Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation.

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.

Decreased insulin receptors but normal glucose metabolism in Duchenne muscular dystrophy.

Compared to matched controls, 17 patients with Duchenne muscular dystrophy showed decreased insulin binding to monocytes due to decreased receptor concentration. These patients showed no signs of altered glucose metabolism and retrospective analysis of the clinical records of a further 56 such patients revealed no modification in carbohydrate metabolism. These data suggest that reduced insulin receptor number does not produce overt modifications of glucose metabolism in Duchenne muscular dystrophy.

Binding properties of the insulin receptor of human placenta under different conditions of incubation.

The effect of incubation temperature and media on insulin receptor has been investigated employing membranes (4,000 x g pellet; particulate receptor) and solubilized material (solubilized receptor) from normal human placenta. Particulate receptor concentration (Ro) and affinity (Ke) were seen to be affected by buffer employed in binding experiments, both at 24 degrees C as well as at 4 degrees C; solubilized receptor affinity, on the other hand, is affected by buffer at 24 degrees C but not at 4 degrees C and receptor concentration does not vary at either temperature. Solubilization induced an increase in receptor affinity or in receptor concentration depending upon the buffer used in binding experiments. Insulin binding increases at the lowest incubation temperature; this phenomenon is due entirely to the greater receptor affinity on both particulate and solubilized receptor. From these data it appears that buffers induce variations in binding properties of the human placenta insulin receptor, thus, at ths present state of our knowledge, no definite conclusions can be drawn on the effect of procedures, such as solubilization, on human placenta insulin receptor affinity and/or concentration.

Erythrocyte insulin receptors in non-insulin-dependent diabetes mellitus.

Insulin binding was studied on circulating erythrocytes isolated from 12 normal and 12 untreated, adult, nonobese, nonketotic, non-insulin-dependent diabetic subjects. Insulin binding in normal subjects was higher than in diabetics (P less than 0.01); binding variation was caused mainly by a reduction in insulin receptor concentration. Insulin binding was inversely correlated with fasting serum insulin levels (R = 0.49; P less than 0.01). The close agreement between the present data and previous data on other cell populations suggests that isolated erythrocytes may be a useful tool in clinical investigations on the human insulin receptor.