Fernández-Veledo Sonia, Hernandez Rosario, Teruel Teresa, Mas Jose Antonio, Ros Manuel, Lorenzo Margarita
Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad Complutense, 28040, Madrid, Spain.
Arch Physiol Biochem. 2006 Feb;112(1):13-22. doi: 10.1080/13813450500508137.
Tumour necrosis factor (TNF)-alpha impaired insulin induction on GLUT4 mRNA in foetal brown adipocytes, as demonstrated by quantitative RT-PCR and Northern blot. We have explored the hypothesis that some effects of TNF-alpha could be mediated by the generation of ceramide, since TNF-alpha treatment induced the production of ceramide in these primary cells. A short-chain ceramide analogue, C2-ceramide, precluded insulin-induced GLUT4 mRNA accumulation and GLUT4-chloramphenicol acetyltransferase (CAT) full promoter activation. Moreover, inhibition of the ceramide biosynthesis with fumonisin B, which inhibits ceramide synthase, completely restored insulin-induced GLUT4 mRNA and protein accumulation as well as GLUT4-CAT transactivation in the presence of TNF-alpha. In consequence, TNF-alpha-induced insulin resistance on glucose uptake was completely alleviated. In addition, TNF-alpha down-regulated insulin-induced CCAAT/enhancer binding protein (C/EBP)-alpha gene expression and DNA binding activity, but fumonisin B precludes these effects. Furthermore, co-transfection with a wild-type C/EBP-alpha construct transactivates GLUT4-CAT construct. Our results indicate that de novo ceramide produced by TNF-alpha-induced insulin resistance on GLUT4 gene expression in brown adipocytes by interfering C/EBP-alpha expression, a transcription factor essential for the expression of GLUT4.
定量逆转录聚合酶链反应（RT-PCR）和Northern印迹法表明，肿瘤坏死因子（TNF）-α可损害胎儿棕色脂肪细胞中胰岛素对葡萄糖转运蛋白4（GLUT4）信使核糖核酸（mRNA）的诱导作用。我们探讨了一种假说，即TNF-α的某些作用可能由神经酰胺的生成介导，因为TNF-α处理可诱导这些原代细胞中神经酰胺的产生。一种短链神经酰胺类似物C2-神经酰胺可阻止胰岛素诱导的GLUT4 mRNA积累以及GLUT4-氯霉素乙酰转移酶（CAT）全启动子激活。此外，用伏马菌素B抑制神经酰胺生物合成（伏马菌素B可抑制神经酰胺合酶），在存在TNF-α的情况下，可完全恢复胰岛素诱导的GLUT4 mRNA和蛋白积累以及GLUT4-CAT反式激活。因此，TNF-α诱导的葡萄糖摄取胰岛素抵抗得到完全缓解。此外，TNF-α下调胰岛素诱导的CCAAT/增强子结合蛋白（C/EBP）-α基因表达和DNA结合活性，但伏马菌素B可阻止这些作用。此外，与野生型C/EBP-α构建体共转染可反式激活GLUT4-CAT构建体。我们的结果表明，TNF-α诱导产生的新生神经酰胺通过干扰C/EBP-α的表达，导致棕色脂肪细胞中GLUT4基因表达的胰岛素抵抗，而C/EBP-α是GLUT4表达所必需的转录因子。
