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Original Articles |
Division of Endocrinology (L.P., D.S.-Y., A.S.), Beth Israel Medical Center and Albert Einstein College of Medicine, New York, New York 10003; and Division of Endocrinology (L.P., S.D., M.M.) and Center for Reproductive Medicine and Infertility (L.P., H.-C.L., M.C.Y., Z.R.), Weill Medical College of Cornell University, New York, New York 10021
Address all correspondence and requests for reprints to: Leonid Poretsky, M.D., Division of Endocrinology, Beth Israel Medical Center, First Avenue at 16th Street, New York, New York 10003. E-mail: Lporetsk{at}bethisraelny.org
Abstract
Hyperandrogenism observed in women with a variety of insulin-resistant states is thought to be due to a stimulatory effect of insulin on ovarian steroid hormone production. However, it is not known what mechanisms could allow the ovary to remain sensitive to insulin while classical target organs for insulin action (liver, fat, and muscle) exhibit insulin resistance. One hypothesis proposed to explain this paradox suggests that a postbinding divergence of insulin receptor signaling occurs in the ovary and that signaling pathways for steroid hormone synthesis and other ovarian effects of insulin may be distinct from classical glucose signaling pathways. We now report that activation of phosphatidyl-inositol-3 (PI-3) kinase, which is crucial for glucose transport, is not necessary for the insulin-induced stimulation of progesterone production or for the insulin-induced inhibition of insulin-like growth factor binding protein 1 (IGFBP-1) production in cultured human ovarian cells.
Human granulosa cells obtained during in vitro fertilization procedures were cultured with 10, 102, 103, or 104 ng/mL insulin with or without preincubation with 100 nM wortmannin, a specific irreversible inhibitor of PI-3 kinase. IGFBP-1 concentration in the conditioned medium was measured using immunoradiometric assay or by Western blot analysis. Progesterone concentration was measured using RIA. Additional studies were carried out in cultures of human ovarian cells prepared from homogenized whole ovarian tissue of a woman with a family history of breast cancer and a mutation of BRCA-1 gene who underwent bilateral oophorectomy. These cells were cultured with 103 ng/mL insulin with or without preincubation with 100 nM wortmannin.
Two-way ANOVA was used to compare mean values of IGFBP-1 and progesterone according to insulin dose and the use of wortmannin. In cultured granulosa cell medium, progesterone production was stimulated by insulin in a dose-related manner up to 175% of control (P < 0.0001). In tissue culture medium from ovarian cells obtained from a patient with BRCA-gene mutation, concentration of progesterone in the tissue culture medium increased from 2.5 ± 0.2 ng/mL for control to 5.4 ± 0.3 ng/mL for cells incubated with insulin (P < 0.001). IGFBP-1 production in tissue culture medium from human granulosa cells was inhibited by insulin to the nadir of 45% of control (P < 0.0001). Preincubation with wortmannin, despite complete inhibition of PI-3 kinase in both cell systems confirmed by Western blot analysis, failed to significantly alter these results.
We conclude that inhibition of PI-3 kinase by wortmannin fails to abolish stimulatory effect of insulin on progesterone production or inhibitory effect of insulin on IGFBP-1 production in cultured human ovarian cells. These findings suggest that activation of PI-3 kinase, an enzyme crucial for insulin-stimulated glucose transport, is not necessary for the above effects of insulin in the ovary. These data provide evidence for the presence of PI-3 kinase-independent insulin signaling pathway(s) in human ovarian cells.
OVARIAN HYPERANDROGENISM IS associated with a variety of insulin-resistant states, such as the type A and B syndromes of insulin resistance and acanthosis nigricans, lipoatrophic diabetes, leprechaunism, and polycystic ovary syndrome (1, 2, 3). The cause of hyperandrogenism in insulin-resistant states remains unknown, but a hypothesis suggesting that androgen production in the ovaries of insulin-resistant women is stimulated by high levels of circulating insulin has gained popularity (4, 5). However, it is unclear how the ovary can remain sensitive to insulin while classical target organs for insulin action (liver, fat, and muscle) exhibit significant, sometimes extreme, insulin resistance (3).
