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Selective Insulin Resistance in the Polycystic Ovary Syndrome¹

Division of Women’s Health (A.D.), Departments of Medicine and Obstetrics and Gynecology, Brigham and Women’s Hospital, Boston, Massachusetts 02115; and the Department of Medicine, Pennsylvania State University College of Medicine (C.-B.B., A.D.), Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Andrea Dunaif, M.D., Division of Women’s Health, PBB 5, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02155. E-mail: adunaif{at}bics.bwh.harvard.edu

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia that is amplified by insulin in the presence of resistance to insulin’s action to stimulate glucose uptake in muscle and fat. To explore the mechanisms for this paradox, we examined the metabolic and mitogenic actions of insulin and insulin-like growth factor I (IGF-I) in cultured skin fibroblasts from PCOS (n = 16) and control (n = 11) women. There were no significant decreases in the number or affinity of insulin- or IGF-I-binding sites in PCOS compared to control fibroblasts. Basal rates were similar, but there were significant decreases in insulin-stimulated (control, 51.8 ± 7.0; PCOS, 29.5 ± 2.9 nmol/10⁶ cells·2 h at 1,000,000 pmol/L; P < 0.005) and IGF-I-stimulated (control, 48.9 ± 6.7; PCOS, 33.0 ± 3.2 PCOS nmol/10⁶ cells·2 h at 100,000 pmol/L IGF-I; P < 0.05) glucose incorporation into glycogen in PCOS fibroblasts, a metabolic action of insulin. Stimulation of thymidine incorporation, a mitogenic action of insulin, was similar in PCOS and control fibroblasts in response to both insulin and IGF-I. There were also no significant differences in insulin- or IGF-I-stimulated insulin receptor substrate-1-associated phosphatidylinositol-3-kinase activity in PCOS compared to control fibroblast cells. We conclude that 1) there is a selective defect in insulin action in PCOS fibroblasts that affects metabolic, but not mitogenic, signaling pathways; 2) there is a similar defect in IGF-I action, suggesting that insulin and IGF-I stimulate glycogen synthesis by the same postreceptor pathways; and 3) insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activation by insulin and IGF-I is similar to the control value, suggesting that the metabolic signaling defect is in another pathway or downstream of this signaling step in PCOS fibroblasts.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
POLYCYSTIC ovary syndrome (PCOS) is among the most common endocrinopathies of premenopausal women (1, 2). The disorder is characterized by hyperandrogenism, chronic anovulation, and hyperinsulinemia (1). Its etiology remains unknown, but it is clear that hyperinsulinemia secondary to insulin resistance plays an important role in the pathogenesis of reproductive abnormalities by stimulating ovarian androgen production (1, 3, 4). It has long been proposed that this insulin action on steroidogenesis is mediated by spillover occupancy of the type 1 insulin-like growth factor (IGF) receptor (IGF1R) (1, 5, 6). Circulating insulin levels in PCOS, however, are usually not high enough to activate the IGF1R in nonovarian tissues (7, 8). Recent studies have supported this observation by demonstrating that insulin is acting through its cognate receptor to modulate steroidogenesis in normal and in PCOS ovaries (9, 10). It is not known how this insulin action on steroidogenesis is preserved when there is profound resistance to insulin action on both carbohydrate (1) and lipid metabolism (11) in PCOS.

Insulin acts via its cell surface receptor, which is a ligand-activated protein tyrosine kinase (1, 12, 13). After tyrosine autophosphorylation, the activated receptor phosphorylates endogenous substrates, such as insulin receptor substrate-1 and -2 (IRS-1 and IRS-2) (12, 13). Phosphorylated tyrosine residues on these substrates then bind to a variety of molecules to activate signaling pathways, such as those mediating carbohydrate metabolism, lipogenesis, protein synthesis, gene transcription, and cell growth/differentiation (12, 13). The IGF1R shares substantial sequence and structural homology with the insulin receptor and uses many of the same signaling pathways (13, 14). The mechanisms of insulin receptor signal specificity are the subject of intense investigation (12, 13, 14). Current evidence suggests that IRS-1/2-mediated activation of phosphatidylinositol 3-kinase (PI3-kinase) controls insulin-mediated glucose transport and carbohydrate metabolism (15, 16, 17, 18, 19). Separate pathways regulated through mitogen-activated protein kinase activation appear to control mitogenesis (12, 13, 14, 19, 20). Activation of PI3-kinase may also be involved in these pathways (13, 17).

It is possible to experimentally block certain insulin receptor signaling pathways while others are preserved, producing selective insulin resistance (18, 20). Clinical examples of selective insulin resistance have been reported in pseudoacromegaly (21) and in diabetic patients with a strong family history of type 2 diabetes (22). Resistance to insulin action on carbohydrate metabolism with preserved insulin stimulation of mitogenesis has been demonstrated in cultured skin fibroblasts from such patients (21, 22). We performed this study to determine whether a similar selective defect in insulin action was present that might explain the persistent insulin-mediated changes in reproductive function despite profound resistance to insulin effects on carbohydrate metabolism in PCOS. We also examined the actions of IGF-I on these metabolic and mitogenic pathways to determine whether analogous defects existed. We used cultured skin fibroblasts that had been removed from the in vivo environment for generations so that we could examine intrinsic, rather than acquired, defects in insulin action (13, 21, 22, 23, 24, 25, 26, 27, 28, 29). We report for the first time that there is a selective defect in hormone action on carbohydrate metabolism in PCOS that affects pathways used by both insulin and IGF-I.

