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

T. Lin, J. Haskell, N. Vinson, Louis Terracio

Research output: Contribution to journalArticle

Abstract

Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were 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 K(a) values of 1.08 x 109 and 1.1 x 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 α-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 x 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 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 μg/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-stimulated 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.

Original languageEnglish (US)
Pages (from-to)1641-1647
Number of pages7
JournalEndocrinology
Volume119
Issue number4
StatePublished - 1986

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IGF Type 1 Receptor
Leydig Cells
Insulin
Insulin-Like Growth Factor I
Testosterone
Insulin Receptor
8-Bromo Cyclic Adenosine Monophosphate
LH Receptors
Eagles
Protein Synthesis Inhibitors
Dithiothreitol
Cycloheximide
Autoradiography
Atmosphere
Adenylyl Cyclases
Sprague Dawley Rats
Binding Sites
Air
Cell Membrane
Food

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{d2fe3eaa0a0444ad9e26f350eb1095c9,
title = "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",
abstract = "Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were 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 K(a) values of 1.08 x 109 and 1.1 x 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 α-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 x 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 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 μg/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-stimulated 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.",
author = "T. Lin and J. Haskell and N. Vinson and Louis Terracio",
year = "1986",
language = "English (US)",
volume = "119",
pages = "1641--1647",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "4",

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TY - JOUR

T1 - Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture

T2 - A comparative study

AU - Lin, T.

AU - Haskell, J.

AU - Vinson, N.

AU - Terracio, Louis

PY - 1986

Y1 - 1986

N2 - Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were 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 K(a) values of 1.08 x 109 and 1.1 x 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 α-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 x 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 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 μg/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-stimulated 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.

AB - Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were 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 K(a) values of 1.08 x 109 and 1.1 x 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 α-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 x 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 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 μg/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-stimulated 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.

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