Solomon S S, Mishra S K, Palazzolo M R, Postlethwaite A E, Seyer J M
Department of Research, Veterans Affairs Medical Center and the University of Tennessee, Memphis 38104, USA.
J Lab Clin Med. 1997 Aug;130(2):139-46. doi: 10.1016/s0022-2143(97)90090-1.
Data from a number of laboratories support a potential role for tumor necrosis factor-alpha (TNF-alpha) in the loss of insulin sensitivity and the pathogenesis of insulin resistance (IR) in diabetic animal models and human patients. We designed experiments to establish a dose-response relationship for TNF-alpha and IR in H-411E cells in culture. IR was measured by inhibition of the ability of graded amounts of insulin to stimulate expression of calmodulin (CaM) mRNA in these cells. This was assessed by autoradiographs of Northern blot(s) of CaM mRNA probed with labeled oligonucleotide cDNA for rat CaM. We found that TNF-alpha at 0.1, 1.0, and 10.0 ng/ml opposed 10,000 microU/ml insulin (i.e., %IR = 20%, 67%, and 88%, respectively). At 1.0 ng/ml TNF-alpha, insulin at the concentration of 1000 microU/ml (0.006 micromol/L) stimulated CaM mRNA at a 41% level and at 10,000 microU/ml (0.06 micromol/L) at a 63% level. Furthermore, oligopeptide TNF-alpha homologs (at 1000 x the molar concentration of TNF-alpha) TNF-alpha 69-100 and TNF-alpha 133-157 conferred 66% and 101% IR, respectively, while all other peptide fragments of TNF-alpha were essentially without effect. Studies done with both monoclonal and polyclonal antibodies to the TNF-alpha receptor demonstrated blocking activity by polyclonal but not by monoclonal anti TNF-alpha receptor antibody. This supports the concept that the activity of the peptide fragments occurs through the TNF-alpha receptor and not through nonspecific translocation across the plasma membrane. These data suggest that the epitopes on TNF-alpha that mediate IR reside in two regions of the molecule spanning amino acid residues 69-100 and 133-157.
来自多个实验室的数据表明，在糖尿病动物模型和人类患者中，肿瘤坏死因子-α（TNF-α）在胰岛素敏感性丧失及胰岛素抵抗（IR）发病机制中可能发挥作用。我们设计实验以确定培养的H-411E细胞中TNF-α与IR之间的剂量反应关系。通过抑制不同剂量胰岛素刺激这些细胞中钙调蛋白（CaM）mRNA表达的能力来测量IR。这通过用标记的大鼠CaM寡核苷酸cDNA探针检测CaM mRNA的Northern印迹放射自显影片进行评估。我们发现，0.1、1.0和10.0 ng/ml的TNF-α可对抗10,000 μU/ml胰岛素（即，IR百分比分别为20%、67%和88%）。在1.0 ng/ml TNF-α存在时，1000 μU/ml（0.006 μmol/L）浓度的胰岛素刺激CaM mRNA的水平为41%，10,000 μU/ml（0.06 μmol/L）时为63%。此外，TNF-α寡肽同系物（TNF-α摩尔浓度的1000倍）TNF-α 69-100和TNF-α 133-157分别导致66%和101%的IR，而TNF-α的所有其他肽片段基本无作用。用抗TNF-α受体的单克隆和多克隆抗体进行的研究表明，多克隆抗体具有阻断活性，而单克隆抗TNF-α受体抗体则没有。这支持了肽片段的活性是通过TNF-α受体而非通过非特异性跨质膜转运发生的概念。这些数据表明，TNF-α上介导IR的表位位于分子的两个区域，即跨越氨基酸残基69-100和133-157的区域。