	Subjects and Methods

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Subjects

Sixteen women with PCOS and 11 reproductively normal control women were studied. All women were between the ages of 18–43 yr, in good health, and taking no medications known to alter sex hormone or carbohydrate metabolism for at least 1 month before the study, except for contraceptive agents that were stopped 3 months before the study. The studies were approved by the institutional review boards of the Mt. Sinai School of Medicine (Mt. Sinai) and the Pennsylvania State University College of Medicine (Penn State); all women gave written informed consent before the study. PCOS was diagnosed by the combination of 6 or fewer menses/yr and an elevation of total and/or nonsex hormone-binding globulin-bound testosterone levels greater than 2 SD above the control range established in women aged 18–40 yr with 27- to- 35-day regular menstrual cycles and no hirsutism or acne (28, 29, 30, 31, 32, 33, 34). Disorders of the pituitary, adrenals, and ovaries were excluded by appropriate tests before the diagnosis of PCOS was made (30). No PCOS women had diabetes (35). Control women had 27- to 35-day regular menstrual cycles, no hirsutism or acne, no personal history of diabetes or hypertension, and no first degree relative with diabetes (28, 29, 32, 33, 34, 36, 37). Total testosterone, unbound testosterone, and dehydroepiandrosterone sulfate levels were within the normal range in control women.

A 75-g glucose load oral glucose tolerance test was performed in all women after a 3-day 300-g carbohydrate/day diet and a 10-h or longer overnight fast (35). Blood for glucose and insulin levels was obtained every 30 min for 2 h, and glucose tolerance was assessed according to WHO criteria (35). All subjects had participated in our studies of insulin action in PCOS (28, 29, 31, 32, 33, 34, 36, 37), and their androgen levels, glucose tolerance, and insulin sensitivity data have been previously reported. Glucose tolerance data, fasting insulin levels, and insulin sensitivity (SI), as determined by modified, frequently sampled, iv glucose tolerance (4, 34), are summarized with clinical features in Table 1. The methodology for the insulin assay was not the same as that for Mt. Sinai (30) and Penn State (4) subjects; thus, the fasting insulin and SI results were not directly comparable. However, the PCOS women were not selected for this study on the basis of their individual insulin sensitivity measurements. Normal ranges for insulin and SI measurements for each site are given in Table 1. All subjects had a skin biopsy performed under sterile conditions after local anesthesia with 1% lidocaine with epinephrine (28, 29). A 2-mm punch biopsy was taken from the volar aspect of the forearm. Table 2 summarizes the studies in which each subject’s fibroblast cell lines were used.

DMEM (4 g/L glucose), low glucose (1 g/L) DMEM, penicillin-streptomycin solution, trypsin, ethylenediamine tetraacetate (EDTA), and silica gel 60 thin layer chromatography (TLC) plates were obtained from Fisher Scientific (Pittsburgh, PA). FBS was purchased from Life Technologies, Inc./BRL (Gaithersburg, MD). Isoton II was obtained from Coulter Corp. (Miami, FL). HEPES, Triton X-100, and dithiothreitol were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Protein A-Sepharose CL-4B was obtained from Pharmacia LKB (Piscataway, NJ). Human insulin was purchased from Eli Lilly & Co. (Indianapolis, IN), and IGF-I was purchased from Bachman (Torrance, CA). A-14-tyrosine-[¹²⁵I]insulin, [methyl-³H]thymidine, and D-[U-¹⁴C]glucose were purchased from DuPont (Wilmington, DE). [-³²P]ATP was obtained from ICN Biochemicals, Inc. (Costa Mesa, CA), and [¹²⁵I]IGF-I was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). The anti-IRS-1 antibody (JD66) was a gift from Dr. Morris White of the Joslin Diabetes Center (Boston, MA). The remaining reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Skin fibroblast cultures

Skin was cultured in DMEM (4 g/L glucose) supplemented with 10% FBS and 1% penicillin-streptomycin solution in a humid 37 C incubator with 5% CO₂ (28, 29). In 7–14 days, fibroblast outgrowth from the primary biopsy was evident and was allowed to grow until near confluence before further subculture. Cells were used for study between the 5th and 15th passage.

Cells were counted with each of the assays performed. Because these assays required slightly different culture conditions, each cell line was counted after exposure to the various conditions. All cells were rinsed twice with ice-cold PBS (pH 7.4), solubilized in trypsin, suspended in Isoton II, and immediately counted with a Coulter counter (Coulter Electronics, Inc., Hialea, FL). The mean value of triplicate wells was used to normalize results from different fibroblast cultures. Cells from a control and a PCOS woman were always run together in each assay; hence, assays were repeated in several control subjects because we had fewer well characterized control than PCOS cell lines.

