CHARACTERIZATION OF INSULIN AND INSULIN-LIKE GROWTH FACTOR-I RECEPTORS OF PURIFIED LEYDIG-CELLS AND THEIR ROLE IN STEROIDOGENESIS IN PRIMARY CULTURE - A COMPARATIVE-STUDY
CHARACTERIZATION OF INSULIN AND INSULIN-LIKE GROWTH FACTOR-I RECEPTORS OF PURIFIED LEYDIG-CELLS AND THEIR ROLE IN STEROIDOGENESIS IN PRIMARY CULTURE - A COMPARATIVE-STUDY
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DOI：
10.1210/endo-119-4-1641
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发表时间：
1986-10-01
期刊：
影响因子：
4.8
通讯作者：
TERRACIO, L
中科院分区：
文献类型：
--
作者：
LIN, T;HASKELL, J;TERRACIO, L
用纯化的成年SD大鼠Leydig细胞评价胰岛素和I型胰岛素样生长因子（IGF-I）受体的特征以及胰岛素和IGF-I对类固醇生成的影响。发现纯化的Leydig细胞含有对胰岛素的高和低亲和力结合位点，Ka值为1.08 × 10 - 6。109和1.1 ×107 M-1，分别。使用[125 I]碘胰岛素与质膜胰岛素受体的亲和交联，在非还原条件下通过放射自显影法鉴定了几条带，分子量为230，000、280，000和300，000。在用50 mM二硫苏糖醇还原后，仅鉴定出一条分子量为130，000的条带，与α-二硫苏糖醇的分子量一致。胰岛素受体亚单位。纯化的Leydig细胞还含有特异性I型IGF受体，其估计结合亲和力为0.6 × 10 - 6。109 M-1在非还原条件下通过亲和交联鉴定了多个高分子量条带（> 250，000）。在还原条件下，鉴定出一条分子量约为135，000的条带。将纯化的Leydig细胞（105个细胞/ml）在含有0.1%胎牛血清的Dulbecco ′ s Modified Spille ′ s Medium-Ham ′ s F-12营养混合物（1：1）中于37 ℃培养。在5%CO2 -95%空气的潮湿气氛中。胰岛素和IGF-I早在给药后3小时就刺激睾酮形成，并且它们的作用被加入蛋白质合成抑制剂放线菌酮（1 μ g/ml）完全阻断。胰岛素和IGF-I也显著增强hCG和8-溴-cAMP诱导的睾酮形成。此外，胰岛素和IGF-I增强hCG刺激的cAMP形成。这表明胰岛素和IGF-I在LH受体位点和腺苷酸环化酶以外的步骤都有作用。胰岛素和IGF-I刺激的睾酮形成的ED 50值相当（25 ng/ml）。总之，我们发现Leydig细胞含有特异性胰岛素和I型IGF受体，并且胰岛素和IGF-I都能够调节Leydig细胞类固醇生成。
Characteriation of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis was evaluated by using purified adult Leydig cells from Sprague-Dawley rats. Purified Leydig cells were found to contain both high and low affinity binding sites for insulin, with Ka values of 1.08 .times. 109 and 1.1 .times. 107 M-1, respectively. Using affinity cross-linking of [125I]iodoinsulin to plasma membrane insulin receptor, several bands were identified by autoradiography under nonreduced conditions with mol wt of 230,000, 280,000, and 300,000. After reduction with 50 mM dithiothreitol, only one band was identified with a mol wt of 130,000, consistent with the .alpha.-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 .times. 109 M-1. Multiple high mol wt bands (> 250,000) were identified under nonreduced conditions by affinity cross-linking. Under reduced conditions, one band with an approximate mol wt of 135,000 was identified Purified Leydig cells (105 cells/ml) were cultured in Dulbecco''s Modified Eagle''s Medium-Ham''s F-12 Nutrient Mixture (1:1) containing 0.1% fetal calf serum at 37.degree. C in a humidified atmosphere of 5% CO2-95% air. Insulin and IGF-I stimulated testosterone formation as early as 3 h after administration, and their effects were completely blocked by the addition of a protein synthesis inhibitor, cycloheximide (1 .mu.g/ml). Insulin and IGF-I also significantly potentiated hCG- and 8-bromo-cAMP-induced testosterone formation. Furthermore, insulin and IGF-I potentiated hCG-stimulated cAMP formation. This suggests that insulin and IGF-I have effects at both the LH receptor sites and the steps beyond adenylate cyclase. The ED50 values of insulin and IGF-I-stmulated testosterone formation were comparable (25 ng/ml). In conclusion, we found that Leydig cells contain specific insulin and type I IGF receptors, and both insulin and IGF-I are capable of modulating Leydig cell steroidogenesis.