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Endocrinology, doi:10.1210/en.2003-0329
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Endocrinology Vol. 145, No. 1 175-183
Copyright © 2004 by The Endocrine Society

Insulin Augmentation of 17{alpha}-Hydroxylase Activity Is Mediated by Phosphatidyl Inositol 3-Kinase But Not Extracellular Signal-Regulated Kinase-1/2 in Human Ovarian Theca Cells

Iqbal Munir, Hui-Wen Yen, David H. Geller, Donna Torbati, Rebecca M. Bierden, Stacy R. Weitsman, Sanjay K. Agarwal and Denis A. Magoffin

Departments of Obstetrics and Gynecology (I.M., H.-W.Y., D.T., R.M.B., S.R.W., S.K.A., D.A.M.) and Pediatrics (D.H.G.), Cedars-Sinai Burns and Allen Research Institute, Cedars-Sinai Medical Center/The David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Denis A. Magoffin, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Davis 2066, Los Angeles, California 90048. E-mail: magoffin{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome, characterized by hyperandrogenism and chronic anovulation, is frequently associated with insulin resistance. Ample evidence implicates a role for insulin in the genesis of ovarian hyperandrogenism. The objective of this study was to begin to define the intracellular signaling pathway(s) that mediates insulin regulation of 17{alpha}-hydroxylase activity in human ovarian theca cells. Third-passage theca cells, isolated from the ovaries of regularly cycling premenopausal women, were used. Insulin alone had no effect on 17{alpha}-hydroxylase activity or CYP17 mRNA expression but required costimulation with forskolin. At the insulin concentration used (10 ng/ml), a neutralizing antibody to the insulin receptor (but not an antibody to the type I IGF receptor) blocked the insulin stimulation of 17{alpha}-hydroxylase activity, demonstrating that the effects were mediated by the insulin receptor. Insulin stimulated both phosphatidylinositol-3-kinase (PI3-kinase) and extracellular signal-regulated kinase-1/2 (MAPK) pathways. Specific inhibition of MAPK kinase (MEK) with PD98059 or I0126 did not decrease the 17{alpha}-hydroxylase activity stimulated by forskolin or forskolin plus insulin. In contrast, the PI3-kinase inhibitor LY294002 completely blocked insulin-stimulated 17{alpha}-hydroxylase activity. Our data demonstrate that insulin stimulates PI3-kinase and extracellular signal-regulated kinase-1/2 activities in human theca cells, but only PI3-kinase mediates the insulin augmentation of forskolin-stimulated 17{alpha}-hydroxylase activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is the most common reproductive disorder of premenopausal women, affecting approximately 5% of the general population. PCOS is characterized by chronic anovulation and hyperandrogenism, and approximately 50% of women with PCOS are insulin resistant (1, 2). Although the insulin-resistant women maintain normal circulating glucose concentrations, higher insulin concentrations are required to do so (3). The observation that insulin, acting through ovarian insulin receptors, augments thecal androgen production (4) supports the hypothesis that insulin stimulation plays a significant role in the etiology of hyperandrogenism in insulin-resistant women with PCOS.

This concept is further supported by studies using the insulin-sensitizing agents metformin (5) and troglitazone (6) in women with PCOS. The disubstituted biguanide, metformin, reduces fasting plasma glucose levels and increases oral glucose tolerance in PCOS (7). Despite certain reports to the contrary, the preponderance of recent studies confirms that metformin improves peripheral insulin sensitivity and attenuates hyperandrogenemia in both lean and obese women with PCOS (8, 9). Troglitazone, a member of the thiazolidenedione class of insulin-sensitizing agents, acts as a ligand for the nuclear peroxisome proliferator-activated receptor {gamma} that, when activated, increases the transcription of factors that assist in glucose disposal in skeletal muscle (10). Treatment with troglitazone resulted in dramatic reductions in androgen excess and insulin resistance in women with PCOS, simultaneously reversing both metabolic and reproductive endocrine dysfunction (6, 11). In a small proportion of previously amenorrheic women, metformin or troglitazone has been shown to restore menses (9, 10, 11). Thus, clinical evidence supports an important role for insulin resistance as a cause of ovarian hyperandrogenism.

The mechanism of insulin action in women with PCOS has been studied in fibroblasts, adipocytes, and skeletal muscle cells. Examination of the insulin receptor in women with PCOS has revealed no mutations or structural abnormalities (12). Therefore, it seems that postreceptor defects in the insulin signaling pathway play important roles in the etiology of the insulin resistance.