Insulin and IGF-I binding

The numbers and affinities of insulin and IGF-I receptors in each fibroblast cell line were determined as described previously (28, 29). Cultured skin fibroblasts from control and PCOS women were grown to confluence in six-well trays before incubation for 48 h in serum-free DMEM containing 0.1% BSA. Cells were incubated for 4 h at room temperature with 0.1 µCi ¹²⁵I-labeled insulin or IGF-I, increasing concentrations (1,000,000 pmol/L) of unlabeled ligands, and binding buffer (50 mmol/L HEPES, 50 mmol/L Tris, 10 mmol/L MgCl₂, 2 mmol/L EDTA, 10 mmol/L dextrose, 50 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L CaCl₂, and 1% BSA, pH 7.4). Reactions were terminated by washing the cells with ice-cold PBS (pH 7.4), solubilizing them in 0.2 N NaOH, and counting the amount of ¹²⁵I-labeled ligand in a -counter. Receptor number and affinity were determined per 10⁴ cells by Scatchard analysis using the Ligand computer program (28).

Thymidine incorporation

Mitogenic effects of insulin in fibroblasts were assessed by the cellular incorporation of [methyl-³H]thymidine (38, 39). Cultured skin fibroblasts from control and PCOS women were grown to approximately 80% confluence in six-well trays before incubation for 48 h in serum-free DMEM containing 0.1% BSA. Cells then were incubated overnight at 37 C with DMEM containing 0.3% FBS and increasing doses of insulin (1,000,000 pmol/L) or IGF-I (100,000 pmol/L). Radioactive thymidine (1.5 µCi) was added to each well for an additional 4 h at 37 C. Incorporation of [methyl-³H]thymidine was terminated by aspirating media and rapidly washing the cells sequentially with ice-cold solutions of PBS (pH 7.4), 10% trichloroacetic acid, and 80% ethanol. Cells were solubilized with 1 N NaOH, neutralized with 1 N HCl, and quantitated in duplicate by liquid scintillation counting. The percentage of [methyl-³H]thymidine incorporated at each dose of insulin or IGF-I was reported per million cells.

Glucose incorporation into glycogen

Metabolic effects were estimated by the incorporation of glucose into glycogen in fibroblasts (40). Cultured skin fibroblasts from control and PCOS women were grown to confluence in six-well trays using low glucose DMEM with 10% FBS before incubation overnight at 37 C in serum-free low-glucose DMEM containing 0.5% BSA. Cells then were incubated in low glucose DMEM containing 1% BSA, increasing doses of insulin (1,000,000 pmol/L) or IGF-I (100,000 pmol/L), and D-[U-¹⁴C]glucose (0.4 µCi). After 2 h at 37 C, cells were washed with ice-cold PBS (pH 7.4) and solubilized in 2 N NaOH. Glycogen was extracted by repeated ethanol precipitations, and the amount of incorporated D-[U-¹⁴C]glucose was determined in duplicate by liquid scintillation counting. Results were expressed as nanomoles of radioactive glucose incorporated into glycogen per million cells.

PI3-kinase activity

Insulin and IGF-I signaling were assessed in PCOS fibroblasts by PI3-kinase activity associated with IRS-1 (41, 42). Cultured skin fibroblasts from control and PCOS women were grown to confluence in 150-mm dishes before overnight incubation in serum-free DMEM with 0.5% BSA. Cells were treated with 0–100,000 pmol/L doses of insulin or 0–10,000 pmol/L doses of IGF-I for 10 min at 37 C, rinsed twice in ice-cold PBS (pH 7.4) to terminate the reaction, and solubilized in 20 mmol/L Tris-HCl (pH 8.0) containing 137 mmol/L NaCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 10% glycerol, 1% Nonidet P-40, 150 µmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonylfluoride, and 10 µg/mL leupeptin. Equal protein concentrations of clarified lysates (1 mg) were immunoprecipitated with anti-IRS-1 antibody overnight at 4 C. Antibody-antigen complexes were incubated for 2 h at 4 C with protein A-Sepharose beads, and the beads were washed sequentially in PBS (pH 7.4) with 1% Nonidet P-40 (three times); 100 mmol/L Tris-HCl (pH 7.6) with 500 mmol/L LiCl (twice); 10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, and 1 mmol/L EDTA (once); and finally once in PI3-kinase assay buffer (20 nmol/L Tris, pH 7.4, containing 100 mmol/L NaCl, 10 mmol/L MgCl₂, 0.5 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 120 µmol/L adenosine). Washed beads were preincubated for 10 min at 30 C in 40 µL PI3-kinase assay buffer and 5 µL lipid mixture containing phosphatidylserine and phosphatidylinositol in 20 mmol/L HEPES and 1 mmol/L EDTA (pH 7.4). Kinase reactions were started by the addition of 100 µmol/L ATP containing 15 µCi [-³²P]ATP. After 10 min at 30 C, reactions were terminated by the addition of 1 N HCl, and phosphorylated lipids were extracted twice in chloroform-methanol (1:1). Products of PI3-kinase (15 µL) were spotted onto 1,2-cyclohexanediaminetetraacetic acid-treated silica gel 60 TLC plates and were resolved using the chromatography system of Walsh et al. (43). Labeled PI-3-phosphate was detected by autoradiography, scraped from the TLC plates, and quantified by Cherenkov scintillation counting. Results were expressed as counts per min of ³²P/mg protein.

