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Troglitazone, an Insulin-Sensitizing Thiazolidinedione, Represses Combined Stimulation by LH and Insulin of de Novo Androgen Biosynthesis by Thecal Cells in Vitro

Division of Endocrinology, Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, M.D., Endocrinology Division, Department of Internal Medicine, P.O. Box 800202, University of Virginia School of Medicine, Charlottesville, Virginia 22908. E-mail: . jdv{at}virginia.edu

Abstract

Polycystic ovarian syndrome (anovulatory hyperandrogenism) is marked by adolescent onset of systemic hyperinsulinism, oligoovulation, hirsutism, excessive LH and androgen secretion, and variable reduction in fertility. Insulin and LH are believed to act in concert to promote ovarian androgen hypersecretion in this disorder. Administration of troglitazone, an insulin-sensitizing agent and putative PPAR agonist, can decrease hyperinsulinism, suppress T production, and ameliorate oligoovulation in some women with this endocrinopathy. The present study tests the hypothesis that troglitazone directly inhibits de novo androgen biosynthesis stimulated jointly by LH and insulin in primary cultures of (porcine) thecal cells. We show that troglitazone dose-dependently antagonizes LH/insulin’s combined stimulation of androstenedione and T production by thecal cells in vitro. Consistent steroidogenic inhibition of 80–95% was achieved at drug concentrations of 3–6.8 µM (P < 0.001). Exposure of thecal cells to the thiazolidinedione derivative also blocked bihormonally stimulated accumulation of CYP17 (cytochrome P450 17 -hydroxylase/C_17–20 lyase) gene expression, as reflected by decreased accumulation of cognate heterogeneous nuclear RNA and mRNA (by 30–65%; P < 0.05). Moreover, troglitazone suppressed LH/insulin-induced phosphorylation of the 52-kDa immunoprecipitated CYP17 enzyme by 88% (P < 0.001). A putative natural agonist of PPAR nuclear transcription, 15-deoxy--12,14-prostaglandin J₂, also inhibited LH/insulin-driven androstenedione biosynthesis and CYP17 gene expression in thecal cells. In conclusion, a synthetic thiazolidinedione (troglitazone) and a natural ligand of PPAR (15-deoxy--12,14-prostaglandin J₂) effectively impede the concerted stimulation by LH and insulin of in vitro thecal cell androgen production, CYP17 gene expression, and CYP17 protein phosphorylation. This ensemble of inhibitory actions on LH/insulin-stimulated steroidogenesis offers a plausible mechanistic basis for at least part of the observed clinical efficacy of troglitazone in mitigating androgen excess in women with polycystic ovarian syndrome.

THE PATHOPHYSIOLOGICAL mechanisms that sustain heightened T production in adolescents and women with the polycystic ovary syndrome (PCOS) are not known (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Clinical studies and in vitro experiments indicate that both LH and insulin augment androgen biosynthesis, inasmuch as 1) potent GnRH agonists initially enhance and then down-regulate LH and T secretion in parallel in patients with PCOS (1, 2, 3, 4); 2) amelioration of systemic hyperinsulinism by diverse interventions, such as fasting, weight loss, or administration of troglitazone (below), D-chiroinositol, metformin, diazoxide, or octreotide, reduces androgen overproduction in this syndrome (5, 6, 7); and 3) in vitro stimulation of thecal cells with LH and insulin amplifies T or androstenedione biosynthesis synergistically (8).

Recent in vitro experiments suggest that selected chemical mediators of tissue insulin action, such as inositol glycans, can modulate basal and/or hormonally stimulated thecal and granulosa cell steroidogenesis (9, 10, 11). In addition, clinical studies have disclosed that troglitazone, an insulin-sensitizing thiazolidinedione derivative, is able to suppress systemic hyperandrogenism in patients with PCOS (12, 13, 14). Troglitazone also appears to inhibit granulosa cell progesterone production in vitro (15). The present investigation examines the hypothesis that this presumptive PPAR agonist (16) can repress combined LH- and insulin-enhanced androgen biosynthesis in primary cultures of (porcine) thecal cells and explores the corresponding mechanisms underlying such inhibition.