In adipocytes from women with PCOS, a 30% decrease in insulin receptor autophosphorylation correlating with insulin resistance has been reported (13), whereas responses were normal in PCOS fibroblasts (14). In approximately 50% of women with PCOS, increased serine phosphorylation of the insulin receptor seems to perturb insulin signaling (15). In women with PCOS whose receptor phosphorylation is unaffected, signaling mechanisms downstream of the receptor are likely to be aberrant. Insulin receptor substrate (IRS)-2 levels are increased in muscle cells of women with PCOS, whereas the levels of IRS-1 remain unchanged (16). In addition, IRS-1-associated phosphatidylinositol-3-kinase (PI3-kinase) activity is lower in PCOS muscle cells after insulin stimulation, compared with control. In contrast, no difference in IRS-1-associated PI3-kinase activity was detected in PCOS fibroblasts, compared with control (17). Taken together, these studies indicate that there are alterations of signal transduction downstream from the level of the receptor binding and that the defects may be tissue specific. Thus, to understand the role that insulin plays in ovarian hyperandrogenism, it is important that insulin signaling be examined in human theca cells.

Little is known regarding the cellular mechanisms by which insulin regulates androgen biosynthesis by human ovarian theca cells. Understanding the insulin signaling mechanisms that regulate androgen production in theca cells is a prerequisite to defining abnormalities in PCOS and to establishing targets for therapeutic intervention. The purpose of this study was to begin to define the intracellular signaling pathways that link the insulin receptor to androgen biosynthesis in human ovarian theca cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Human theca interna tissue was obtained from the follicles of regularly cycling premenopausal women undergoing ovariectomy for reasons not related to this study. There was no ovarian pathology, and the women were not taking any medication known to affect sex hormone metabolism. Women with hirsutism, acne, hypertension, or type I or type II diabetes mellitus or who had a first-degree relative with type I or type II diabetes were excluded from the study. All women had regular menstrual cycles every 27–35 d and normal serum androgen concentrations. The study was approved by the Cedars-Sinai Medical Center Institutional Review Board, and informed consent was obtained from all subjects.

Theca cell isolation and propagation
Individual follicles were dissected away from the ovarian stroma. Small antral follicles, 8–9 mm in diameter, were identified under a dissecting microscope and hemisected. The granulosa cells were gently removed with a platinum loop, and the theca interna was microdissected from the follicle wall. The theca shells were dispersed with collagenase (0.5 mg/ml), collagenase IA (0.5 mg/ml), and deoxyribonuclease (0.1 mg/ml) (Sigma Chemical Co., St. Louis, MO) in HEPES-buffered DMEM containing 4.5 g/liter D-glucose supplemented with 10% (vol/vol) fetal bovine serum (FBS) and antimicrobial agents (200 IU/ml penicillin, 200 µg/ml streptomycin, 0.50 µg/ml amphotericin B, and 100 µg/ml gentamycin). The cells from each follicle were plated individually in two six-well plates containing growth medium [DMEM (without HEPES)/F-12 medium (1:1) supplemented with 5% (vol/vol) FBS, 5% (vol/vol) horse serum (Omega Scientific, Tarzana, CA), 2% (vol/vol) Ultroser G (Life Technologies, Paisley, Scotland, UK), 20 nM sodium selenite, 1 µM vitamin E, 100 IU/ml penicillin, 100 µg/ ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamycin] and at 37 C in a 5% CO2-5% O2-90% N2 humidified atmosphere. When the theca cells reached subconfluence, they were removed from the dish with neutral protease (Sigma Chemical Co.) in DMEM/F12 (1:1) and were frozen, and the first-passage cells were stored in liquid nitrogen in culture medium containing 20% FBS and 10% dimethylsulfoxide. For experiments, the cells were thawed and propagated to the third passage in growth medium. Before initiating experimental treatments the cells were cultured for 3 d in DMEM/F-12 (1:1) supplemented with 20 nM sodium selenite, 1 µM vitamin E, 100 µg/ml transferrin, 0.1% BSA (ICN Biomedicals, Costa Mesa, CA; fraction V), 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamycin.

17{alpha}-Hydroxylase assay
To measure 17{alpha}-hydroxylase activity, cultured theca cells were incubated with DMEM/F-12 (1:1) containing 0.5 mg/ml BSA, 1 µM unlabeled progesterone, and 0.2 µCi [1,2,6,7-3H]progesterone (NEN Life Science Products, Boston, MA; 114 Ci/mmol). After 3 h of incubation at 37 C in a 5% CO2-5% O2-90% N2 humidified atmosphere, the medium was harvested and was extracted twice with diethyl ether, and the pooled extracts were dried under a stream of air. The extracted steroids were dissolved in ethyl acetate:isooctane (1:1 by volume), and the samples were spotted on thin-layer chromatography plates (PE SIL G/UV, Whatman, Clifton NJ). The plates were developed in chloroform:ethyl acetate (3:1 by volume). The thin-layer chromatography plates were sprayed with EN3HANCE (NEN Life Science Products) and then exposed to autoradiographic film. After developing of the films, the areas on the plate corresponding to the labeled progesterone substrate and 17{alpha}-hydroxyprogesterone product were cut from the plate and counted in a scintillation counter. The identity of the spots was confirmed by comparing their migration with authentic [3H]progesterone and [3H]17{alpha}-hydroxyprogesterone standards.