Data analysis

Because considerable evidence suggests that defects in target tissue insulin action secondary to obesity, hyperinsulinemia, and/or hyperglycemia are acquired rather than intrinsic in both normal and PCOS individuals (1, 13, 24, 25, 26, 27, 28, 29, 44, 45, 46, 47), we pooled the data from obese and nonobese subjects. However, we also analyzed the data in a homogeneous subgroup of obese non-Hispanic white PCOS (n = 10) and control (n = 7) women. Simple linear correlations were assessed between parameters of fibroblast insulin/IGF-I action and body mass index (BMI), glucose tolerance, glucose/insulin ratio (G:I) (36), and insulin sensitivity. As different insulin assays were used at Mt. Sinai and at Penn State, the correlations between fibroblast insulin/IGF-I action and fasting insulin levels, G:I, or SI were examined only in the Penn State subjects who had the larger dataset. Statistical analyses were performed using SAS software (SAS Institute, Inc., Cary, NC), and all data are reported as the mean ± SE, except for reference data for insulin levels and SI in Table 1, which are reported as the mean ± SD.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical features

There were no significant differences in age between the PCOS and control subjects (data not shown). There were no significant differences in BMI in the obese non-Hispanic white PCOS compared to the obese non-Hispanic white control women (data not shown). Five of 16 PCOS women had impaired glucose tolerance, but none had diabetes mellitus by WHO criteria (Table 1) (35). There was a broad range of SI values in the PCOS women, with some values overlapping the normal range, as the PCOS subjects were not selected on the basis of insulin resistance.

Binding

There were no significant differences in insulin binding (control, 0.07 ± 0.04%; PCOS, 0.03 ± 0.01% bound/total/10⁴ cells; P = NS) or binding affinity (data not shown) between control and PCOS fibroblasts, consistent with previous studies (28, 29, 48). The amount of IGF-I binding in both control and PCOS fibroblasts was approximately 10-fold higher than the amount of insulin binding. There was no significant difference in IGF-I binding (control, 0.91 ± 0.30%; PCOS, 0.97 ± 0.30% bound/total/10⁴ cells; P = NS) or binding affinity (data not shown) between control and PCOS cells, similar to a previous report (48).

Insulin and IGF-I action

Basal rates of glycogen synthesis and thymidine incorporation did not differ significantly in PCOS compared to control cells (Figs. 1 and 2). Maximal rates of insulin-stimulated glucose incorporation into glycogen were significantly decreased in PCOS compared to control cells (P < 0.005; Fig. 1). These results remained significant in the subgroup analysis of obese non-Hispanic white PCOS vs. control women (P < 0.05; data not shown). Fold stimulation by insulin of glucose incorporation into glycogen was 4.2 ± 0.4-fold in control cells and 2.7 ± 0.4-fold in PCOS cells (P < 0.05). The insulin dose-response curves in PCOS women were so flat that the ED₅₀ could not be reliably estimated. The ED₅₀ insulin in control women for glycogen synthesis was 1565 ± 440 pmol/L. There were no significant differences in maximal rates of insulin-stimulated thymidine incorporation in PCOS compared to control cells in the entire sample (Fig. 2) or in the obese non-Hispanic white subgroup. The ED₅₀ insulin for thymidine incorporation was 4018 ± 841 pmol/L in controls vs. 5,122 ± 2,065 pmol/L in PCOS women (P = NS).

View larger version (18K): [in this window] [in a new window]   Figure 1. Insulin (PCOS, n = 12; control, n = 7) and IGF-I (PCOS, n = 11; control, n = 7) stimulation of glucose incorporation into glycogen in fibroblasts. Basal rates of glycogen synthesis are similar in PCOS and control cells. Stimulated responses are significantly decreased for both insulin at 1,000,000 pmol/L (*, P < 0.005) and IGF-I at 100,000 pmol/L (**, P < 0.05) in PCOS compared to control fibroblasts.

View larger version (19K): [in this window] [in a new window]   Figure 2. Insulin and IGF-I stimulation of thymidine incorporation in PCOS (n = 10) and control (n = 6) fibroblasts. There are no significant differences noted in basal rates or insulin/IGF-I-stimulated rates of thymidine incorporation in PCOS compared to control fibroblasts.