Several hypotheses have been proposed to explain this paradox (3, 6). Among these is a hypothesis that postulates that postbinding divergence of insulin signaling pathways occurs in the ovary (3), so that insulin-signaling pathways mediating ovarian effects of insulin may be distinct from the classical insulin signaling pathways involved in glucose transport. However, experimental evidence for this hypothesis has been lacking. In the present study we examined whether activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), which is crucial for insulin-induced glucose transport (7, 8), is necessary for two ovarian effects of insulin: stimulation of progesterone synthesis and inhibition of insulin-like growth factor binding protein 1 (IGFBP-1) production.
Materials and Methods
All of the studies described in this report were approved by the Institutional Review Board at Beth Israel Medical Center and the Weill Medical College of Cornell University.
Human granulosa cells
Human granulosa cells (which are a byproduct of ovum retrieval procedures) were obtained in the course of in vitro fertilization (IVF) and purified on Percoll gradients as previously described (9, 10). The cells were obtained over the course of 2 yr from 42 women and pulled from several patients at a time to assure adequate cell number for the experiments. The diagnoses included male factor infertility, ovulatory dysfunction, tubal factor infertility, and uterine factor infertility.
After removal of the ovum, the content of aspirated follicles was centrifuged at 2200 revolutions per minute (rpm) for 10 min in a J-6B centrifuge (Beckman Coulter, Inc., Fullerton, CA). The follicular fluid was then aspirated and the cells were resuspended in HBSS and layered on 50% Percoll (diluted with HBSS). The Percoll was centrifuged at 4000 rpm for 30 min at room temperature. The interface cells were removed and washed with HBSS, sedimented with the same centrifugation force, resuspended in McCoy’s 5A tissue culture medium, and counted in a hemocytometer. The cells were then incubated for 48 h at 37 C, 5% CO2, 90% humidity in McCoy’s 5A tissue culture medium supplemented with 10% FCS, 100 µg/mL penicillin, and 100 µg/mL streptomycin in 24-well Falcon tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ). The density of the cells was approximately 2 x 105/mL. After 48 h of incubation, the medium supplemented with calf serum was removed and replaced by serum-free medium. The cells were incubated in serum-free medium for an additional 24-h period under similar conditions. Experiments outlined below were then carried out in serum-free medium.
Whole ovarian cell culture
Human ovarian tissue was obtained during bilateral salpingoophorectomy from a woman with a family history of breast cancer and a mutation of BRCA gene. The cell culture was established as previously described (11). The tissue was minced with fine scissors into small fragments (1–2 mm) and then rinsed with HBSS. After the addition of 4 mg/mL collagenase, the cells were incubated in water bath for 5 min at 37 C and then mixed vigorously. The supernatant was then removed, the tissue fragments were transferred into fresh tubes containing tissue culture medium, and the procedure was repeated for the remaining tissue fragments. The cells were then sedimented by centrifugation at 2200 rpm for 5 min in a J-6B Beckman Coulter, Inc. centrifuge and washed with medium. The cells were plated in 24-well Falcon tissue culture plates in McCoy’s 5A tissue culture medium supplemented with 10% FCS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The cells grew to a density of 2 x 105/mL at 37 C, 5% CO2, 90% humidity. After incubation in serum-free medium for 24 h, the cells’ cultures were used for the studies outlined below in serum-free medium.
Assessment of PI-3 kinase activity
Assay for PI-3 kinase activity was carried out as previously
described (12) after the experiments with or without
insulin and wortmannin were completed. Cell cultures were washed with
freshly prepared buffer A (137 mM NaCl; 20 mM
Tris-HCl, pH 7.4; 1 mM CaCl2; 1
mM MgCl2; and 0.1 mM
sodium orthovanadate) and solubilized in ice-cold lysis buffer B
(buffer A containing 1% Nonidet P-40 and 1 mM
phenylmethylsulfonyl fluoride). Lysates were centrifuged at 14,000
x g for 10 min at 4 C to sediment-insoluble material.
Supernatant fraction was immunoprecipitated with the anti-insulin
receptor substrate-1 polyclonal antibody (Upstate Biotechnology, Inc.) followed by the addition of protein
A-Sepharose. The immunoprecipitate complexes were collected by
centrifugation and washed with buffer B, then with buffer C (100
mM Tris-HCl, pH 7.4; 5 mM
LiCl; and 0.1 mM sodium orthovandate), and then
with buffer D (containing 10 mM Tris-HCl, pH 7.4;
150 mM NaCl; 5 mM EDTA).
