression of either UCP-1 or MnSOD. Similar to TNF-alpha, overexpression of wild-type ASK1 increased serine phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation of IRS-1, whereas overexpression of dominant-negative ASK1 ameliorated these TNF-alpha-induced events. In addition, TNF-alpha activated c-jun NH(2)-terminal kinases (JNKs), and this observation was partially inhibited by overexpression of UCP-1, MnSOD, or dominant-negative ASK1. These results suggest that TNF-alpha increases mitochondrial ROS and activates ASK1 in Huh7 cells and that these TNF-alpha-induced phenomena contribute, at least in part, to impaired insulin signaling.
肿瘤坏死因子（TNF）-α可抑制胰岛素作用，但其确切机制尚不清楚。据报道，TNF-α可增加线粒体活性氧（ROS）的产生，且凋亡信号调节激酶1（ASK1）被认为是TNF-α诱导凋亡所必需的。在此，我们研究了线粒体ROS和ASK1在TNF-α诱导的培养人肝癌（Huh7）细胞胰岛素信号受损中的作用。使用还原型MitoTracker Red探针，我们证实TNF-α增加了线粒体ROS的产生，而解偶联蛋白-1（UCP）-1或锰超氧化物歧化酶（MnSOD）的过表达可抑制这种增加。TNF-α显著激活ASK1，增加胰岛素受体底物（IRS）-1的丝氨酸磷酸化，并降低胰岛素刺激的IRS-1酪氨酸磷酸化和Akt的丝氨酸磷酸化，而UCP-1或MnSOD的过表达均可抑制所有这些效应。与TNF-α相似，野生型ASK1的过表达增加了IRS-1的丝氨酸磷酸化并降低了胰岛素刺激的IRS-1酪氨酸磷酸化，而显性负性ASK1的过表达则改善了这些TNF-α诱导的事件。此外，TNF-α激活了c-jun氨基末端激酶（JNKs），而UCP-1、MnSOD或显性负性ASK1的过表达可部分抑制这一现象。这些结果表明，TNF-α增加了Huh7细胞中的线粒体ROS并激活了ASK1，且这些TNF-α诱导的现象至少部分导致了胰岛素信号受损。