Materials and Methods

Reagents and oligonucleotides

Ovine LH (NIDDK oLH-25, 2.3 U/mg, 1 U = activity of NIH-LH-S1) was provided by the National Hormone and Pituitary Program of NIDDK. Insulin (from porcine pancreas) was obtained from Sigma (St. Louis, MO). 15-Deoxy-^12,14-prostaglandin J₂ (PGJ₂) was obtained from Cayman Chemical (Ann Arbor, MI). Oligo(deoxythymidine)₁₅ primer and dNTPs were purchased from Roche Molecular Biochemicals (Indianapolis, IN); ribonuclease inhibitor, murine leukemia virus reverse transcriptase, and AmpliTaq Gold DNA polymerase from Perkin-Elmer Corp. (Brandsberg, NJ); and PicoGreen DNA assay reagents from Molecular Probes, Inc. (Eugene, OR). Oligonucleotides were obtained from Operon Technologies (Alameda, CA). The sequences selected for forward and reverse oligonucleotide primers included 5'-TCCGAGAGGTGCTTCGATTC-3' and 5'-GGCGCTCCTTGATCTTCACT-3' for CYP17 mRNA (13), 5'-GATGGCATCCTGGATGCTGT-3' and 5'-CACTCTCGGCAGAAGGCAAT-3' for CYP17 heterogeneous nuclear RNA (hnRNA) (14), and 5'-CGATGCTCTTAGCTGAGTGT-3' and 5'-GGAACTACGACGGTATCTGA-3' for 18S rRNA (17, 18, 19, 20). PCR products for CYP17 mRNA, hnRNA, and 18S rRNA comprised 420, 305, and 315 bp, respectively. Degenerate reverse primer sequences were selected from the third introns of the pig CYP17 genes for RT-PCR of hnRNA (below). Porcine 18S ribosomal RNA (18S rRNA) sequences were not available in GenBank. Thus, we identified fully conserved sequences among human, rat, mouse, rabbit, and/or cow to serve as primer templates. The PCR products were sequenced three times for confirmation (8).

Porcine thecal cell culture

Thecal cells were isolated from porcine ovaries as described previously (8). Briefly, combined thecal-granulosa membranes were removed from immature follicles (diameter, <5 mm) using forceps, and granulosa cells were detached by mechanical agitation for 3 min. Thecal-basal lamina linings were allowed to settle at unit gravity, and the supernatant containing granulosa cells was discarded. Residual tissue was digested with collagenase (1 mg/ml) and deoxyribonuclease (10 µg/ml), recovered via a 0.2-µm pore size nylon screen (Millipore Corp., Bedford, MA), and washed three times in Ham’s F-12/DMEM (1:1) by centrifugation for 5 min at 1000 rpm.

Thecal cells were purified further via a Percoll density gradient, as reported previously (21). Cells were recovered, washed, resuspended in F-12/DMEM (1:1), and plated in 96-well plates (Costar, Cambridge, MA) at a density of 10⁵ cells/well in a humidified 95% air/5% CO₂ incubator at 37C. After an initial 40 h of culture to allow anchorage, attached thecal cells were exposed to LH and insulin (100 ng/ml each). Culture medium was collected at 48 h for analysis of steroid hormone concentrations. Androstenedione and T were assayed by commercially available RIAs (ICN Pharmaceuticals, Inc., Costa Mesa, CA), which have a sensitivity of 0.17 ng/ml and exhibit low cross-reactivity with progesterone (<0.02%). The androstenedione RIA also cross-reacts minimally with T (0.2%). DNA was measured as described previously (22). Androgen measurements were normalized per unit DNA per culture.

RNA isolation

Total RNA was isolated from cells pooled from eight wells using the Tri-Reagent total RNA isolation kit (Molecular Research Center, Inc., Cincinnati, OH) and was quantified by absorbance at 260/280 nm (model 601, Spectronics, Milton Roy, Rochester NY).