Stimulation of the cells for MAPK and protein kinase B (PKB) activation
After reaching semiconfluence, the theca cells were cultured in serum-free DMEM/F-12 medium containing antibiotics for 3 d. The cells were then incubated for 30 min at 37 C in Krebs-Ringer phosphate (KRPH) buffer containing 30 mM HEPES (118 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 30 mM HEPES, pH 7.4). The KRPH buffer was aspirated and replaced with KRPH buffer with and without PD98059 (Calbiochem, San Diego, CA) (0.3–50 µM) and LY294002 (Calbiochem) (0.1–10 µM) for another 30 min. The cells were then stimulated with 25 µM forskolin (Sigma Chemical Co.) and/or 10 ng/ml recombinant human insulin (Sigma Chemical Co.) in KRPH for 5 min. After stimulation, the KRPH buffer was rapidly aspirated from the dishes, and the monolayers were quickly frozen in liquid nitrogen and stored at -80 C.

Western blot analysis
The frozen theca monolayers, prepared as described above, were lysed in solubilizing buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1% Triton X-100, 1 mM Na3VO4, 100 nM calyculin A, 1 mM phenylmethylsulfonylfluoride, 100 µM leupeptin, 20 µg/ml aprotinin, 7.5 µM pepstatin, pH 7.4). The lysates were scraped from the dishes and homogenized with hand homogenizers in microfuge tubes. The homogenates were centrifuged to remove insoluble materials, and the supernatant was used for Western blot analysis. An aliquot of the lysate was used to quantitate protein concentration. Fifty micrograms of protein from each sample were separated on 4–12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and electrophoretically transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were blocked overnight at 4 C with TBST [10 mM Tris (pH 8), 150 mM NaCl, 0.05% Tween 20] containing 5 mg/ml nonfat dry milk. Blots were incubated with rabbit polyclonal phospho-extracellular signal-regulated kinase (phospho-ERK)-1/2 antibody that binds both phospho-ERK-1 and phospho-ERK-2 or phospho-PKB antibody (Cell Signaling Technology, Beverly, MA) for 4 h at room temperature (1:1000 in blocking buffer). After incubation, the membranes were washed three times with TBST and incubated with alkaline phosphatase conjugated donkey antirabbit IgG antibody (Chemicon International, Temecula, CA) for 1 h at room temperature (1:10,000 in blocking buffer). After three washes in TBST at room temperature, the immunoreactive proteins were detected by incubating in 10 mM Tris (pH 9.5), 0.4 µg/ml 7-hydroxy-9H-(1,3-dichloro-9,9-dimethyl-acridin-2-one) (DDAO) phosphate, 1 mM MgCl2 at room temperature for 5 min. Alkaline phosphatase converts the soluble nonfluorescent DDAO phosphate to the insoluble fluorescent product DDAO. Blots were subsequently scanned and quantified with a fluorescence imaging system (Typhoon 8600, Molecular Dynamics, Sunnyvale, CA).

To determine the total amount of MAPK and PKB in each sample, membranes were washed with methanol, to remove the fluorescent DDAO precipitate, and then stripped of the primary antibody by treating with 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (wt/vol), 62.4 mM Tris (pH 6.7), for 30 min at 50 C. After washing three times with TBST, the membrane was blocked overnight, then incubated with MAPK or PKB antibody (1:1000 in blocking buffer) that does not discriminate the phospho and de-phospho forms (Cell Signaling Technology). The proteins were detected as described above.

DNA assay
Total cellular DNA was isolated from theca cells using Tri-Reagent (MRC, Cincinnati, OH) according to the manufacturer’s protocol. The DNA pellet was dissolved in 100 µl of 40-mM NaOH at 4 C for 24 h. The DNA concentration of the samples was measured by a sensitive fluorescence assay (PicoGreen dsDNA quantitation kit, Molecular Probes, Eugene, OR). Briefly, 5 µl of sample was diluted with 200 µl PicoGreen solution in a 96-well plate. The fluorescence was measured on a Molecular Dynamics variable mode imager (Typhoon 8600). Sample concentrations were interpolated from a standard curve calculated by linear regression of the fluorescence of known concentrations of lambda DNA standard.

Protein assay
Total cellular protein was measured in theca cell lysates using a sensitive fluorescent assay (NanoOrange protein quantitation kit, Molecular Probes). Briefly, 1 µl of sample was diluted with 250 µl NanoOrange solution, denatured by heating for 10 min at 95 C in sealed tubes, then transferred to a 96-well plate. The fluorescence was measured on a Molecular Dynamics variable mode imager (Typhoon 8600). Protein concentrations were interpolated from a standard curve calculated by linear regression of the fluorescence of known concentrations of a BSA standard.

