Wu Shi-Bei, Wei Yau-Huei
Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan.
Biochim Biophys Acta. 2012 Feb;1822(2):233-47. doi: 10.1016/j.bbadis.2011.09.014. Epub 2011 Oct 6.
We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.
我们报告称，在患有肌阵挛性癫痫伴破碎红纤维综合征（MERRF）患者的皮肤成纤维细胞原代培养物中，能量代谢转变为无氧糖酵解，作为对氧化应激的适应性反应。为了阐明氧化应激下能量代谢改变所涉及的分子机制，我们用亚致死剂量的过氧化氢（H₂O₂）处理正常人皮肤成纤维细胞（CCD - 966SK细胞）。结果显示，在H₂O₂处理的正常人皮肤成纤维细胞中，包括己糖激酶II型（HK II）、乳酸脱氢酶（LDH）和葡萄糖转运蛋白1（GLUT1）在内的几种糖酵解酶上调。此外，H₂O₂通过激活AMP激活的蛋白激酶（AMPK）及其下游靶点磷酸果糖激酶2（PFK2）的磷酸化，以剂量依赖性方式增加皮肤成纤维细胞的糖酵解通量。而且，我们发现H₂O₂通过AMPK介导的糖酵解通量增加伴随着细胞内NADPH含量的增加。分别用糖酵解抑制剂、AMPK抑制剂处理细胞或对AMPK进行基因敲低后，H₂O₂诱导的NADPH增加被消除，导致细胞内活性氧（ROS）过量产生和细胞死亡。值得注意的是，我们发现MERRF皮肤成纤维细胞中AMPK和糖酵解的磷酸化水平上调，以赋予生存优势。综上所述，我们的研究结果表明，AMPK介导的糖酵解通量增加导致的NADPH产量增加有助于MERRF皮肤成纤维细胞和H₂O₂处理的正常人皮肤成纤维细胞适应氧化应激。
