Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3beta -Hydroxysteroid Dehydrogenase*

Wiebke ArltDagger , Richard J. Auchus§, and Walter L. MillerDagger

From the Dagger  Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, California 94143-0978 and the § Department of Medicine, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-8857

Received for publication, January 2, 2001, and in revised form, February 6, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Androgen biosynthesis requires 3beta -hydroxysteroid dehydrogenase type II (3beta HSDII) and the 17alpha -hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3beta HSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3beta HSDII (KI = 25.4 ± 5.1 µM) and both activities of P450c17 (KI for 17alpha -hydroxylase, 8.4 ± 0.6 µM; KI for 17,20-lyase, 5.3 ± 0.7 µM). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3beta HSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3beta HSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The first and rate-limiting step in the biosynthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the mitochondrial cholesterol side chain cleavage enzyme, P450scc, which is thus the quantitative regulator of steroidogenesis (1). The qualitative regulator is microsomal P450c17, which sequentially catalyzes both 17alpha -hydroxylase and 17,20-lyase activities (2-4). In the absence of either activity of P450c17, adrenal pregnenolone is directed toward the biosynthesis of mineralocorticoids. When only 17alpha -hydroxylase activity is present, the resulting 17alpha -hydroxypregnenolone (17-Preg)1 is converted to cortisol; when both the 17alpha -hydroxylase and 17,20-lyase activities are present, the C19 steroid dehydroepiandrosterone (DHEA) is produced. DHEA then can be converted to androstenedione by 3beta -hydroxysteroid dehydrogenase type II (3beta HSDII), and androstenedione is converted to testosterone and estradiol by isozymes of 17beta -hydroxysteroid dehydrogenase and by aromatase (P450aro) (5, 6). The biosynthesis of all sex steroids proceeds through DHEA because human P450c17 does not convert 17alpha -hydroxyprogesterone (17OHP) to androstenedione (4, 7, 8). Thus P450c17 and 3beta HSDII are key enzymes required for the synthesis of all androgens (Fig. 1A).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Structures and chemical reactions. A, only two enzymes are needed to catalyze the steroid biosynthetic reactions shown, P450c17 and 3beta HSDII. The 17alpha -hydroxylase activity of P450c17 catalyzes the conversion of pregnenolone to 17-Preg and of progesterone to 17OHP with equivalent Km and Vmax values (8); the 17,20-lyase activity of P450c17 catalyzes the conversion of 17-Preg to DHEA with much lower efficiency (8). 3beta HSDII converts pregnenolone to progesterone, 17-Preg to 17OHP, and DHEA to androstenedione, all with equivalent Km and Vmax values (21). In this study, 3beta HSD activity is measured as conversion of pregnenolone to progesterone, 17alpha -hydroxylase activity is measured as conversion of progesterone to 17OHP, and 17,20-lyase activity is measured as conversion of 17-Preg to DHEA. B, structures of the inhibitors used. Top, metformin, shown as the hydrochloride; middle, the thiazolidinedione nucleus (note the R group in the ether linkage); bottom, structures of the thiazolidinedione R group in troglitazone (TRO), rosiglitazone (ROSI), and pioglitazone (PIO).

Polycystic ovary syndrome (PCOS) is the most frequent cause of female infertility, affecting ~5-10% of women of reproductive age (9, 10). The insulin-sensitizing drugs metformin and troglitazone decrease hyperandrogenemia and increase fertility in women with PCOS (11-14). These agents might decrease circulating androgens indirectly by lowering insulin levels, which may secondarily inhibit steroidogenesis by an unknown mechanism. Alternatively, metformin and troglitazone also might inhibit steroidogenic enzymes directly (12). Metformin and troglitazone are members of two fundamentally different drug families (Fig. 1B). Metformin, a biguanide, decreases hepatic gluconeogenesis and enhances peripheral glucose uptake, either secondarily by alleviating glucose toxicity (15) or possibly by inhibiting complex 1 of the mitochondrial respiratory chain (16, 17). Troglitazone, a thiazolidinedione drug, decreases both hepatic gluconeogenesis and peripheral insulin resistance, probably through its action as a ligand for the nuclear peroxisome proliferator-activated receptor gamma  (PPARgamma ) (18-20).