Measurement of mRNA expression
CYP17 mRNA was measured by RT-PCR assay as previously described (18). Total RNA was isolated with Tri-reagent, resuspended in 30 µl diethylpyrocarbonate-treated water and transcribed into cDNA by incubation for 30 min in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM deoxy (d)-ATP, 1 mM dCTP, 1 mM dGTP, 1 mM thymidine 5'-triphosphate, 5 µg oligo(deoxythymidine)12–18 (Amersham Pharmacia Biotech), 20 U RNAsin (Promega Corp., Madison, WI), and 200 IU Moloney murine leukemia virus reverse transcriptase (Invitrogen) in a total vol of 100 µl. One picogram of mutant control DNA, 50 pmol of each PCR primer, 8 µl of 10x PCR buffer [100 mM Tris-HCl (pH 8.3), and 500 mM KCl], 9.6 µl of 25-mM MgCl2, 10 µCi [32P]dCTP (3000 Ci/mmol; DuPont NEN Life Science Products), and 2.5 U Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) were added to individual aliquots of sample cDNA (4 µl), and the vol was adjusted to 100 µl. cDNA was amplified for 25 cycles (94 C for 1 min, 55 C for 1 min, 72 C for 1 min). Control templates were synthesized by site-directed mutagenesis to create a unique Msp I restriction site by substituting T for C at position 268. After amplification, the products were ethanol precipitated and digested with Msp I to cut the control products, then separated on a 2% agarose gel. The DNA was visualized with ethidium bromide staining, and the bands were cut from the gel and counted in a scintillation counter. The counts per min in the bands amplified from the cellular mRNA were normalized to the counts per minute in the bands amplified from the mutant cDNA to control for procedural variations. The data were also normalized to total cellular DNA to control for variations in the number of cells in each sample. The amount of mRNA in each sample was interpolated from the standard curve and expressed as picograms of full-length mRNA per microgram of total cellular DNA. The standard concentrations were chosen to obtain a linear range spanning 2 orders of magnitude. The volumes of sample were adjusted to yield data within the linear range of the standard curve.

Statistical analysis
Each experiment was repeated three to four times with triplicate wells/treatment in each experiment. Multiple comparisons were performed using one-way ANOVA with post hoc comparisons employing Tukey’s test. Statistical significance was considered to be P at or below 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because third-passage human theca cells have not been validated as a model for investigating insulin action and insulin signaling, it was important to determine that the third-passage cultures exhibit responses that correspond to in vivo results and results obtained with freshly isolated theca cells in culture.

Insulin stimulation of 17{alpha}-hydroxylase activity in cultured human theca cells
Insulin has been found to enhance LH-stimulated androgen biosynthesis in freshly isolated ovarian theca cells of rodent, porcine and human origin. The key enzyme for androgen biosynthesis is 17{alpha}-hydroxylase/C17–20 lyase. Therefore we measured 17{alpha}-hydroxylase activity in our studies. The data in the literature imply that insulin stimulation of 17{alpha}-hydroxylase activity is dependent upon concomitant activation of the cAMP signaling pathway (19). To test this concept in this model, cultured human theca cells were treated with 10, 30, or 100 ng/ml of insulin with and without 25 µM forskolin to activate the cAMP signaling pathway (Fig. 1AGo). Basal 17{alpha}-hydroxylase activity was unchanged with insulin alone at any of the three concentrations. Basal 17{alpha}-hydroxylase activity was increased by forskolin treatment and further increased by concomitant treatment with insulin. These findings show that insulin stimulation of 17{alpha}-hydroxylase activity depends on concurrent activation of the cAMP signaling pathway.



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  		FIG. 1. Insulin stimulation of 17{alpha}-hydroxylase activity in cultured human theca cells. A, Third-passage human theca cells were cultured for 3 d with and without 25 µM forskolin in the presence of increasing concentrations (0–100 ng/ml) of recombinant human insulin. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. B, Third-passage human theca cells were cultured for up to 3 d with and without 25 µM forskolin and 10 ng/ml of recombinant human insulin. At each time point, replicate cultures were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Fsk, Forskolin; Ins, insulin. Data are the mean ± SEM. Data points with different letters are significantly different (P < 0.05).

 
Insulin induction of 17{alpha}-hydroxylase activity was time dependent (Fig 1BGo). At 24 h neither insulin nor forskolin increased 17{alpha}-hydroxylase activity. Although forskolin stimulated 17{alpha}-hydroxylase activity at 48 h, insulin treatment did not increase 17{alpha}-hydroxylase activity in the presence of forskolin until 72 h.

