Studies on insulin receptor Part 2 Effect of insulin antibodies on insulin receptors

Radioreceptorassay using particulate membrane of human placenta, liver and kidney of guinea pigs were carried out to investigate the effect of insulin antibodies on insulin receptors. (125)I-insulin binding to insulin receptors at 24℃ increased slowly, binding in an apparent steady-state was reached within 1 h and continued for 3 h. (125)I-insulin binding to insulin antibodies at 24℃ increased rapidly, binding in an apparent steady-state was reached within 0.5 h and continued for 3 h. (125)I-insulin incubated with buffer at 24℃ was not degraded until 2.5 h, but (125)I-insulin incubated with 300μg of particulate membrane of human placenta at 24℃ was degraded and its degradation rate was 37.5% at 1 h, 50.9% at 2 h, 60.4% at 3 h. The degradation rate of particulate membrane of human placenta in the same condition was 4.3% at 0.5 h, 1.8% at 1 h, 19.6% at 2 h. After these studies, the following four experiments were performed. Group 1: (125)I-insulin was preincubated with insulin antibodies for 90 min., then insulin receptor was added to the incubation material. The mixture was incubated for 120 min.. Group 2: (125)I-insulin, insulin antibodies and insulin receptor were incubated together for 120 min.. Group 3: (125)I-insulin was preincubated with insulin receptor for 120 min., and insulin antibodies were added to the incubation material. The mixture was incubated for 90min.. Group 4: Insulin antibodies were preincubated with insulin receptor for 30 min., then the mixture was washed once, (125)I-insulin was added, and the mixture was incubated for 120 min.. (125)I-insulin binding to insulin receptor was inhibited in group 1, group 2, and group 4, but was not inhibited in group 3. The inhibition rate correlated positively with the titers of insulin antibodies. Different insulin target organs: human placental insulin receptor, and hepatic and renal insulin receptors of guinea pigs showed different inhibition rates. Insulin antibodies obtained in the insulin resistant period showed the most inhibition, but insulin antibodies from a patient with insulin resistant diabetes in the remissive state and from a patient with steroid induced diabetes showed decreased inhibition. The dissociation rate of (125)I-insluin from the insulin receptor was increased by insulin antibodies. Scatchard analysis showed that affinity constants of insulin antibodies (high affinity-low capacity component (K(1)), low affinity-high capacity component (K(2)) and average affinity constant (K(e)) in the insulin resistant period) were 1.810×10(9) M(-1) (K(1)), 0.046×10(9) M(-1) (K2), 0.083×10(9) M(-1) (K(e)), respectively, and K(1), K(2) and K(e) of insulin antibodies in the remissive state of the insulin resistant diabetic were 0.542×10(9) M(-1), 0.025×10(9) M(-1), 0.060×10(9) M(-1), respectively. K(1), K(2) and K(e) of insulin receptor in the presence of insulin antibodies were 0.600×10(9) M(-1), 0.132×10(9) M(-1), 0.176×10(9) M(-1), respectively. The numbers of insulin receptors decreased in the presence of insulin antibodies. The inhibition rate is thought to be influenced by affinity constants as well as titers of insulin antibodies. Comparing the affinity constants of insulin receptors and insulin antibodies in the insulin resistant period, the ratio of K(2) of insulin receptor to K(1) of insulin antibodies was 1:8.30 and the ratio of K(1) of insulin receptor to K(2) of insulin antibodies was 1:0.058. Comparing with affinity constants of insulin receptors and insulin antibodies in the remissive state, the ratio of K(2) of insulin receptor to K(1) of insulin antibodies was 1:2.49 and the ratio of K(1) of insulin receptor to K(2) of insulin antibodies was 1:0.032. Therefore binding in-hibition of (125)I-insulin to insulin receptor is thought to be caused mainly by insulin antibodies of high affinity-low capacity component.