RT-PCR of CYP17 cDNA

Semiquantitive RT-PCR was used to assess mRNA concentrations by first reverse transcribing 4 µl (1 µg) total RNA/sample in a final volume of 40 µl. Each reaction contained 1 mM of each dNTP, 2.5 µM oligo(deoxythymidine)₁₅, 40 U ribonuclease inhibitor, 5 mM MgCl₂, 4 µl 10 x RT-PCR buffer, and 50 U murine leukemia virus reverse transcriptase. RT was performed at 42 C for 15 min, followed by 99 C for 5 min.

PCR reaction mixtures of 100 µl final volume contained 2.5 mM MgCl₂, 100 pmol each of forward and reverse gene-specific primers, 0.2 mM of each dNTP, 10 µl 10 x RT-PCR buffer, one fifth of the total RT volume for gene specific products, and 2.5 U AmpliTaq Gold DNA polymerase. PCR amplification was optimized as follows: 95 C for 12 min, then 28 cycles at 94 C for 30 sec, 62 C for 2 min, followed by 62 C for 10 min.

To amplify hnRNA for CYP17 and 18S rRNA, 2 pmol reverse gene-specific primer was used to synthesize cDNA, which was primed by intron-specific reverse primers (above). In the PCR step, the following thermal cycling protocol was used: 30 sec at 95 C, 35 sec at 62 C, and 1 min 30 sec at 67 C.

To control for potential DNA contamination, RT-PCR reactions were also conducted using total RNA without reverse transcriptase. To normalize relative recoveries, porcine 18S ribosomal RNA was amplified in parallel. Results are thus expressed as the ratio of specific gene product to that of 18S ribosomal RNA.

The identities of the foregoing RT-PCR products were confirmed in multiple (at least three) independent samples from different batches of ovaries by automated gene sequencing (University of Virginia Molecular Core Laboratory).

PicoGreen quantification of PCR products

The amount of amplified cDNA was quantified using the PicoGreen ultrasensitive fluorometric assay (Molecular Probes, Inc.). Twenty microliters of PCR product per sample were prepared in 1 x TE buffer in a final volume of 100 µl, and added in a 96-well microplate. One hundred microliters of 1:200 PicoGreen in 1 x TE were then mixed with each PCR sample. After incubation at room temperature for 2–5 min, microtiter plates were scanned at an excitation wavelength of 488 nm and an emission wavelength of 530 nm using a FluorImager 595 optical scanner, version 5.01 (Molecular Dynamics, Inc., Sunnyvale, CA). The image was analyzed using ImageQuant version 5.0 (Molecular Dynamics, Inc.). The amount of PCR product was quantitated by extrapolation from DNA standards, as previously reported (23). The assay has a sensitivity of 25 pg/ml with a linear range to 1000 ng/ml. double-stranded DNA was used as standard. Intra- and interassay coefficients of variation were 5.1% and 8.3%, respectively.

In vitro metabolic labeling and immunoprecipitation of CYP17 protein

Thecal cells were labeled in monolayer culture with [³²P]orthophosphate (150 mCi/ml; NEN Life Science Products, Boston, MA) for 4 h in phosphate-free DMEM (Life Technologies, Inc., Gaithersburg, MD). Labeled cells were exposed to vehicle or hormones for 15 min at 37 C and lysed in PBS containing 1.5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, and 10% glycerol in the presence of protease inhibitors (34 µg/ml phenylmethysulfonylfluoride, 1 µg/ml pepstatin, and 5 µg/ml leupeptin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium vanadate). The lysates were clarified by centrifugation at 15,000 x g for 10 min, and 80 µg total protein from each sample were mixed overnight at 4 C with antiporcine CYP17 antiserum (a gift from Dr. D. B. Hales, Chicago, IL) at a dilution of 1:500 (24). Protein A-Sepharose was added, and samples were mixed at room temperature for an additional 2 h. The immune complexes were washed six times, and an equal number of counts for each sample was applied and analyzed on 10% SDS-polyacrylamide gels. Gels were exposed overnight and up to 3 d to Fuji Photo Film Co., Ltd. RX film (Tokyo, Japan) at -70 C with intensifying screens. Immunoprecipitated bands were quantitated by laser densitometry using ImageQuant software and densitometer (Molecular Dynamics, Inc.).

Statistical analysis

One-way ANOVA of logarithmically transformed data was used with Tukey’s post-hoc test to contrast means at P < 0.05. Data are given as the mean ± SEM (n = 3 independent experiments).