Basal and insulin-stimulated IRS-1-associated PI3-kinase did not differ in PCOS and control cells at any insulin concentration (Fig. 3). Consistent with the approximately 10-fold greater number of IGF1Rs compared to insulin receptors, IGF-I-stimulated, IRS-1-associated PI3-kinase was about 10-fold greater than insulin-stimulated, IRS-1-associated PI3-kinase activity, but did not differ at 0–10,000 pmol/L IGF-I in PCOS compared to control cells (Fig. 3). These results did not differ in the obese non-Hispanic white subgroup. The anticipated significant inverse correlations between BMI and SI or G:I were present (data not shown) (34, 36). There were no significant correlations, however, between fibroblast insulin/IGF-I action and BMI, glucose levels, insulin levels, G:I, or SI.

View larger version (20K): [in this window] [in a new window]   Figure 3. Insulin (PCOS, n = 10; control, n = 9) and IGF-I (PCOS, n = 5; control, n = 5) stimulation of IRS-1-associated PI3-kinase activation. There are no significant differences noted in basal or insulin/IGF-I-stimulated IRS-1-associated PI3-kinase activity in PCOS compared to control fibroblasts. However, there is an approximately 10-fold greater IGF-I-stimulated than insulin-stimulated IRS-1-associated PI3-kinase activity, consistent with the approximately 10-fold greater amount of IGF-I- compared to insulin-binding sites.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

We have previously shown reduced insulin-stimulated receptor autophosphorylation and substrate phosphorylation in receptors isolated from PCOS skin fibroblasts (29). This appeared to be secondary to constitutive serine phosphorylation of the receptor, which inhibits its tyrosine kinase activity. This decrease in receptor tyrosine kinase activity would be predicted to result in diminished phosphorylation of endogenous receptor substrates, such as IRS-1, which would result in decrease activation of PI3-kinase (1, 12, 13). We have shown decreased insulin-stimulated PI3-kinase activation in association with decreased glucose uptake in PCOS skeletal muscle (52). In the present study, however, there were no significant decreases in PI3-kinase activation at physiological insulin doses in PCOS compared to control fibroblasts despite significant decreases in insulin-stimulated glycogen synthesis. This suggests that this signaling pathway was not impaired by the defect in insulin receptor phosphorylation that we have previously reported (29), and that the defect impairing glycogen synthesis is in another signaling pathway or at a locus downstream of IRS-1-mediated PI3-kinase activation.

Fibroblasts do not contain the insulin-stimulated glucose transporter isoform, GLUT4, and manifest minimal insulin-stimulated glucose transport (21). It is thus possible that tissue differences explain our failure to detect decreases in IRS-1-associated PI3-kinase activation in fibroblasts, whereas such abnormalities are present in PCOS skeletal muscle (52). Indeed, recent studies suggest that IRS-2 mediates insulin’s metabolic effects in many tissues (53), and IRS-2 messenger ribonucleic acid is present in human fibroblasts (Dunaif, A., unpublished observations). As IRS-2-associated PI3-kinase activation was not examined in the present study, we were unable to address this possibility. A recent report in patients with pseudoacromegaly found decreased stimulation of IRS-1-associated PI3-kinase activity in cultured fibroblasts at submaximal (1,000 pmol/L), but not maximal (100,000 pmol/L), insulin doses (50). However, insulin stimulation of glycogen synthesis was normal in these patients, supporting the hypothesis that IRS-1-associated PI3-kinase activation is not involved in the regulation of glycogen synthesis in human fibroblasts. Further, in our PCOS fibroblasts there were no significant decreases in PI3-kinase activation at any insulin dose, suggesting that the mechanisms of insulin resistance differ from those in pseudoacromegaly (50) despite the fact that PCOS women occasionally have acromegaloid features (54).

Ciaraldi and colleagues failed to find decreases in insulin-stimulated glycogen synthesis in fibroblasts from insulin-resistant PCOS compared to those from control women (48). Otherwise, their results were similar to ours. They studied a smaller sample of PCOS (n = 6) and control (n = 5) women than we did, but we still found significant decreases in insulin-stimulated glycogen synthesis in our subgroup analysis of obese non-Hispanic white PCOS and control women with a similar sample size to that in their study (48). Their subjects had very low mean SI measurements, whereas some of our PCOS women had values within the normal range. However, there were no significant correlations between fibroblast insulin/IGF-I action and BMI, glucose levels, insulin levels, or insulin sensitivity in our study. Our subjects were recruited from different geographic regions compared to those of Ciaraldi et al. (48), so it remains possible that more subtle ethnic differences explain the discrepant findings between our studies.

The failure to detect a relationship between insulin action in the cultured fibroblasts and adiposity or glycemia is compatible with evidence indicating that these factors produce reversible changes in insulin sensitivity (13, 24, 26, 44, 45, 46, 47). The failure to detect a relationship between in vivo insulin (fasting insulin levels or SI) and fibroblast (glycogen synthesis) parameters of insulin action may reflect the fact that fibroblasts are not target tissues for insulin-mediated glucose uptake (55). We also failed to find a significant relationship between fibroblast insulin receptor phosphorylation and in vivo insulin action in our previous study (29). Insulin action in vivo has been significantly correlated with glycogen synthesis in cultured muscle cells, the major target tissue for insulin-mediated glucose uptake (56). Alternatively, other factors, such as a relatively small sample size or a more complex relationship between insulin action in vivo and in fibroblasts, may have accounted for our failure to find significant associations. It is also possible, as suggested by Ciaraldi et al. (48), that there are tissue differences in insulin action in PCOS.

