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Division of Endocrinology, Beth Israel Medical Center and Albert Einstein College of Medicine (D.S.-Y., J.Z., L.P.), New York, New York 10003; and Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University (H.-C.L., Z.R.), New York, New York 10021
Address all correspondence and requests for reprints to: Leonid Poretsky M.D., Division of Endocrinology, Beth Israel Medical Center, 317 East 17th Street, Fierman Hall, 7th Floor, New York, New York 10003. E-mail: lporetsk{at}bethisraelny.org.
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Abstract |
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Human granulosa cells were obtained during in vitro fertilization. Cells (0.5–1 x 105) were incubated for 24 h in the presence of 0, 10, 102, or 103 ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 µM PD98059, a specific inhibitor of ERK1/2 MAPK. The progesterone concentration in the tissue culture medium was measured by RIA (Pantex, Santa Monica, CA), and the IGFBP-1 concentration was measured by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX). MAPK activity was assessed using the MAPK IP-Kinase assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). ANOVA was used to compare mean values of progesterone or IGFBP-1 concentrations.
MAPK was stimulated by insulin up to 350% of the baseline value. Progesterone production in human granulosa cells was stimulated by insulin in a dose-related manner to 123% of the control value (P < 0.001), and IGFBP-1 production was inhibited to 25% of the baseline value (P < 0.001). Despite inhibiting MAPK activity by 99%, PD98059 (1 µM) did not interfere with insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production.
MAPK was stimulated by IGF-I to 730% of the baseline value, with maximal stimulation achieved at 0.5 ng/ml IGF-I. Progesterone production in granulosa cells was stimulated by IGF-I to 130% of the control value (P < 0.001), whereas IGFBP-1 production was inhibited to 44% of the control value (P < 0.001). PD98059 (1 µM) inhibited IGF-I-induced MAPK activity by 94%. In the presence of 1 µM PD98059, IGF-I-induced stimulation of progesterone production was inhibited by 96% (P < 0.001). The inhibitory effect of IGF-I on IGFBP-1 production was reduced in the presence of 1 µM PD98059 by 45% at 5 ng/ml IGF-I and was completely abolished in the presence of 1 µM PD98059 at concentrations of IGF-I ranging from 0.5–2.5 ng/ml (P < 0.001). We conclude that, under conditions of our experiments, insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells does not require MAPK activation, whereas similar effects of IGF-I are largely MAPK dependent.
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Introduction |
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The effects of IGFs in the ovary are similar to those of insulin, but both IGF-I and IGF-II, acting via type I IGF receptors, appear to be much more potent regulators of ovarian function than insulin (7). The interaction between insulin and the IGF system in the ovary is complex. Some of the effects of insulin in the ovary, for example, may involve activation of the IGF system. Insulin inhibits ovarian IGFBP-1 production and up-regulates ovarian IGF-I-binding sites (5, 7, 8), thus increasing the bioavailability of IGFs and at the same time increasing the sensitivity of ovarian cells to IGF action. We recently demonstrated that activation of phosphatidylinositol 3-kinase (PI-3-kinase), the enzyme whose activation is crucial for glucose transport, is not necessary for certain insulin effects in the human ovary, thus providing experimental evidence for the divergence of insulin signaling pathways in the ovary (9). We now examined whether activation of MAPK in human granulosa cells is necessary to mediate the effects of insulin or IGF-I on progesterone and IGFBP-1 production.
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Materials and Methods |
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Human granulosa cells
Human granulosa cells were obtained during in vitro fertilization over the course of 12 months and were taken from several patients at a time to assure adequate cell number for the experiments. The diagnoses included male factor infertility, nonandrogenic ovulatory dysfunction, tubal factor infertility, and uterine factor infertility. None of the women from whom the cells were obtained received insulin-sensitizing agents.
