Identification of specific sites in the TNF-α molecule promoting insulin resistance in H-411E cells

Abstract

Data from a number of laboratories support a potential role for tumor necrosis factor-α (TNF-α) 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-α 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-α at 0.1, 1.0, and 10.0 ng/ml opposed 10,000 μU/ml insulin (i.e., %IR = 20%, 67%, and 88%, respectively). At 1.0 ng/ml TNF-α, insulin at the concentration of 1000 μU/ml (0.006 μmol/L) stimulated CaM mRNA at a 41% level and at 10,000 μU/ml (0.06 μmol/L) at a 63% level. Furthermore, oligopeptide TNF-α homologs (at 1000 × the molar concentration of TNF-α) TNF-α 69–100 and TNF-α 133–157 conferred 66% and 101% IR, respectively, while all other peptide fragments of TNF-α were essentially without effect. Studies done with both monoclonal and polyclonal antibodies to the TNF-α receptor demonstrated blocking activity by polyclonal but not by monoclonal anti TNF-α receptor antibody. This supports the concept that the activity of the peptide fragments occurs through the TNF-α receptor and not through nonspecific translocation across the plasma membrane. These data suggest that the epitopes on TNF-α that mediate IR reside in two regions of the molecule spanning amino acid residues 69–100 and 133–157.