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Original Studies |
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
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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.
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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.
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Results |
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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).
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). The results remained similar in the obese
non-Hispanic white subgroup, but the difference no longer achieved
statistical significance. IGF-I-stimulated thymidine incorporation was
similar in PCOS and control cells (Fig. 2). These results did not
change in the obese non-Hispanic white subgroup. As maximally
stimulating concentrations of IGF-I were not examined, the
ED50 for IGF-I action could not be calculated. The
dose-response curves for insulin and IGF-I action on glycogen synthesis
and thymidine incorporation indicated that stimulation of glycogen
synthesis and thymidine incorporation occurred at physiological
concentrations of these hormones (22, 28, 29, 31, 32, 33, 49, 50). This is
consistent with each ligand acting through its cognate receptor
(8).
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.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Footnotes |
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Received March 2, 1999.
Revised June 2, 1999.
Accepted June 8, 1999.
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K.-M. Seow, C.-C. Juan, L.-Y. Wu, Y.-P. Hsu, W.-M. Yang, Y.-L. Tsai, J.-L. Hwang, and L.-T. Ho Serum and adipocyte resistin in polycystic ovary syndrome with insulin resistance Hum. Reprod., January 1, 2004; 19(1): 48 - 53. [Abstract] [Full Text] [PDF]
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 I. Munir, H.-W. Yen, D. H. Geller, D. Torbati, R. M. Bierden, S. R. Weitsman, S. K. Agarwal, and D. A. Magoffin 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 Endocrinology, January 1, 2004; 145(1): 175 - 183. [Abstract] [Full Text] [PDF]
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 V. De Leo, A. la Marca, and F. Petraglia Insulin-Lowering Agents in the Management of Polycystic Ovary Syndrome Endocr. Rev., October 1, 2003; 24(5): 633 - 667. [Abstract] [Full Text] [PDF]
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C. C. L. Wang, I. Gurevich, and B. Draznin Insulin Affects Vascular Smooth Muscle Cell Phenotype and Migration Via Distinct Signaling Pathways Diabetes, October 1, 2003; 52(10): 2562 - 2569. [Abstract] [Full Text] [PDF]
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D. Dereli, G. Ozgen, F. Buyukkececi, E. Guney, and C. Yilmaz Platelet Dysfunction in Lean Women with Polycystic Ovary Syndrome and Association with Insulin Sensitivity J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2263 - 2268. [Abstract] [Full Text] [PDF]
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R. Azziz Polycystic Ovary Syndrome, Insulin Resistance, and Molecular Defects of Insulin Signaling J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4085 - 4087. [Full Text] [PDF]
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M. Li, J. F. Youngren, A. Dunaif, I. D. Goldfine, B. A. Maddux, B. B. Zhang, and J. L. Evans Decreased Insulin Receptor (IR) Autophosphorylation in Fibroblasts from Patients with PCOS: Effects of Serine Kinase Inhibitors and IR Activators J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4088 - 4093. [Abstract] [Full Text] [PDF]
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L. Haddad, J. C. Evans, N. Gharani, C. Robertson, K. Rush, S. Wiltshire, T. M. Frayling, T. J. Wilkin, A. Demaine, A. Millward, et al. Variation within the Type 2 Diabetes Susceptibility Gene Calpain-10 and Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2606 - 2610. [Abstract] [Full Text] [PDF]
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A. Dunaif, X. Wu, A. Lee, and E. Diamanti-Kandarakis Defects in insulin receptor signaling in vivo in the polycystic ovary syndrome (PCOS) Am J Physiol Endocrinol Metab, August 1, 2001; 281(2): E392 - E399. [Abstract] [Full Text] [PDF]
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M.W. Elting, T.J.M. Korsen, P.D. Bezemer, and J. Schoemaker Prevalence of diabetes mellitus, hypertension and cardiac complaints in a follow-up study of a Dutch PCOS population Hum. Reprod., March 1, 2001; 16(3): 556 - 560. [Abstract] [Full Text] [PDF]
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J. W. M. Martens, D. H. Geller, W. Arlt, R. J. Auchus, V. S. Ossovskaya, H. Rodriguez, A. Dunaif, and W. L. Miller Enzymatic Activities of P450c17 Stably Expressed in Fibroblasts from Patients with the Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4338 - 4346. [Abstract] [Full Text]
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