Cell culture
The granulosa cells were purified on Percoll gradients and cultured as previously described with some modifications (9, 10, 11). The cells were incubated for 48 h at 37 C in 5% CO2 at 90% humidity in McCoy’s 5A tissue culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products, Woodland, CA), 100 µg/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin in 24-well Falcon tissue culture dishes (BD Biosciences, Franklin Lakes, NJ). The density of the cells was approximately 0.5–1 x 105/ml. After 48 h of incubation, the medium supplemented with 10% FBS was replaced by medium with 1% FBS, and the cells were incubated for another 48 h. For the experiments that examined the effects of IGF-I on progesterone and IGFBP-1 production, the cells were preincubated with 10 ng/ml insulin (8, 12, 13) added to 1% FBS medium for another 48 h. Finally, the cells were incubated for an additional 24 h in FBS-free medium in the presence of 0, 10, 102, or 103 ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I in the presence or absence of 1 µM PD98059, a specific inhibitor of ERK1/2 MAPK. Cell viability was assayed using the Trypan Blue method. PD98059 and IGF-I were obtained from Calbiochem (San Diego, CA). Insulin was obtained from Roche (Indianapolis, IN).
Progesterone and IGFBP-1 determinations
The IGFBP-1 concentration in the conditioned tissue culture medium was assayed by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX), and the progesterone concentration was measured using an RIA kit (Pantex, Santa Monica, CA). The data were normalized per 1 x 105 cells/ml.
Assessment of MAPK activity
Approximately 2–4 x 106 cells were cultured in six-well Falcon tissue culture dishes (BD Biosciences) in the presence of 0, 10, 102, or 103 ng/ml insulin or 0, 0.5, 1, or 5 ng/ml IGF-I and in the presence or absence of 1 µM PD98059. After 24-h incubation, the medium was aspirated, and the cells were lysed with buffer A [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM sodium orthovanadate, 1% Nonidet P-40, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, 0.8 µM aprotinin, 50 µM bestain, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A]. Lysates were centrifuged at 14,000 x g for 10 min to sediment insoluble material, and the supernatant fraction was immunoprecipitated with 2.5 µg anti-MAPK antibody immobilized with agarose (50 µg/300 µl buffer A, 
15 µl/reaction). The mixtures were incubated at 4 C for 15 h, and the immunoprecipitate complexes were collected by centrifugation and washed first with 1 ml buffer A; then with the buffer containing 20 mM Tris/HCl (pH 7.4) and 500 mM NaCl; then with one buffer containing 100 mM Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate; and finally with buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA. The phosphorylation assay was carried out using myelin basic protein as a substrate, and the procedure was performed as recommended by the manufacturer (Upstate Biotechnology, Inc., Lake Placid, NY).
Immunoblot assay
The phosphorylated myelin basic protein was separated by 12% SDS-PAGE and transferred to nitrocellulose paper (wet transfer in the buffer containing 25 mM Tris/HCl, 192 mM glycine, and 10% methanol, pH 8.3). The immunoblot was probed with phospho-specific myelin basic protein antibodies (0.25 µg/ml; mouse monoclonal immunoglobulin G2
) and then with a second antibody conjugated with horseradish peroxidase (0.5 µg/ml; goat antimouse immunoglobulin G-horseradish peroxidase conjugate). The immunoblot procedure was carried out as described by the manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA) in Tris-buffered salline buffer with 5% dry milk as the blocking reagent. The bands were detected by chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). The densities of the bands were determined using PhotoShop 5.5 (Adobe Systems, Mountain View, CA) and were calculated using the Scion imaging program.
Statistical analysis
All experiments were carried out in quadruplicate and were repeated 7–13 times. Two-way ANOVA was used to compare mean values according to insulin or IGF-I concentrations in the presence or absence of PD98059. The statistical interactions between the data sets obtained with or without PD98059 were examined. Pairwise, Bonferroni-adjusted contrasts were analyzed to determine statistical significance.
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Results |
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A representative experiment demonstrating insulin-induced MAPK activity in the presence or absence of 1 µM PD98059 is shown in Fig. 1A
. MAPK activity was induced in a dose-responsive manner by increasing concentrations of insulin and was inhibited by 1 µM PD98059. Insulin stimulated MAPK up to 300% of the baseline (Fig. 1B), and this effect was inhibited in the presence of 1 µM PD98059 by 99%. A representative experiment demonstrating IGF-I-induced MAPK activity in the presence or absence of 1 µM PD98059 is shown in Fig. 2A. MAPK activity was increased up to 730% of the baseline value, and maximal stimulation was achieved at 0.5 ng/ml IGF-I (Fig. 2B).
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. Insulin stimulated progesterone production in a dose-dependent manner up to 123% of the control value (P < 0.001), with the most robust response occurring between 1–10 ng/ml insulin. No statistically significant effect of 1 µM PD98059 on this interaction between insulin and progesterone was observed.