After removing the last wash completely, 50 µL buffer D, 10 µL
phosphatidylinositol (PI); and 10 µL of 100 mM
MgCl2 were added to each test tube. Reaction was
initiated by addition of 5 µL [
-32P]ATP
(0.88 mM ATP containing 30 µCi of
[-32P]ATP, 3000 µCi/mmol, 10 Ci/mL, and 20
mM MgCl2). Incubation with
agitation for 10 min at 37 C was carried out. The reaction was
terminated by the addition of 20 µL of 6 M HCl
and 160 µL CHCl3-MeOH (1:1). The organic phase
was extracted, spotted on a silica gel thin layer chromatography
plate (Analytical Technology, Newark, NJ) and developed in
CHCl3-MeOH-H2O-NH4OH
(60:47:11:3.2). Plates were dried and subsequently visualized by
autoradiography.
Experimental procedures
Human granulosa cell cultures were incubated in serum-free McCoy’s 5A culture medium with or without 10, 102, 103, and 104 ng/mL insulin at 37 C, 5% CO2, 90% humidity. Samples of whole ovarian cell cultures from a patient with BRCA gene mutation were incubated with or without 103 ng/mL insulin. Some cell samples underwent a 30-min preincubation with 100 nM wortmannin (Sigma, St Louis, MO), a potent irreversible inhibitor of PI-3 kinase (13, 14, 15). After 24 h incubation, the medium was removed, and the new set of serum-free medium with insulin was added. After an additional 24 h (total of 48 h) of incubation, the medium was collected and stored at -20 C. IGFBP-1 was assayed by immunoradiometric assay (specific immunoradiometric assay kit from DSL-7800, Diagnostics Systems Laboratories, Inc., Webster, TX) or Western blot analysis. Progesterone concentration in the conditioned medium was measured using RIA (ACS:180 kit from Bayer Corp., Tarrytown, NY or Pantex, Santa Monica, CA).
Western blot assay
The effect of insulin (103 ng/mL) and wortmannin on IGFBP-1 production in human granulosa cells was also assessed using Western blot analysis. The medium was collected and proteins were precipitated with ethanol. Equal amounts of proteins were loaded on to a 12% SDS-PAGE and transferred onto nitrocellulose membrane using a wet transfer system (Bio-Rad Laboratories, Inc., Hercules, CA). The transfer procedure was performed according to the manufacturer’s recommendations. The membranes were incubated with a 1:500 dilution of primary anti- IGFBP-1 antibody (goat polyclonal IgG, epitope mapping at the carboxy terminus of IGFBP-1 of human origin, specific for IGFBP-1; not cross-reactive with IGFBP-2, 3, 4, 5, 6, and 7) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The second antibody was rabbit antigoat IgG conjugated with horse peroxidase (1:2000 dilution) (Santa Cruz Biotechnology, Inc.). The bands were detected by chemiluminescence. The protein measurement was carried out using the Bio-Rad Laboratories, Inc. detergent-compatible protein assay (Bio-Rad Laboratories, Inc.).
Statistical analysis
Two-way ANOVA was used to compare means according to insulin concentration (0, 10, 102, 103, and 104 ng/mL) and presence or absence of wortmannin. The statistical interactions between the two sets of data obtained with or without preincubation with wortmannin were examined. Upon finding a difference for insulin, pairwise Bonferroni-adjusted contrasts were analyzed to determine which insulin concentrations differed from each other. The Sigma plot and regression analysis were used to develop best-fit insulin dose-response curve for progesterone.