To determine whether metformin or troglitazone inhibits either P450c17 or 3beta HSDII, and if so by what mechanism, we examined the actions of these two drugs using our humanized yeast expression system (8, 21), in which individual human steroidogenic enzymes are examined in native microsomal environments without interfering factors that are often present in whole-cell experiments.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Drug Preparations-- Reagent-grade metformin was purchased from Sigma and dissolved in water; reagent-grade troglitazone, a generous gift of Dr. Andrea Dunaif (Harvard University), was dissolved in 100% Me2SO. Tablets of troglitazone, rosiglitazone, and pioglitazone were purchased as Rezulin (Parke-Davis), Avandia (Lilly), and Actos (Smith Kline Beecham), respectively, and were dissolved in 100% Me2SO and centrifuged to remove insoluble tablet material. The chemical equivalence and purity of the troglitazone solutions prepared from reagent-grade powder and tablets were confirmed by liquid chromatography/mass spectrometry. Reagent-grade metformin and troglitazone were used in all experiments except for the comparison of troglitazone to rosiglitazone and pioglitazone in which troglitazone prepared from tablets was used to control for any variations in preparation.

Expression Vectors and Yeast Microsome Preparation-- Saccharomyces cerevisiae strain W303B (22) was transformed by the lithium acetate procedure (23) with the yeast expression vector V10 (22) containing the cDNA sequences for human P450c17 or 3beta HSDII as described (8, 21). For P450c17 transformation, we cotransformed the yeast cultures with the vector pYcDE2 (24) expressing human P450 oxidoreductase cDNA (8). Yeast was grown, microsomes were prepared, and the microsomal P450 content was measured as described (8).

Yeast Enzyme Assays-- Yeast microsomes were incubated in the presence and absence of various concentrations of thiazolidinediones or metformin added in 4 µl of Me2SO or water to 196 µl of 50 mM potassium phosphate buffer (pH 7.4). Enzymatic assays were performed with 0.5-4 µM progesterone or 17alpha -hydroxypregnenolone for P450c17 activities and 1-20 µM pregnenolone for 3beta HSDII activity. Each reaction also contained 20,000 cpm of [14C]pregnenolone (55.4 mCi/mmol) (Amersham Pharmacia Biotech) for analysis of 3beta HSDII activity, 20,000 cpm of [14C]progesterone (55.4 mCi/mmol, PerkinElmer Life Sciences) for 17alpha -hydroxylase activity, or 50,000 cpm of [3H]17-Preg (21.1 Ci/mmol, PerkinElmer Life Sciences) for 17,20-lyase activity. Catalysis was initiated by adding 1 mM NADPH for P450c17 or 1 mM NAD+ for 3beta HSDII activity, and assays were done in the linear time range of the enzymatic reaction. Assays of 17,20-lyase activity were performed with and without addition of purified recombinant human cytochrome b5 (PanVera, Madison, WI) in 10-fold molar excess to the total P450 content of the microsomes.

Steroids were extracted from the reaction mixtures with 400 µl of ethyl acetate/isooctane (1:1) concentrated by evaporation under continuous nitrogen flow and assayed by thin layer chromatography on phosPE SIL G/UV silica gel plates (Whatman) using 3:1 chloroform/ethyl acetate as the solvent system (8, 25). The radiolabeled steroids were quantified by phosphorimaging analysis on a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). All assays were performed in triplicate, and data are presented as means ± S.D. Kinetic behavior was approximated as a Michaelis-Menten system, and data were plotted as described by Lineweaver and Burk and by Dixon and Webb (26). KI values were calculated from the equation for competitive inhibition, Vapp = Vmax × [S]/(Km (1 + [I]/KI) + [S]), whereas data from noncompetitive-competitive inhibition were approximated by the formula for generic inhibition, Vapp = Vmax/(1 + [I]/KI) at each [S] (27). Data fitting was carried out by LEONORA version 1.0 for analysis of steady-state enzyme kinetics (27).