The effects of forskolin on insulin stimulation of 17{alpha}-hydroxylase activity were concentration dependent (Fig. 2Go). The optimal concentration of forskolin for further studies with a physiological concentration of insulin (10 ng/ml) was determined to be 25 µM.



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  		FIG. 2. Forskolin stimulation of 17{alpha}-hydroxylase activity in cultured human theca cells. Third-passage human theca cells were cultured for 3 d with 10 ng/ml insulin and increasing concentrations (0–30 µM) of forskolin. Control cells (C) were cultured in the absence of added treatments. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Data are the mean ± SEM. *, Significantly different from control (P < 0.05).

 
Insulin action on target tissues, including human theca cells, can be initiated at the cell surface by binding either to its cognate receptor or to the structurally homologous type I IGF receptor (4, 19, 20). As shown in Fig. 3Go, detailed analysis of the insulin concentration-response curve, in the presence of 25 µM forskolin, revealed a biphasic increase in 17{alpha}-hydroxylase activity in the cultured human theca cells. The apparent EC50 for the initial increase was 0.52 ± 0.20 nM. The insulin concentrations spanning the initial increase (1–30 ng/ml) are well within the physiological range and are consistent with insulin actions mediated by the insulin receptor (dissociation constant = 0.56 nM) (21). The apparent EC50 for the second increase was 8.9 ± 2.7 nM, consistent with insulin activation of the type I IGF receptor (dissociation constant = 150 nM) (22). Based on these data, 10 ng/ml insulin, which lies in the upper part of the postabsorptive insulin concentration curve, was used in the subsequent experiments to maximally activate the insulin receptor without activation of the type I IGF receptor.



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  		FIG. 3. Insulin dose-response curve for stimulation of 17{alpha}-hydroxylase activity in cultured human theca cells. Third-passage human theca cells were cultured for 3 d with 25 µM forskolin and increasing concentrations (0–1000 ng/ml) of recombinant human insulin. Control cells (C) were cultured in the absence of added treatments. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Data are the mean ± SEM. Data points with different letters are significantly different (P < 0.05).

 
To demonstrate that the chosen concentration of insulin increases 17{alpha}-hydroxylase activity by selectively activating the insulin receptor but not the type I IGF receptor, cultured human theca cells were treated with insulin plus forskolin in the presence and absence of neutralizing antibodies selective against either the insulin receptor or the type I IGF receptor. The insulin receptor blocking antibody used (5D9) was previously shown to block the insulin receptor-mediated effects of insulin on human theca cell testosterone production without affecting the type I IGF receptor (4, 23). As shown in Fig. 4Go, addition of insulin receptor blocking antibody (5D9) completely abolished the insulin stimulation of 17{alpha}-hydroxylase activity without altering the stimulatory effect of forskolin. The type I IGF receptor blocking antibody ({alpha}IR3) completely abolishes receptor binding at the concentration chosen (5 µg/ml), whereas there is no effect on insulin receptor binding even at 100-fold higher concentrations (24). The neutralizing antibody against the type I IGF receptor ({alpha}IR3) had no effect on either forskolin or insulin stimulation of 17{alpha}-hydroxylase activity (Fig. 4Go). These data demonstrate that 10 ng/ml insulin increases 17{alpha}-hydroxylase activity by activating the insulin receptor but not the type I IGF receptor.



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  		FIG. 4. Physiological concentrations of insulin stimulate 17{alpha}-hydroxylase activity through the insulin receptor in cultured human theca cells. Third-passage human theca cells were cultured for 3 d with medium alone (C), 10 ng/ml insulin, and/or 25 µM forskolin, in the presence or absence of anti-insulin receptor neutralizing antibody (5D9, 1.25 µg/ml) or anti-type I IGF receptor antibody ({alpha}IR3, 5 µg/ml). After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Data are the mean ± SEM. Within treatment groups, bars with different letters are significantly different (P < 0.05).

 
Effect of forskolin and insulin on CYP17 mRNA expression
We next examined the effects of selected insulin concentrations, in combination with forskolin, on CYP17 mRNA expression. As expected, forskolin alone caused a significant increase in CYP17 mRNA expression (Fig. 5Go). At an insulin concentration that activates only the insulin receptor (10 ng/ml), there was an increase in CYP17 mRNA expression, compared with treatment with forskolin alone. CYP17 mRNA was further enhanced by increasing the insulin concentration to a level that activates both the insulin and type I IGF receptors (100 ng/ml). These observations demonstrate that this model of cultured human theca cells responds to insulin in a physiologically relevant manner that makes it a useful model for defining the molecular mechanisms by which insulin regulates CYP17 mRNA expression and 17{alpha}-hydroxylase activity.



