Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [³H]thymidine incorporation were investigated. Insulin stimulated [³H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 μg/ml insulin, a secondary increase in [³H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [³H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [³H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [³H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alphaIR-3 interfered with the incremental increase in [³H]thymidine incorporation observed at 10-100 μg/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.