Cell Culture Enzyme Assay-- COS-1 monkey kidney cells were grown as monolayers in 10-cm Petri dishes in 10 ml of Dulbecco's modified Eagle's medium-H21 containing 4.5 g/liter glucose, 10% fetal bovine serum, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. For transient transfection, COS-1 cells were grown to 80% confluence and split into six-well plates 24 h prior to transfection. Plasmid pcDNA3 containing the wild-type human P450c17 cDNA (28) was transfected into COS-1 cells at ~60% confluence using the calcium phosphate/DNA coprecipitation method. Thirty-six hours after transfection, cells were incubated with 2 ml of fresh Dulbecco's modified Eagle's medium-H21 containing 20,000 cpm [14C]progesterone and 0-100 µM troglitazone; each troglitazone concentration was added in 10 µl of Me2SO to triplicate wells. The steroids then were extracted with 8 ml of ethyl acetate/isooctane (1:1), concentrated, and analyzed as described above for the microsomal assays. Transfection efficiency was monitored by cotransfecting COS-1 cells with the pRL-CMV plasmid (Promega, Madison, WI) containing the Renilla luciferase gene driven by the cytomegalovirus promoter. After removing the medium, the cells were lysed and assessed for luciferase activity by the dual luciferase reporter assay system (Promega).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Metformin and Troglitazone on P450c17-- Both 17alpha -hydroxylase and 17,20-lyase activities require the interaction of P450c17 with its electron donor P450 oxidoreductase (OR); 17,20-lyase activity is enhanced further by allosteric interaction of the P450c17-OR complex with cytochrome b5 (8). Microsomes from yeast expressing human P450c17 cDNA and OR catalyze the conversion of pregnenolone to 17-Preg and of progesterone to 17OHP equally well, but the 17,20-lyase reaction is 100 times more efficient with 17-Preg as the substrate than with 17OHP (7, 8). Thus, 17alpha -hydroxylase activity was measured as the conversion of progesterone to 17OHP, because this product is not metabolized further, and 17,20-lyase activity was measured as the conversion of 17-Preg to DHEA in the presence of exogenously added cytochrome b5 (8, 21).

Concentrations of metformin up to 100 µM did not inhibit either activity of P450c17, but troglitazone readily inhibited both activities of P450c17 (Fig. 2). At a substrate concentration of 1 µM progesterone, troglitazone inhibited 17alpha -hydroxylase activity with an IC50 of 12 µM, and at 1 µM 17-Preg, troglitazone inhibited 17,20-lyase activity with an IC50 of 11 µM. These 1 µM steroid-substrate concentrations are above the apparent Km of about 0.5 µM for both the 17alpha -hydroxylase reaction and the 17,20-lyase reaction in this system (8). The presence or absence of cytochrome b5 did not affect the IC50 of the lyase reaction (data not shown). Thus, the inhibitory effect of troglitazone on 17,20-lyase activity does not depend on interaction with cytochrome b5. Kinetic analysis using both the Lineweaver-Burk method (1/V plotted against 1/S) and the Dixon method (1/V plotted against inhibitor concentration) revealed a competitive mode of inhibition with KI values of 8.4 ± 0.6 µM for 17alpha -hydroxylase activity and of 5.3 ± 0.7 µM for 17,20-lyase activity (Fig. 3). Thus, troglitazone inhibits both activities of P450c17 by the same mechanism.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of metformin and troglitazone on the differential activities of human P450c17. A, 17alpha -hydroxylase activity. Microsomes prepared from yeast coexpressing human P450c17 and human P450 oxidoreductase were incubated with 1 µM [14C]progesterone (Prog) and 0-100 µM metformin (MET) and troglitazone (TRO). 17alpha -Hydroxylase activity was assessed as the conversion rate of [14C]progesterone to 17OHP. B, 17,20-lyase activity. Microsomes coexpressing P450c17 and OR were incubated with 1 µM 17-Preg and 0-100 µM metformin and troglitazone. 17,20-Lyase activity was assessed as the conversion rate of [3H]17alpha -hydroxypregnenolone to DHEA. Each data point represents the mean ± S.D. of triplicate determinations.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Kinetic analysis of the mode of inhibition of P450c17 activities by troglitazone (TRO). Lineweaver-Burk plots of 17alpha -hydroxylase activity (A) and 17,20-lyase activity (B) and Dixon plot of 17alpha -hydroxylase activity (C) in the absence and presence of troglitazone (0-20 µM) and with 0.5, 1, 2, and 4 µM [14C]progesterone (Prog) and 17-Preg for the assessment of 17alpha -hydroxylase and 17,20-lyase activity, respectively. Incubations were carried out with yeast microsomes (40 µg of total protein) coexpressing human P450c17 and human P450 oxidoreductase. Each data point represents the mean ± S.D. of triplicate determinations.