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  		FIG. 5. Expression of CYP17 mRNA after insulin and forskolin stimulation. Third-passage human theca cells were cultured for 3 d with and without 25 µM forskolin and increasing concentrations (0–100 ng/ml) of insulin. Total RNA was extracted from the cells, and CYP17 mRNA was measured by competitive RT-PCR. Data are the mean ± SEM. *, Significantly different from control (P < 0.05).

 
The role of PI3-kinase in insulin stimulation of 17{alpha}-hydroxylase activity
Having demonstrated the utility of the model, we began to define the intracellular signaling pathways that mediate insulin stimulation of thecal androgen biosynthesis. A principal intracellular signaling pathway mediating insulin action in target cells involves activation of PI3-kinase, which can phosphorylate and activate PKB (or Akt). Insulin, binding to its cognate receptor, results in phosphorylation of IRS docking proteins that recruit PI3-kinase to the membrane and stimulate its activation. Because protein PKB phosphorylation is dependent on PI3-kinase activation, an increase in phospho-PKB provides an indirect marker of PI3-kinase activation. As shown in Fig. 6Go, 10 ng/ml insulin markedly increased the phospho-PKB concentration in cultured theca cells. Forskolin alone or in combination with insulin did not stimulate PKB phosphorylation. A specific PI3-kinase inhibitor, LY294002 (25), totally abolished the effect of insulin on PKB phosphorylation. These data support the concept that PI3-kinase is activated by insulin in human theca cells.



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  		FIG. 6. Effect of LY 294002 on insulin stimulation of PKB in cultured human theca cells. Third-passage theca cells were incubated in KRPH medium for 30 min, then pretreated with 10 µM LY294002 (LY) for 30 min in KRPH medium. The cells were then stimulated with and without 10 ng/ml Ins and 25 µM Fsk for 5 min. The cells were lysed, and Western blot analysis was performed with antibodies specific for total and phospho-PKB. A is representative of two experiments, and B is the mean ± SEM of the cumulative data from fluorescent quantitation of the bands.

 
To test the role of PI3-kinase in mediating the effects of insulin on 17{alpha}-hydroxylase activity, cultured theca cells were treated with forskolin plus insulin in the presence of increasing concentrations of LY294002. LY294002 had no effect on 17{alpha}-hydroxylase activity by itself (data not shown). As expected, forskolin alone increased 17{alpha}-hydroxylase activity (Fig. 7Go). LY294002 (10 µM) did not alter the forskolin effect. Addition of insulin further increased 17{alpha}-hydroxylase activity, and LY294002 caused a concentration-dependent inhibition of the insulin effect. These results indicate that insulin stimulation of 17{alpha}-hydroxylase is mediated by PI3-kinase.



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  		FIG. 7. Effect of LY294002 on insulin stimulation of 17{alpha}-hydroxylase activity. Third-passage theca cells were cultured for 3 d with 25 µM Fsk with and without 10 ng/ml Ins in the presence or absence of increasing concentrations (0–10 µM) of LY294002. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Data are the mean ± SEM. Bars with different letters are significantly different (P < 0.05).

 
The role of ERK-1/2 in insulin stimulation of 17{alpha}-hydroxylase activity
Another major intracellular signaling pathway that can be activated by insulin independent of PI3-kinase is the MAPK pathway. Phosphorylated IRS proteins can also initiate a well-defined signaling cascade culminating in the phosphorylation and activation of ERKs, which include 44-kDa ERK-1 and 42-kDa ERK-2 (26). To determine whether ERK-1 and -2 are activated by insulin in human theca cells, phosphorylation of ERK-1 and -2 was assessed by Western blot using an antibody that selectively detects the phosphorylated forms of ERK-1 and -2. As shown in Fig. 8Go, insulin alone induced an approximately 5-fold increase in ERK-1/2 phosphorylation. A similar degree of ERK-1/2 phosphorylation was stimulated by forskolin. PD98059, a specific inhibitor of MEK (27), the kinase that phosphorylates ERK-1 and -2, blocked phosphorylation of ERK-1/2 by insulin (Fig. 8Go). LY294002 did not affect stimulation of ERK-1/2 phosphorylation by insulin, indicating that PI3-kinase is not required for insulin stimulation of the MAPK pathway in human theca cells.



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  		FIG. 8. Insulin stimulation of ERK-1/2 phosphorylation in cultured human theca cells. A, Third-passage theca cells were incubated in KRPH medium for 30 min, then pretreated with 10 µM LY294002 (LY) for 30 min in KRPH medium. The cells were then stimulated with and without 10 ng/ml Ins and 25 µM Fsk for 5 min. The cells were lysed, and Western blot analysis was performed with antibodies specific for total and phospho-ERK1/2. B, Third-passage theca cells were incubated in KRPH medium for 30 min, then pretreated with 10 µM PD98059 (PD) for 30 min in KRPH medium. The cells were then stimulated with and without 10 ng/ml Ins for 5 min. The cells were lysed, and Western blot analysis was performed with antibody specific for total and phospho-ERK1/2. The data in A and B are representative of four experiments. C represents the mean ± SEM of data from fluorescent quantitation of the bands.