The persistence of defects in insulin action in cultured cells that have been removed from the in vivo environment for generations is consistent with a genetic defect. In our previous report (29), abnormalities in insulin receptor phosphorylation were also found in cultured cells, suggesting a genetically programed abnormality. Most recently, we found familial clustering of hyperandrogenemia in PCOS sisters (57). Taken together, these observations provide further support for our hypothesis that both the insulin resistance and the reproductive abnormalities of PCOS have a genetic component (1).

There is now considerable evidence to indicate that hyperinsulinemia amplifies hyperandrogenemia in PCOS by stimulating ovarian and adrenal androgen production (1, 3, 4, 6, 10) as well as pituitary LH release (1, 4). It had been suggested that this insulin action was mediated via spillover occupancy of the IGF1R (1, 6). Studies in normal and PCOS ovarian cells (9, 10), however, strongly suggest that insulin is acting through its own receptor to stimulate steroidogenesis. The current study provides a mechanism by which there could be resistance to insulin’s action on carbohydrate metabolism, but preserved insulin action on steroidogenesis in PCOS: a selective defect affecting metabolic, but not mitogenic, signaling pathways. This hypothesis will require investigation in steroidogenic tissues, such as thecal cells, from PCOS women.

In summary, we have shown that cultured PCOS fibroblasts are insulin resistant. Moreover, this resistance affects the metabolic, but not the mitogenic, actions of insulin. IGF-I action is similarly impaired, suggesting that the same postreceptor signaling pathways are used for insulin and IGF-I action on glycogen synthesis in human fibroblasts. Activation of IRS-1-associated PI3-kinase does not appear to be impaired. This observation suggests that other signaling pathways or pathways downstream of IRS-1-mediated PI3-kinase activation are defective and that the abnormality we have reported in PCOS fibroblast insulin receptor phosphorylation does not impair IRS-1 activation of PI3-kinase in this cell type. PCOS will provide an excellent model in which to further investigate the mechanisms of insulin receptor signal specificity.

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The authors thank Dr. Morris White for the generous gift of the anti-IRS-1 antibody, and Dr. Theodore Ciaraldi for helpful discussions concerning assay methods.

	Footnotes

 
¹ Preliminary findings were presented as part of the 56th Annual Meeting of the American Diabetes Association, San Francisco, CA, June 1996 (Abstract 317). This work was supported by NIH Postdoctoral Training Grants T32-DK-07684 (to C.-B.B.) and R01-DK-40605 (to A.D.).

Received March 2, 1999.

Revised June 2, 1999.

