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

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Abstract
Summary: 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): < 1; proinsulin: 5; guinea‐pig insulin: 2; hCG, ovine prolactin and bovine GH: O. High and low affinity binding sites for insulin were identified on purified Leydig cells with Ka values of 1.2 × 109 and 0.3 × 108 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.