 
To investigate whether ERK-1 and -2 are involved in mediating insulin stimulation of 17{alpha}-hydroxylase activity, theca cells were incubated with the MEK inhibitors PD98059 and U0126 (28). The inhibitors alone had no effect on 17{alpha}-hydroxylase activity by theca cells (data not shown). As shown in Fig. 9Go, forskolin treatment alone increased 17{alpha}-hydroxylase activity; however, concomitant treatment with PD98059 did not block the forskolin effect. As expected, addition of insulin further increased thecal 17{alpha}-hydroxylase activity; however, PD98059 did not inhibit the insulin stimulation. Interestingly, PD98059 increased forskolin- and forskolin-plus-insulin-stimulated 17{alpha}-hydroxylase activity at a concentration that totally abolished forskolin and insulin stimulation of ERK-1/2 phosphorylation in theca cells. Similar results were obtained with U0126, a MEK inhibitor that is chemically distinct from PD98059 (Fig. 9Go). These data support the conclusion that ERK-1 and -2 do not mediate insulin or forskolin stimulation of 17{alpha}-hydroxylase activity in human theca cells.



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  		FIG. 9. Effect of MEK inhibitors on insulin stimulation of 17{alpha}-hydroxylase activity in cultured human theca cells. A, Third-passage theca cells were cultured for 3 d with 25 µM Fsk with and without 10 ng/ml Ins in the presence of increasing concentrations (0–50 µM) of PD98059. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. B, Third-passage theca cells were cultured for 3 d with 25 µM Fsk with and without 10 ng/ml Ins in the presence of 10 µM I0126. After the 3-d culture period, the cells were incubated (3 h) with [3H]progesterone to measure 17{alpha}-hydroxylase activity. Data are the mean ± SEM. Data points with different letters are significantly different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study validates the human theca culture system as a model for investigating insulin signaling. Our data confirm previous data showing that insulin can directly stimulate ovarian androgen production at physiological concentrations (4). Our data further confirm that the insulin effect is mediated through the insulin receptor, and not the type I IGF receptor, at the concentrations found in women with PCOS (4, 29). The data extend our knowledge by demonstrating that insulin regulation of 17{alpha}-hydroxylase activity is mediated by PI3-kinase and that the MAPK cascade is not involved in stimulation of 17{alpha}-hydroxylase activity.

Previously reported evidence supports the concept that insulin regulation of ovarian androgen biosynthesis is mediated by its cognate receptor and not by a so-called spill-over effect through the structurally homologous type I IGF receptor (4, 29). Our concentration-response data indicate that both the insulin receptor and the type I IGF receptor are linked to regulation of 17{alpha}-hydroxylase activity in human theca cells. By careful selection of the insulin concentration, we were able to ensure that the effects under study were mediated by the insulin receptor without significant contribution of the type I IGF receptor. Two lines of evidence support this conclusion. First, the optimal concentration of insulin (10 ng/ml) induced a maximal increase in 17{alpha}-hydroxylase activity on the first plateau of the concentration-response curve, which corresponds to activation of the insulin receptor. Second, the use of previously characterized selective neutralizing antibodies to the insulin (23) and type I IGF (24) receptors confirms that the effects we observed at 10 ng/ml of insulin were mediated by the insulin receptor and not the type I IGF receptor.

In many tissues, insulin/type I IGF hybrid receptors are present as a significant percentage of the total receptor (40% in liver and spleen, 70% in the placenta, and 85–90% in skeletal muscle and heart) (30). The neutralizing antibody against the insulin receptor, 5D9, is known to effectively block insulin effects but can also block the effects of IGF-I that are mediated by insulin/type I IGF hybrid receptors (31). Conversely, the antitype I IGF receptor antibody, {alpha}IR3, effectively blocks the effects of IGF-1 but blocks insulin action only weakly (32). The observation that 5D9 blocked only the effects of insulin, and not IGF-I, indicates that hybrid receptors are not significantly involved in mediating the insulin stimulation of thecal 17{alpha}-hydroxylase activity at physiological concentrations.

The stimulatory effects of insulin on both 17{alpha}-hydroxylase activity and CYP17 mRNA expression in human theca cells were dependent on the coactivation of the cAMP signaling pathway. This pattern of response is different from the effects of insulin on glucose metabolism, where insulin alone stimulates glucose uptake (33). These differences raise the possibility that insulin regulation of glucose metabolism is mediated by intracellular mechanisms distinct from those mediating insulin regulation of thecal androgen production. Further studies will be required to compare and contrast the intracellular mediators of the insulin signals that regulate glucose metabolism and androgen biosynthesis.

