Characterization of insulin binding and comparative action of insulin and insulin-like growth factor I on purified Leydig cells from the adult rat.

Insulin binding and insulin action were characterized in adult rat Leydig cells, purified on discontinuous Percoll gradients. Binding of [125I]-porcine insulin was found to be dependent on time, temperature, cell concentration and Leydig cell specific gravity. Competition relative to porcine insulin (100) was as follows: insulin-like growth factor I (IGF-I) : less than 1; proinsulin : 5; guinea-pig insulin : 2; hCG, ovine prolactin and bovine GH : 0. High and low affinity binding sites for insulin were identified on purified Leydig cells with Ka values of 1.2 X 10(9) and 0.3 X 10(8) M-1, with 10,300 and 34,000 binding sites per cells, respectively. Using primary cultures of Leydig cells in serum-free medium, the action of insulin on steroidogenesis was studied and compared with IGF-I action. Insulin and IGF-I used at 1-35 nM enhanced basal testosterone production in a dose-dependent manner; the effect was significant 4 h after administration. Insulin or IGF-I also potentiated the effect of hCG on steroidogenesis during short-term incubation (4 h). Insulin was shown to improve hCG responsiveness without modifying sensitivity to hCG. Moreover, neither cell number nor hCG-binding was altered by insulin, IGF-I or a combination of the two. Concomitant treatment with insulin and IGF-I at half-maximal and maximally effective doses, in the presence or absence of hCG, indicated that the two factors synergized in the stimulation of testosterone production via a common saturable mechanism.