The inhibitory effect of troglitazone was seen both in the yeast microsome system and in mammalian cells expressing P450c17. Troglitazone inhibited 17alpha -hydroxylase activity in transiently transfected COS-1 cells in a dose-dependent fashion with an IC50 of 16 µM (Fig. 4), which is very similar to the IC50 of 12 µM observed in yeast microsomes.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibition of 17alpha -hydroxylase activity by troglitazone (TRO) in whole cells (solid line) and in yeast microsomes (dotted line). COS-1 cells transiently expressing human P450c17 and yeast microsomes expressing both P450c17 and P450 oxidoreductase were incubated with [14C]progesterone (Prog), and 17alpha -hydroxylase activity was determined as the conversion rate of [14C]progesterone to 17OHP. Each data point represents the mean ± S.D. of triplicate determinations.

Effects of Metformin and Troglitazone on 3beta HSDII Activity-- The two forms of human 3beta HSD are catalytically equivalent (29, 30). Because 3beta HSDII is the only form expressed in human adrenals and gonads (30), we examined the potential inhibitory effects of metformin and troglitazone on this isozyme. We assayed 3beta HSD activity in microsomes from yeast transformed with an expression vector for human 3beta HSDII. Because all three substrates for 3beta HSD (pregnenolone, 17-Preg, and DHEA) have the same apparent Km values and similar Vmax values when analyzed in this yeast expression system (21), we measured the conversion of pregnenolone to progesterone, which reflects the rate of conversion of DHEA to androstenedione.

Metformin had no effect on 3beta HSDII activity, but troglitazone inhibited 3beta HSDII activity in a dose-dependent fashion with an IC50 of 24 µM (Fig. 5). To determine the mechanism by which troglitazone inhibited 3beta HSDII activity we performed Lineweaver-Burk and Dixon kinetic analyses. In contrast to the competitive inhibition of P450c17, Lineweaver-Burk plots showed that troglitazone inhibited 3beta HSDII activity by a noncompetitive mode with a KI of 25.4 ± 5.1 µM (Fig. 6A). Pure noncompetitive enzymatic competition yields a linear dose-response curve in Dixon analysis (26). However, instead of showing a linear curve, Dixon plots of 3beta HSDII inhibition by troglitazone yielded a hyperbolic curve (Fig. 6B). This indicates that there are separate modes of inhibition occurring at low inhibitor concentrations (a more potent noncompetitive mode) and at high inhibitor concentrations (an additional competitive mode). Such behavior might be explained by the simultaneous binding of two molecules of inhibitor to one molecule of enzyme (26). Thus, the mode by which troglitazone inhibits 3beta HSDII activity is clearly different from the mode by which it inhibits P450c17.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of metformin and troglitazone on 3beta HSDII activity. Microsomes prepared from yeast expressing human 3beta HSDII were incubated with 5 µM [14C]pregnenolone (Preg) and 0-100 µM metformin (MET) and troglitazone (TRO). 3beta HSDII activity was assessed as the conversion rate of [14C]pregnenolone to progesterone (Prog). Each data point represents the mean ± S.D. of triplicate determinations.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Kinetic analysis of the mode of inhibition of 3beta HSDII activity by troglitazone (TRO). Lineweaver-Burk plot (A) and Dixon plot (B) of 3beta HSDII activity. Incubations were carried out with yeast microsomes (40 µg total protein) expressing human 3beta HSDII in the absence and presence of troglitazone (0-60 µM) with 1, 5, 10, and 20 µM [14C]pregnenolone (Preg). Each data point represents the mean ± S.D. of triplicate determinations.

