Effects of anti-insulin antibody on insulin binding to liver membranes: evidence against antibody-induced enhancement of insulin binding to the insulin receptor

Abstract

In the presence of anti-insulin antibody, 2- to 3-fold enhancement of 125I-insulin binding to liver membranes was observed when binding was estimated by the radioactivity of 125I-insulin bound to the membrane pellets. However, after 125'I-insulin was covalently cross-linked to liver membranes using disuccinimidyl suberate in the presence of anti-insulin antibody, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that 125I-insulin bound to the α-subunit of the insulin receptor was inhibited by antiinsulin antibody in an dose-dependent manner. More importantly, at an anti-insulin antibody dilution range between 1:50 and 1:5,000, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two 125I-labelled bands of mol wt 62,000 and 27,000, while only one band of mol wt 130,000 was revealed in the absence of anti-insulin antibody. These Mr=62,000 and Mr=27,000 bands were found to be the heavy and the light chain of anti-insulin IgG molecules respectively. Pepsin digested anti-insulin serum had only an inhibitory effect on 125I-insulin binding to liver membranes. Non-immunized guinea pig serum or IgG completely abolished the enhanced effect of anti-insulin antibody. Further, this enhanced effect was inhibited by Fc fragment-specific anti-IgG serum or H&L-chain-specific anti-IgG serum in a dosedependent manner. Protein A also inhibited the effect of antiinsulin antibody. In IM-9 lymphocytes and human red blood cell ghosts, which have no Fcy receptors, enhancement of insulin binding was not observed in the presence of anti-insulin antibody. These data suggest that anti-insulin antibody-induced enhancement of insulin binding to liver membranes is not due to the enhanced binding to the insulin receptor itself but probably due to the binding of insulin-anti-insulin antibody complex to the Fcγ receptor. © 1986 Springer-Verlag.