IDENTIFICATION OF SPECIFIC SITES IN THE TNF-ALPHA MOLECULE PROMOTING INSULIN-RESISTANCE IN H-411E CELLS

Data from a number of laboratories support a potential role for tumor necrosis factor-alpha (TNF-alpha) in the loss of insulin sensitivity a nd the pathogenesis of insulin resistance (IR) in diabetic animal mode ls and human patients. We designed experiments to establish a dose-res ponse relationship for TNF-alpha and IR in H-411E cells in culture. IR was measured by inhibition of the ability of graded amounts of insuli n to stimulate expression of calmodulin (CaM) mRNA in these cells. Thi s was assessed by autoradiographs of Northern blot(s) of CaM mRNA prob ed with labeled oligonucleotide cDNA for rat CaM. We found that TNF-al pha at 0.1, 1.0, and 10.0 ng/ml opposed 10,000 mu U/ml insulin (i.e., %IR = 20%, 67%, and 88%, respectively). At 1.0 ng/ml TNF-alpha, insuli n at the concentration of 1000 mu U/ml (0.006 mu mol/L) stimulated CaM mRNA at a 41% level and at 10,000 mu U/ml (0.06 mu mol/L) at a 63% le vel. Furthermore, oligopeptide TNF-cx homologs (at 1000 x the molar co ncentration of TNF-alpha) TNF-alpha 69-100 and TNF-alpha 133-157 confe rred 66% and 101% IR, respectively, while all other peptide fragments of TNF-alpha were essentially without effect. Studies done with both m onoclonal and polyclonal antibodies to the TNF-alpha receptor demonstr ated blocking activity by polyclonal but not by monoclonal anti TNF-al pha receptor antibody. This supports the concept that the activity of the peptide fragments occurs through the TNF-alpha receptor and not th rough 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 133-15 7.