Results
Representative assays of PI-3 kinase activity in human whole
ovarian cell cultures from a patient with BRCA gene mutation and in
cultured human granulosa cells obtained during IVF are shown in Fig. 1
, A and B, respectively. As expected,
PI-3 kinase activity was stimulated by insulin and completely inhibited
by wortmannin in both systems.
|
. Under the influence of insulin,
progesterone production increased up to 175% of control
(P < 0.0001). No statistically significant effect of
wortmannin on this interaction between insulin and progesterone was
noted.
|
. The progesterone
concentration was 2.5 ± 0.2 ng/mL in the tissue culture medium
from the control cells; 5.4 ± 0.3 ng/mL in the tissue culture
medium from the cells incubated with 103 ng/mL
insulin (P < 0.001 compared with control cells); and
4.8 ± 0.1 ng/mL in the tissue culture medium from the cells which
were incubated with 103 ng/mL insulin after
preincubation with wortmannin (P = 0.2, not
significant, compared with the cells incubated with insulin without
preincubation with wortmannin).
|
. Under influence of insulin IGFBP-1
production in these cells was reduced to a nadir of 45% of control
(P < 0.0001). Preincubation with wortmannin failed to
abolish this inhibitory effect of insulin on IGFBP-1 production.
|
). Insulin (103
ng/mL) inhibited IGFBP-1 production in cultured human granulosa cells
to undetectable levels. Preincubation with wortmannin failed to abolish
this inhibitory effect of insulin on IGFBP-1 production.
|
Description of hyperandrogenism and polycystic ovaries or ovarian hyperthecosis in women with syndromes of extreme insulin resistance due to insulin receptor mutations or anti-insulin receptor autoantibodies lead to a hypothesis that insulin, when circulating in high concentrations, may stimulate ovarian growth, androgen production, and cyst development (1, 2, 3, 4, 16, 17). Demonstration of the presence of insulin resistance in patients with garden variety polycystic ovary syndrome (18) suggested that insulin resistance and subsequent hyperinsulinemia may be instrumental in the development of this common disorder. Improvement of hyperandrogenism in patients treated with insulin sensitizing agents (which lead to a reduction in circulating insulin concentrations) further strengthened this hypothesis (19, 20, 21, 22, 23). However, it remains unclear how insulin can stimulate ovarian androgen production, growth, and cyst formation when insulin resistance, sometimes of extreme nature, is present in classical target organs for insulin action.
To explain this paradox, at least two hypotheses have been proposed (3, 4). The first hypothesis postulates that in insulin-resistant states ovarian effects of insulin are mediated by the type I IGF receptor (4). This mechanism of insulin action in the ovary is likely to operate in patients with extreme forms of insulin resistance (like the type A and B syndromes of insulin resistance and acanthosis nigricans) whose circulating insulin levels are extraordinarily high (1, 2). However, it seems unlikely that this mechanism is operable in patients with garden variety polycystic ovary syndrome whose circulating insulin levels are only moderately increased (3, 6, 18). Besides, studies of insulin action in human ovarian cells that examined the nature of receptors mediating insulin effects in the ovary produced evidence of insulin action via its own receptor rather than the type I IGF receptor (24, 25, 26).
The second hypothesis, which attempts to explain the paradox of insulin-induced ovarian stimulation in patients with insulin resistance, proposes that postbinding insulin signaling pathways in the ovary may diverge. According to this hypothesis, when one of these pathways (for example, insulin signaling pathway responsible for glucose transport) is deficient, thus producing a state of insulin resistance, other pathways may continue to operate normally (3). In fact these alternate pathways may become hyperactive because of hyperinsulinemia that develops due to the presence of a defect in the glucose-transport pathway. Although this latter hypothesis achieved substantial popularity, the experimental evidence for an alternate pathway of insulin action in the ovary has been minimal. We are aware of only one study in human ovarian cells that demonstrated that insulin signaling pathway mediating androgen production in these cells involves inositol-glycan signaling system rather than the classical tyrosine kinase system (26). Inositol-glycans are generated at the cell membrane after insulin binds to the insulin receptor (27). Their generation does not involve activation of the insulin receptor tyrosine kinase or PI-3 kinase activation, both of which are necessary for the glucose transport.
In the current report we present evidence that activation of PI-3 kinase is not necessary for two ovarian effects of insulin in the ovarian cells. These effects are the insulin-stimulated progesterone production and insulin-induced inhibition of IGFBP-1 production.