Accepted June 8, 1999.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Dunaif A. 1997 Insulin resistance and the polycystic ovary syndrome: mechanisms and implications for pathogenesis. Endocr Rev. 18:774–800.[Abstract/Free Full Text]
2. Knochenhauser ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots, LR, Azziz R. 1998 Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Enocrinol Metab. 83:3078–3082.[Abstract/Free Full Text]
3. Nestler JE, Barlascini CO, Matt DW, Steingold KA, Plymate SR, Clore JN, Blackard WG. 1989 Suppression of serum insulin by diazoxide reduces serum testosterone levels in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab. 68:1027–1032.[Abstract]
4. Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R. 1996 The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab. 81:3299–3306.[Abstract]
5. Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR. 1997 The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem. 272:6653–6662.[Abstract/Free Full Text]
6. Barbieri RL, Makris A, Randall RW, Daniels G, Kistner RW, Ryan KJ. 1986 Insulin stimulates androgen accumulation in incubations of ovarian stroma obtained from women with hyperandrogenism. J Clin Endocrinol Metab. 62:904–910.[Abstract]
7. Froesch ER, Zapf J. 1985 Insulin-like factors and insulin: comparative aspects. Diabetologia. 28:485–493.[Medline]
8. Fradkin JE, Eastman RC, Lesniak MA, Roth J. 1989 Specificity spillover at the hormone receptor–exploring its role in human disease. N Engl J Med. 320:640–645.[Medline]
9. Willis D, Franks S. 1995 Insulin action in human granulosa cells from normal and polycystic ovaries is mediated by the insulin receptor and not the type-I insulin-like growth factor receptor. J Clin Endocrinol Metab. 80:3788–3790.[Abstract]
10. Nestler JE, Jakubowicz DJ, De Vargas AF, Brink C, Quintero N, Medina F. 1998 Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system. J Clin Endocrinol Metab. 83:2001–2005.[Abstract/Free Full Text]
11. Ek I, Arner P, Bergqvist A, Carlstrom K, Wahrenberg H. 1997 Impaired adipocyte lipolysis in nonobese women with the polycystic ovary syndrome: a possible link to insulin resistance? J Clin Endocrinol Metab. 82:1147–1153.[Abstract/Free Full Text]
12. White MF. 1998 The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res. 53:119–138.
13. Virkamäki A, Kohjiro U, Kahn CR. 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest. 103:931–941.[Medline]
14. Blakesley VA, Scrimgeour A, Esposito D, Le Roith D. 1996 Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev. 7:153–159.[CrossRef][Medline]
15. Kanai F, Ito K, Todaka M, et al. 1993 Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun. 195:762–768.[CrossRef][Medline]
16. Okada T, Kawano Y, Sakakibara T, Osamu H, Ui M. 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipoysis in rat adipocytes. J Biol Chem. 269:3568–3573.[Abstract/Free Full Text]
17. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR. 1994 Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 14:4902–4911.[Abstract/Free Full Text]
18. Marchand-Brustel Y, Gautier N, Cormont M, Van Obberghen E. 1995 Wortmannin inhibits the action of insulin but not that of okadaic acid in skeletal muscle. Comparison with fat cells. Endocrinology. 136:3564–3570.[Abstract]
19. Lawrence JC, Roach PJ. 1997 New insights into the role and mechanism of glycogen synthase activation by insulin. Diabetes. 46:541–547.[Abstract]
20. Takata Y, Webster NJG, Olefsky JM. 1991 Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signaling but enhanced mitogenic signaling properties. J Biol Chem. 266:9135–9138.[Abstract/Free Full Text]
21. Flier JS, Moller DE, Moses AC, et al. 1993 Insulin-mediated pseudoacromegaly: clinical and biochemical characterization of a syndrome of selective insulin resistance. J Clin Endocrinol Metab. 76:1533–1541.[Abstract]
22. Wells AM, Sutcliffe IC, Johnson AB, Taylor R. 1993 Abnormal activation of glycogen synthesis in fibroblasts from NIDDM subjects. Evidence for abnormality specific to glucose metabolism. Diabetes. 42:583–589.[Abstract]
23. Podskalny JM, Kahn CR. 1980 Insulin activates glycogen synthase in cultured human fibroblasts. Diabetes. 29:724–729.[Abstract]
24. Prince MJ, Tsai P, Olefsky JM. 1981 Insulin binding, internalization, and insulin receptor regulation in fibroblasts from type II, non-insulin-dependent diabetic subjects. Diabetes. 30:596–600.[Abstract]
25. Stuart CA, Pietrrzyk RA, Peters EJ, Smith FE, Prince MJ. 1989 Autophosphorylation of cultured skin fibroblast insulin receptors from patients with severe insulin resistance and acanthosis nigricans. Diabetes. 38:328–332.[Abstract]
26. Henry RR, Ciaraldi TP, Mudaliar S, Abrams L, Nikoulina SE. 1996 Acquired defects of glycogen synthase activity cultured human skeletal muscle cells. Diabetes. 45:400–407.[Abstract]
27. Henry RR, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Soo Park K. 1996 Glycogen synthase activity is reduced in cultured skeletal muscle cells of non-insulin-dependent diabetes mellitus subjects. J Clin Invest. 98:1231–1236.[Medline]
28. Sorbara LR, Tang Z, Cama A, et al. 1994 Absence of insulin receptor gene mutations in three women with the polycystic ovary syndrome. Metabolism. 43:1568–1574.[CrossRef][Medline]
29. Dunaif A, Xia J, Book C-B, Schenker E, Tang Z. 1995 Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle: a potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest. 96:801–810.
