PDF(Pubmed)
Abstract：
Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.
摘要：
分化诱导因子1（DIF-1），在盘基网柄菌中发现,在哺乳动物细胞中具有抗增殖和促进葡萄糖摄取的活性。DIF-1是开发抗肿瘤和/或抗肥胖/抗糖尿病药物的潜在线索，但是其行动背后的机制尚未完全阐明。在这项研究中,我们通过使用亲和层析和液相色谱-串联质谱法鉴定人宫颈癌HeLa细胞和小鼠3T3-L1成纤维细胞中的DIF-1结合蛋白,搜索了在哺乳动物细胞中介导DIF-1作用的DIF-1靶分子,并在两种细胞系中发现线粒体苹果酸脱氢酶(MDH2)是DIF-1结合蛋白.由于DIF-1已被证明直接抑制MDH2活性，我们在体外比较了DIF-1和MDH2抑制剂LW6对HeLa和3T3-L1细胞生长以及对融合的3T3-L1细胞葡萄糖摄取的影响.在HeLa和3T3-L1细胞中，DIF-1在10-40μM剂量依赖性地抑制生长，而LW6在20μM，但不是2-10μM，显著抑制这些细胞的生长。在汇合的3T3-L1细胞中，10-40μM的DIF-1显著促进葡萄糖摄取，在20μMDIF-1时作用最强，而LW6在2-20μM时显着促进葡萄糖摄取，在10μMLW6处具有最强的效果。蛋白质印迹分析显示LW6(10μM)和DIF-1(20μM)磷酸化,因此，激活的AMP激酶在3T3-L1细胞。我们的结果表明，MDH2抑制可以抑制细胞生长并促进细胞中的葡萄糖摄取。但似乎比抑制细胞生长更强烈地促进葡萄糖摄取。因此，DIF-1可能促进葡萄糖摄取，至少在某种程度上,通过直接抑制MDH2和随后激活3T3-L1细胞中的AMP激酶。