Results

Figure 1 shows the concentration-dependent inhibitory effects of troglitazone on bihormonally (LH and insulin) stimulated thecal cell production of androstenedione and T in vitro over 48 h of incubation (P < 0.001). Forty-eight hours was selected based on pilot time-course analyses. LH plus insulin (100 ng/ml) stimulated androstenedione (4-fold) and T (2.5-fold) production compared with control (untreated) cultures. In contrast, troglitazone (3–10 µM) suppressed androgen production by 80–95% of LH/insulin-enhanced values. When PGJ₂ was used as a PPAR ligand, LH- and insulin-stimulated androgen output was inhibited by 60–95%. To assess cell viability, thecal cell DNA content was quantitated after 48 h of culture by fluorometric assay using Hoechst 33258 dye. Only the maximum concentration of troglitazone decreased the culture DNA content relative to control (viz. 25%; P < 0.05), whereas PGJ₂ did not decrease DNA content at any concentration tested. Moreover, exposure of thecal cells to troglitazone (10 µM) or PGJ₂ (30 µM) increased progesterone concentrations by 8- and 5-fold relative to control untreated cultures (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Figure 1. Concentration-dependent inhibition by the insulin-sensitizing drug, troglitazone, of combined stimulation by LH and insulin (100 ng/ml each) of androstenedione (top) and T (bottom) production by serum-free cultures of (porcine) thecal cells. Units are nanograms of steroid secreted per µg DNA/48 h (A). Comparable data presentation for PGJ2, a natural agonist and structurally independent activator of PPAR (B). Data are the mean ± SEM of three independently replicated experiments. Different alphabetic superscripts identify significant differences among means (P < 0.001).

View larger version (28K): [in this window] [in a new window]   Figure 2. A and B, Suppressive action of troglitazone on joint LH- and insulin-stimulated accumulation of CYP17 hnRNA and mRNA normalized to 18S rRNA. Thecal cells were exposed to vehicle, LH, and insulin (100 ng/ml each) and/or troglitazone (7 µM) for 48 h. CYP17 hnRNA, CYP17 mRNA and 18S rRNA were quantitated by RT-PCR (see Materials and Methods). Data are the mean ± SEM of three independent experiments. Different superscripts denote statistically distinguishable means (P < 0.05).

View larger version (28K): [in this window] [in a new window]   Figure 3. A and B, Inhibition by PGJ2 (0.8 µM), a putative natural ligand of the PPAR receptor, of combined LH- and insulin-stimulated accumulation of CYP17 hnRNA and mRNA in cultured thecal cells. Data are presented as described in Fig. 2. a, b, and c indicate significant differences in means (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Figure 4. Troglitazone blocks LH/insulin-stimulated accumulation of immunoprecipitable CYP17 phosphoprotein (top panel). Thecal cells cultured in serum-free medium were prelabeled for 4 h with [32P]orthophosphate and then exposed for 15 min to vehicle (C), troglitazone (7 µM; TG), LH plus insulin (100 ng/ml each; LH+I), or all three effectors. Extracted proteins (80 µg) were immunoprecipitated with antiserum to porcine CYP17. An equal number of counts from each sample was applied to and resolved by 10% SDS-PAGE. Visualization was by autoradiography (bottom). Data are the mean ± SEM OD units at 52 kDa normalized to control values (no interventions) in three independent experiments. The response greater than control is identified by an asterisk (P < 0.001, by Tukey’s multiple comparison test).