30. Dunaif A, M Graf, Mandeli J, Laumas V, Dobrjansky A. 1987 Characterization of groups of hyperandrogenic women with acanthosis nigricans, impaired glucose tolerance, and/or hyperinsulinemia. J Clin Endocrinol Metab. 65:499–507.[Abstract]
31. Dunaif A, Futterweit W, Segal KR, Dobrjansky A. 1989 Profound peripheral insulin resistance, independent of obesity, in the polycystic ovary syndrome. Diabetes. 38:1165–1174.[Abstract]
32. Dunaif A, Segal KR, Shelley DR, Dobrjansky A, Lincolai T. 1992 Evidence for distinctive and intrinsic defects in insulin action in the polycystic ovary syndrome. Diabetes. 41:1257–1266.[Abstract]
33. Rosenbaum D, Haber R, Dunaif A. 1993 Insulin resistance in polycystic ovary syndrome: decreased expression of GLUT4 glucose transporters in adipocytes. Am J Physiol. 264:E197–E202.
34. Dunaif A, Finegood DT. 1996 B-Cell dysfunction independent of obesity and glucose intolerance in the polycystic ovary syndrome. J Clin Endocrinol Metab. 81:942–947.[Abstract]
35. Modan M, Harris MI, Halkin H. 1989 Evaluation of WHO and NDDG criteria for impaired glucose tolerance. Results from two national samples. Diabetes. 38:1630–1635.[Abstract]
36. Legro RS, Finegood D, Dunaif A. 1998 A fasting glucose to insulin ratio is a useful measure of insulin sensitivity in women with polycystic ovary syndrome. J Clin Endocrinol Metab. 83:2694–2698.[Abstract/Free Full Text]
37. Legro RS, Kunselman A, Dodson WC, Dunaif A. 1999 Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women. J Clin Endocrinol Metab. 84:165–169.[Abstract/Free Full Text]
38. Sasaoka T, Kobayashi M, Takata Y, et al. 1988 Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor. Diabetes. 37:1515–1523.[Abstract]
39. Flier JS, Usher P, Moses AC. 1986 Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts. Proc Natl Acad Sci USA. 83:664–668.[Abstract/Free Full Text]
40. Wong EH-A, Tan C, Khoo H, Ng F, Lim K, Ciaraldi TP. 1995 Rat fibroblast cells overexpressing kinase-inactive human insulin receptors are insulin responsive: influence of growth conditions. Endocrinology. 136:1459–1467.[Abstract]
41. Ruderman NB, Kapellar R, White M, Cantley LC. 1990 Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci USA. 87:1411–1415.[Abstract/Free Full Text]
42. Backer JM, Schroeder GG, Kahn CR, Myers Jr MG, Wilden PA, Cahill DA, White MF. 1992 Insulin stimulation of phosphatidylinositol 3-kianse activity maps to insulin receptor regions required for endogenous substrate phosphorylation. J Biol Chem. 267:1367–1374.[Abstract/Free Full Text]
43. Walsh JP, Caldwell KK, Majerus PW. 1995 Formation of phosphatidylinositol 3-phosphate by insomerization from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci USA. 88:9184–9187.[Abstract/Free Full Text]
44. Freidenberg G, Reichart RD, Pories WJ, Sinha MK. 1988 Reversability of defective adipocyte insulin receptor kinase activity in non-insulin-dependent diabetes mellitus. Effect of weight loss. J Clin Invest. 82:1398–1406.
45. Lima FB, Ties RS, Garvey WT. 1991 Glucose and insulin regulate insulin sensitivity in primary cultured adipocytes without affecting insulin receptor kinase activity. Endocrinology. 128:2415–2426.[Abstract]
46. Bak JF, Moller N, Schmitz O, Saaek A, Pedersen O. 1991 In vivo insulin action and muscle glycogen synthase activity in type 2 (non-insulin-dependent) diabetes mellitus: effects of diet treatment. Diabetologia. 35:777–784.
47. Kiddy DS, Hamilton-Fairly D, Bush A, Short F, Anyaoku V, Reed MJ, Franks S. 1992 Improvement in endocrine and ovarian function during dietary treatment of obese women with polycystic ovary syndrome. Clin Endocrinol (Oxf). 36:105–111.[Medline]
48. Ciaraldi TP, Morales AJ, Hickman MG, Odom-Ford, Yen SSC, Olefsky JM. 1998 Lack of insulin resistance in fibroblasts from subjects with polycystic ovary syndrome. Metabolism. 47:940–946.[CrossRef][Medline]
49. Fiad TM, Smith TP, Cunningham SK, McKenna TJ. 1998 Decline in insulin-like growth factor I levels after clomiphene citrate does not correct hyperandrogenemia in polycystic ovary syndrome. J Clin Endocrinol Metab. 83:2394–2398.[Abstract/Free Full Text]
50. Dib K, Whitehead JP, Humphreys PJ, et al. 1998 Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. J Clin Invest. 101:1111–1120.[Medline]
51. Hawkins M, Barzilai N, Chen W, Angelov I, Hu M, Cohen P, Rossetti L. 1996 Increased hexosamine availability similarly impairs the action of insulin and IGF-I on glucose disposal. Diabetes. 45:1734–1743.[Abstract]
52. Dunaif A, Diamanti E. 1997 Defective insulin receptor signaling in vivo in the polycystic ovary syndrome (PCOS) [Abstract 91]. Proc of the 57th Annual Scientific Meet of the Am Diabetes Assoc. 1997.
53. Withers DJ, Gutierrez JS, Towery H, et al. 1998 Disruption of IRS-2 causes type 2 diabetes in mice. Nature. 391:900–904.[CrossRef][Medline]
54. Mechanick J, Dunaif A. 1990 Masculinization: a clinical approach to the diagnosis and treatment of hyperandrogenic women. In: Mazzaferri E, ed. Advances in endocrinology and metabolism. Chicago: Mosley-YearBook; vol 1:129–173.
55. Kahn BB. 1998 Type 2 diabetes: when insulin secretion fails to compensate for insulin resistance. Cell. 92:593–596.[CrossRef][Medline]
56. Henry RR, Abrams L, Nikoulina S, Ciaraldi TP. 1995 Insulin action and glucose metabolism in nondiabetic control and NIDDM subjects. Diabetes. 44:936–946.[Abstract]
57. Legro RS, Driscoll D, Strauss JF, Fox J, Dunaif A. 1998 Evidence for a genetic basis for hyperandrogenemia in the polycystic ovary syndrome. Proc Natl Acad Sci USA. 95:14956–14960.[Abstract/Free Full Text]

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