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shows the dose-response analysis of insulin-induced inhibition of IGFBP-1 production in granulosa cells. Under the influence of insulin, IGFBP-1 production in granulosa cells was reduced to 25% of the control value (P < 0.001). PD98059 (1 µM) failed to affect this inhibitory effect of insulin on IGFBP-1 production.
Dose-response analysis of IGF-I-stimulated progesterone production in granulosa cells is shown in Fig. 4A. IGF-I stimulated progesterone production to 130% of the control value. Maximal IGF-I stimulation of progesterone was reduced by 95% in the presence of 1 µM PD98059 (P < 0.001).
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shows the dose-response analysis of IGF-I-induced inhibition of IGFBP-1 production in granulosa cells. IGF-I reduced IGFBP-1 production in these cells to 44% of the control value (P < 0.001). In the presence of the MAPK inhibitor PD98059 (1 µM), this inhibitory effect of IGF-I on IGFBP-1 production was reduced by 50% at 5 ng/ml IGF-I and was completely abolished at 0.5–2.5 ng/ml IGF-I (P < 0.001). |
Discussion |
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To explain this paradox, at least two hypotheses have been proposed (2, 4). The first hypothesis postulates that in insulin-resistant states, ovarian effects of insulin are mediated by the type I IGF receptor (2). This mechanism of insulin action in the ovary is likely to operate when extremely high concentrations of insulin are present in circulation, such as in patients with extreme forms of insulin resistance (for example, the type A or B syndrome of insulin resistance and acanthosis nigricans) (2). However, this mechanism is unlikely to explain similar observations in patients with polycystic ovary syndrome, whose circulating insulin levels are only moderately increased (1, 4). Further, studies of insulin action in human ovarian cells indicate that under most circumstances, including in cells from patients with polycystic ovary syndrome, insulin mediates its effects in the ovary via its own receptor, rather than the type I IGF receptor (5, 6). Some of the ovarian effects of insulin, even when insulin acts via its own receptor, however, can be attributed to insulin’s ability to activate the ovarian IGF system (1): insulin inhibits IGFBP-1 production in the ovary (5, 7), thus increasing the amount of bioavailable IGFs, and may increase the number of ovarian type I IGF receptors or hybrid insulin/type I IGF receptors (1, 8).
The second hypothesis that attempts to explain the paradox of insulin-induced ovarian stimulation in patients with insulin resistance proposes that the postbinding insulin signaling pathways in the ovary diverge. It has been shown that inositolglycan signaling system, rather than the tyrosine kinase signaling system, of the insulin receptor may play a role in mediating androgen production in human ovarian cells (14). We recently demonstrated that activation of PI-3-kinase, the enzyme involved in glucose transport, is not necessary for the insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production in the human ovary (9), thus providing experimental evidence for the existence of alternate insulin action pathways in the ovary. Our present report demonstrates that activation of another insulin signaling pathway, the one involving MAPK, is not necessary for the stimulation of progesterone or the inhibition of IGFBP-1 production in human granulosa cells. MAPK activation, however, appears to be important for similar effects of IGF-I.
Our conclusions depend on the ability of PD98059 to inhibit MAPK activity while not affecting cell viability. PD98059 is a potent and selective inhibitor of the MAPK kinase, which is the upstream kinase responsible for dual phosphorylation of MAPK (ERK1/2) (15). In some studies inhibition of this MAPK has been shown to induce apoptosis in human granulosa cells, as evidenced by the blocked nuclear translocation of phosphorylated MAPK and increased appearance of subdiploid apoptotic nuclei after treatment with 100 µM PD98059 (16). However, such effects are not observed consistently (16, 17). We used a much lower concentration of PD98059 (1 µM). At 1 µM PD98059, cell viability ranged from 92–100%, whereas at higher concentrations (2–3 µM) of PD98059, granulosa cell function and viability were affected (data not shown). Thus, 1 µM PD98059 was the lowest concentration at which cell viability was not affected, and yet MAPK activity was inhibited almost completely (1 µM PD98059 inhibited insulin-stimulated MAPK activity by 99% and IGF-I-stimulated MAPK activity by 94%).
Despite the almost complete (99%) inhibition of MAPK activity, 1 µM PD98059 did not interfere with insulin-induced stimulation of progesterone or inhibition of IGFBP-I production in human granulosa cells. Baseline progesterone production was not affected by 1 µM PD98059, thus indicating that the steroidogenic pathways involving progesterone production were not influenced by this MAPK inhibitor at a 1-µM concentration.