Androgen biosynthesis is controlled by the CYP17 (cytochrome P450-containing 17-hydroxylase/C_17–20 lyase) enzyme, which converts progestin precursors to androgens via a two-step biochemical reaction. The first step catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone. The phosphorylated CYP enzyme then mediates oxidative cleavage of the C_17–20 bond to yield DHEA and ⁴-androstenedione, which serve as the primary precursors for T. The present in vitro data corroborate the ability of combined stimulation with LH and insulin to induce expression of the CYP17 gene, increase phosphorylation of CYP17 protein, and drive androgen biosynthesis by 1.9- to 4.3-fold in (porcine) thecal cells (8). More importantly, we demonstrate for the first time that the insulin-sensitizing thiazolidinedione compound, troglitazone, and a putative natural ligand of the PPAR receptor, PGJ₂, antagonize the joint stimulatory actions of LH and insulin on each of the foregoing end points of hormone action in cultured thecal cells. In particular, troglitazone and PGJ₂ repressed LH’s and insulin’s combined induction of CYP17 hnRNA and CYP17 mRNA (by 30–65%), CYP17 protein phosphorylation (by 88%), and androstenedione and T biosynthesis (by >80–95%). These novel findings suggest that the PPAR nuclear signaling pathway can mediate repressive control of LH- and insulin-stimulated CYP17-dependent androgen production by thecal cells.

The ability of troglitazone and PGJ₂ to antagonize the bihormonally (LH/insulin) stimulated accumulation of both hnRNA and mRNA encoding CYP17 in thecal cells would be consistent with transcriptional level inhibitory effects, as homologous hnRNA represents incompletely processed or primary gene transcripts (8). Whereas our data do not exclude concomitant changes in mRNA stability, these observations offer a rationale for additional studies of presumptive transcriptional control of CYP17 gene expression by PPAR effectors in suitable thecal cell models. In the latter regard, recent RT-PCR-based analyses show that the PPAR gene is expressed in ovarian thecal and lutein cells (26, 27), thereby allowing for the idea that this key nuclear signaling pathway may govern the expression of other ovarian genes.

The predominant inhibitory actions of troglitazone on the androgen biosynthetic pathway in thecal cells included marked (>85%) impairment of LH/insulin-stimulated androstenedione and T production and immunoprecipitable CYP17 phosphoprotein accumulation. Effective in vitro concentrations of troglitazone were nontoxic and approached those expected under therapeutic conditions in vivo (28). As serine-directed protein phosphorylation of the CYP17 enzyme enhances lyase activity, nearly complete blockade of LH/insulin-stimulated CYP17 phosphoprotein accumulation by troglitazone would be expected to impair the posthydroxylation cleavage activity of this enzyme. In this regard, whereas troglitazone and PGJ₂ can both enhance precursor progesterone accumulation in LH/insulin-stimulated thecal cells (present study and Ref. 26), direct assay of steroidogenic enzyme activity will be required to establish the precise degree and possible multiplicity of enzymic inhibition achieved by these ligands. Recently, troglitazone was shown to inhibit 3ß-hydroxysteroid dehydrogenase type II enzyme activity as well as both the lyase and desmolase activities of human P450 CYP17 directly in a humanized yeast cell system (29). The direct inhibition of CYP17 lyase activity by troglitazone is consistent with our observation of decreased phosphorylation (and, concomitantly, lyase activity) in thecal cells. An apparent discrepancy in the ability of troglitazone to inhibit CYP17 hydroxylase activity in yeast, but not in thecal cells, probably reflects one or more differences in the test systems studied.

In summary, troglitazone, a peripheral insulin-sensitizing agent, impedes LH’s and insulin’s combined stimulation of androgen biosynthesis, CYP17 hnRNA and mRNA accumulation, and CYP17-immunospecific enzyme phosphorylation in porcine thecal cells in vitro. Comparable repressive effects are exerted by PGJ₂, an alternative natural ligand of the nuclear PPAR transcriptional pathway. The present findings thus provide plausible contributing mechanisms for the ovarian androgen-suppressing activity of thiazolidinedione compounds in patients with PCOS and further suggest the need to explore the possible role of altered PPAR signaling in human thecal cell pathobiology.

Acknowledgments

Footnotes

This work was supported in part by NIH Grants HD-16393 and HD-16806, NIH Grant P30-HD-28934 (Center for Cellular and Molecular Reproduction), and the NIH U-54 Specialized Cooperative Centers Program in Reproductive Research (NICHHD Grant HD-96-008).

Abbreviations: hnRNA, Heterogeneous nuclear RNA; PCOS, polycystic ovarian syndrome; PGJ₂, 15-deoxy--12,14-prostaglandin J₂.

Received July 23, 2001.

Accepted November 28, 2001.

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