The results of our experiments largely depend on the ability of wortmannin to inhibit PI-3 kinase. Wortmannin has been shown to inhibit this enzyme in a variety of studies and in a variety of cell systems (13, 14, 15). Our data confirm wortmannin’s ability to inhibit PI-3 kinase in the two ovarian cell systems that were used in our experiments (namely, human granulosa cell culture and whole ovarian cell culture). Although the former cell system provides a pure preparation of granulosa cells, the latter contains a mixture of cells including stromal, thecal, and granulosa cells. Therefore, it is not clear whether more than one type of cells contributed to the progesterone production observed in the experiment with whole ovarian cell culture. Despite this limitation, PI-3 kinase activity was completely inhibited by wortmannin in this cell system, and yet stimulatory effect of insulin on progesterone production was preserved. Therefore, the experiment with whole ovarian cell culture confirmed our finding in granulosa cells obtained during IVF.
The concentrations of insulin that we used ranged from physiological (10 ng/mL) to moderately (102 ng/mL) or significantly (103 and 104 ng/mL) supraphysiological. The supraphysiological insulin concentrations used in our experiments are comparable to the circulatory serum insulin concentrations in patients with insulin-resistant syndromes. In such patients, circulating insulin concentrations can range from moderately elevated (like in patients with PCOS) to extremely elevated (like in patients with type A and B syndromes of insulin resistance) (1, 2, 3, 6, 18). Thus, the data obtained in our experiments may be relevant to both physiological and pathological conditions observed in women.
As discussed earlier, at higher insulin concentration insulin can bind to the type I IGF-receptor, which also activates PI-3 kinase in the course of its signaling (28, 29). Our data does not allow to determine conclusively whether at high insulin concentrations the observed insulin effects were mediated by type I IGF-receptor. Because the effect of insulin on progesterone production was dose-dependent across the entire range of tested insulin concentrations, and because significant inhibition of IGFBP-1 production was significant at the lowest concentration of insulin that we examined (10 ng/mL) these two effects of insulin are likely to be mediated by the insulin receptor. Previous studies have demonstrated that the effects of insulin on steroidogenesis and IGFBP-1 production in human ovarian cells are mediated by the insulin receptor (24, 25, 26).
In summary, our data demonstrate that across a range of physiologic and supraphysiologic insulin concentrations inhibition of PI-3 kinase by wortmannin does not affect insulin’s ability to stimulate progesterone production or to inhibit IGFBP-1 production in at least one type of human ovarian cells (namely, granulosa cells). Thus, PI-3 kinase-independent insulin action pathway(s) are present in these cells. The components of these pathways, including their receptor components, still need to be elucidated. At this time we can only hypothesize which signaling pathways are important for these two effects of insulin in the ovary. Potential candidate pathways would include inositol-glycan pathway as reported by Nestler et al. (26) or mitogen-activated protein kinase pathway. It is also possible that other, so far unknown, insulin action pathway or pathways may be operating in human ovarian cells to produce these two effects of insulin.
We conclude that PI-3 kinase-independent insulin signaling pathway(s) can mediate stimulatory effect of insulin on progesterone production and inhibitory effect of insulin on IGFBP-1 production in cultured human ovarian cells. Identifying this pathway or pathways will require further study. Knowledge of specific pathways mediating specific effects of insulin in the ovary may ultimately lead to the development of target-specific therapeutic agents, which may be useful in the treatment of ovarian dysfunction commonly observed in women with insulin-resistant states.
Acknowledgments
We thank Dr. Julianne Imperato-McGinley for helpful discussion, Dr. Martin Lesser for statistical analysis, and Ms. May Lui for her assistance in all aspects of this work.
Footnotes
1 This work was supported by NIH Grants M01-RR00047, RO3-NICHD5618
(to L.P.), and by the Singer/Hellman award at Beth Israel Medical
Center (to L.P.). 
Received May 30, 2000.
Revised March 2, 2001.
Revised March 2, 2001.
Accepted March 14, 2001.
References
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,
Cells incubated with insulin without preincubation with wortmannin.
•, Cells incubated with insulin after preincubation with 100
nM wortmannin. Each point represents mean ±
SEM of at least 5 experiments performed in 2–3 replicates
to produce a total number of at least 10 measurements. For insulin
effect on progesterone production P less than 0.0001 by
ANOVA, regardless of preincubation with wortmannin. WM, Wortmannin.
, P < 0.2, compared with insulin alone.
, P < 0.2 compared with control. 
, Control;
,
wortmannin; 
, insulin alone;
, insulin and wortmannin.