A principal goal of this study was to initiate a systematic study to define the role of major insulin signaling pathway(s) in the regulation of ovarian androgen biosynthesis. There is a branch point in insulin signaling postinsulin receptor binding and activation. One of the major branches is the IRS/PI3-kinase pathway, which is known to play a prominent role in regulating glucose uptake (34). Insulin binding also activates an independent pathway involving the MAPK cascade that is important for regulating mitogenesis (35). In our study, a specific inhibitor of PI3-kinase, LY294002, inhibited insulin-induced 17{alpha}-hydroxylase activity in theca cells, implying that the PI3-kinase pathway is a key mediator of the insulin signal involved in regulating androgen production in human theca cells. LY294002 also inhibited PKB activation in theca cells, raising the possibility that PKB may be an important mediator downstream of PI3-kinase mediating the insulin effect. In addition to PKB, other signaling pathways, such as the atypical protein kinase C {zeta} and {lambda}, lie downstream of PI3 kinase (36) and could participate in the transcriptional regulation (36, 37) of the CYP17 gene. Additionally, the PI3-kinase-PKB-mammalian target of rapamycin pathway could potentially be involved in translational regulation (38) of insulin-responsive steroidogenic enzymes. Further experiments are in progress to define the mechanism of insulin regulation of androgen production in human theca cells distal to PI3-kinase.

Although insulin activates the MAPK cascade in theca cells, the evidence does not support a role for the MAPK pathway in mediating insulin stimulation of 17{alpha}-hydroxylase activity. Interestingly, MEK inhibitors not only failed to block the stimulatory effect of insulin on 17{alpha}-hydroxylase activity, there was a concentration-related increase of insulin-stimulated 17{alpha}-hydroxylase activity. Recently, it was reported that stimulation of the ERK cascade inhibited steroidogenesis in granulosa cells and that PD98059 stimulated steroidogenic acute regulatory expression (39). ERK pathways were also found to be inhibitory to the expression of the CYP17 gene in an adrenocortical cell line (40). In the adrenal cells, phosphorylated SF-1 was shown to be a repressor of CYP17 transcription. Similar mechanism(s) may be operating in human theca cells in which the MAPKs ERK-1 and -2 may be coupled to negative regulators of CYP17 transcription. Blocking MEK activity, and consequently ERK activation, may decrease repression of the CYP17 promoter and thereby increase CYP17 gene transcription. Further studies will be required to explore this possibility.

Insulin resistance is a common feature of women with PCOS. To maintain glucose homeostasis, there is a mild compensatory increase in circulating insulin concentrations. Many investigators believe that this increase in insulin can play an important role in causing the ovarian hyperandrogenism that is a hallmark of PCOS. A significant unresolved question relates to how insulin can stimulate ovarian hyperandrogenism in insulin-resistant women. The observation that insulin stimulation of 17{alpha}-hydroxylase activity, unlike glucose uptake, requires coactivation of the cAMP pathway indicates that there may be a divergence in the insulin signal distal to PI3-kinase, with one branch regulating glucose uptake and another branch regulating steroidogenesis. If true, the seeming paradox of insulin stimulation of androgen production in insulin-resistant women could be explained by a selective defect in the glucose uptake pathway distal to PI3-kinase.

Thiazolidinediones have been used to treat the hyperandrogenism of PCOS in both insulin-resistant and insulin-sensitive women, but the mechanism of action remains unknown. Although certain thiazolidinediones, such as troglitazone, can have direct inhibitory action on 17{alpha}-hydroxylase activity, this does not seem to be a general mechanism and certainly doesn’t apply to the alternative insulin sensitizer, metformin (41). Thiazolidinediones have been shown to increase insulin-stimulated PI3-kinase activity in Chinese hamster ovary cells and myotubes (42). It is unclear whether such a mechanism occurs in human theca cells; but, based on our data, it is unlikely to explain the decrease in androgen production accompanying thiazolidinedione treatment in women with PCOS. Understanding the details of insulin signaling in human theca cells will help us to understand how insulin sensitizers act to decrease ovarian hyperandrogenism and may lead to novel therapeutic targets for specifically regulating ovarian androgen biosynthesis.


   Acknowledgments
 
We thank Prof. R. A. Roth (Department of Pharmacology, Stanford University) for generously providing us insulin receptor antibody.


   Footnotes
 
This work was supported by National Institute of Child Health and Human Development Grant HD041610 (to D.M.).

Abbreviations: DDAO, 7-Hydroxy-9H-(1,3-dichloro-9,9-dimethyl-acridin-2-one); ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IRS, insulin receptor substrate; KRPH, Krebs-Ringer phosphate; MEK, MAPK kinase; PCOS, polycystic ovary syndrome; PI3-kinase, phosphatidylinositol-3-kinase; PKB, protein kinase B.

Received March 17, 2003.

Accepted for publication August 29, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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