The concentrations of insulin in our experiments ranged from physiological (10 ng/ml) to moderately (102 ng/ml) or significantly (103 ng/ml) supraphysiological. Although the insulin effect on progesterone production was dose related between 1–100 ng/ml, the most robust effect was achieved between 1–10 ng/ml insulin, and significant inhibition of IGFBP-1 production (to 25% of the control value) occurred at 10 ng/ml insulin, a physiological concentration. Thus, these effects of insulin are likely to be mediated by the insulin receptor rather than the type I IGF receptor, whose activation requires supraphysiological insulin concentrations. In summary, our data demonstrate that across a range of physiological and supraphysiological concentrations of insulin, almost complete inhibition of MAPK by 1 µM PD98059 does not affect the ability of insulin to stimulate progesterone production or inhibit IGFBP-1 production in human granulosa cells.
In contrast to insulin, IGF-I-induced stimulation of progesterone and inhibition of IGFBP-1 production were affected by the inhibition of MAPK activity. Although IGF-I signaling pathways for glucose transport are similar to those for insulin (1), IGF-I stimulation of MAPK as well as other ovarian effects of IGF-I are observed at significantly lower concentrations of IGF-I than those of insulin, e.g. IGF-I is a much more potent stimulus for MAPK activation, steroidogenesis, and inhibition of IGFBP-1 production than insulin (1, 7).
Insulin, nonetheless, may contribute to activation of the IGF-I system in ovary indirectly; in the presence of hyperinsulinemia, which inhibits IGFBP-1 production and may up-regulate ovarian IGF-I receptors, IGF-I action can be amplified (1, 4). Thus, in hyperinsulinemic states, IGF-I effects can be enhanced 1) by insulin activating type I IGF receptor if hyperinsulinemia is extreme, 2) by increased availability of IGF-I due to the reduced IGFBP-1 production, and 3) by up-regulation of type I IGF receptors (8).
The results of the experiments presented in this report extend our previous observation, which demonstrated that activation of the PI-3-kinase insulin signaling pathway, despite of its key role in glucose transport, is not necessary for the ovarian effects of insulin (9). The MAPK pathway, although also activated by insulin, is clearly distinct from the PI-3-kinase pathway and is related to the growth-promoting effects of insulin. The fact that activation of this pathway is also not required for the ovarian effects of insulin strongly supports the hypothesis that alternate pathways of insulin action are involved in these effects. Such pathways can remain active in insulin-resistant states, thus resolving the paradox of preserved ovarian insulin action despite insulin resistance in classical target organs (4). IGFs, although more potent than insulin, may be less pleiotropic than insulin, activating mostly growth-related (MAPK), rather than metabolic (PI-3-kinase or inositolglycan), pathways in the ovary (14). In fact, it is possible that the differences in potency (in relation to ovarian effects) between insulin and IGF-I could be explained at least in part by the fact that IGF-I utilizes MAPK signaling cascades for its ovarian effects, whereas insulin does not.
In conclusion, the data presented in this report together with previously published data (9) indicate that activation of insulin signaling pathways involving either PI-3-kinase or MAPK (ERK1/2-kinase) is not necessary for the stimulatory effect of insulin on progesterone production or the inhibitory effect of insulin on IGFBP-1 production in human granulosa cells. Similar effects of IGF-I require MAPK activation. It is hoped that the knowledge of specific pathways mediating specific effects of insulin or IGF-I in the ovary will ultimately lead to the development of specific therapeutic agents that will be useful in the treatment of the ovarian dysfunction commonly observed in insulin-resistant states.
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Footnotes |
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Abbreviations: FBS, Fetal bovine serum; IGFBP, IGF-binding protein; PI-3-kinase, phosphatidylinositol 3-kinase.
Received December 13, 2002.
Accepted March 26, 2003.
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) or presence (
) of 1 µM PD98059 (mean ± SEM of three experiments).
, 1 µM PD98059. Each point represents the mean ± SEM of 9–13 experiments performed in 2–4 replicates. For the insulin effect on progesterone production, P < 0.001 by ANOVA analysis, regardless of incubation with 1 µM PD98059. For the insulin effect on IGFBP-1 production, P < 0.001 by ANOVA analysis, regardless of incubation with 1 µM PD98059.