Comparison of Troglitazone to Other Thiazolidinediones-- To determine whether the inhibitory effects of troglitazone are unique to this particular drug or are a general characteristic of thiazolidinedione compounds, we compared the effects of troglitazone on P450c17 and 3beta HSDII activities to those of two other thiazolidinediones, rosiglitazone and pioglitazone (Fig. 7). Rosiglitazone inhibited both the 17alpha -hydroxylase (IC50 = 86 µM) and the 17,20-lyase (IC50 = 90 µM) activities of P450c17 and also inhibited 3beta HSDII activity (IC50 = 54 µM) but to a considerably lesser extent than troglitazone (IC50 = 10, 16, and 20 µM, respectively). Pioglitazone had minimal inhibitory effects on all three enzymatic activities with IC50 values of >100 µM. A summary of all the inhibition constants of the three thiazolidinediones on the three enzymatic activities is shown in Table I. Thus, the potential for direct inhibition of both P450c17 and 3beta HSDII seems to be a general characteristic of thiazolidinedione drugs, but the substantial variations in their inhibitory effects indicate that minor changes in the thiazolidinedione structure have major effects on their actions as inhibitors of steroidogenic enzymes.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   Differential inhibition of P450c17 and 3beta HSDII by thiazolidinediones. A, 17alpha -hydroxylase activity. Microsomes prepared from yeast coexpressing human P450c17 and human P450 oxidoreductase were incubated with 1 µM [14C]progesterone (Prog) and 0-100 µM troglitazone (TRO), rosiglitazone (ROSI), and pioglitazone (PIO). 17alpha -Hydroxylase activity was assessed as the conversion rate of [14C]progesterone to 17alpha -hydroxyprogesterone. B, 17,20-lyase activity. Microsomes coexpressing P450c17 and OR were incubated with 1 µM [3H]17alpha -hydroxypregnenolone and 0-100 µM troglitazone, rosiglitazone, or pioglitazone. 17,20-Lyase activity was assessed as the conversion rate of 17-Preg to DHEA. C, 3beta HSDII activity. Microsomes prepared from yeast expressing human 3beta HSDII were incubated with 5 µM [14C]pregnenolone (Preg) and 0-100 µM troglitazone, rosiglitazone, or pioglitazone. 3beta HSDII activity was assessed as the conversion rate of [14C]pregnenolone to progesterone (Prog). Each data point represents the mean ± S.D. of triplicate determinations.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Inhibition constants of thiazolidinediones


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-sensitizing drugs increase insulin sensitivity and decrease hyperandrogenemia in women with PCOS (11-14), and it has been suggested that metformin decreases P450c17 activity in PCOS (12, 31, 32), but this suggestion has been based on serum hormone values rather than on direct biochemical study. In our microsomal system we found no evidence for a direct inhibition of P450c17 or 3beta HSDII by metformin, even in concentrations far exceeding the usual therapeutic concentrations of 10-20 µM (33). Thus the inhibitory effect of metformin on androgen synthesis seems to be mediated only indirectly, most likely by lowering circulating insulin concentrations. Similarly, lowering insulin concentrations without drug treatment, by dieting and weight loss, decreases circulating androgen concentrations in women with PCOS (31, 34, 35).

However, we have now shown that troglitazone, in contrast to metformin, exerts a direct inhibitory effect on the enzymatic activities of P450c17 and 3beta HSDII, thus directly interfering with androgen biosynthesis. This effect probably contributes to the androgen-lowering effect of troglitazone observed in women with PCOS because enzymatic inhibition occurs at pharmacologically relevant concentrations. Therapeutic concentrations of troglitazone in serum are 2-10 µM (36). If the intracellular concentrations are equivalent, 2-10 µM troglitazone would inhibit 20-50% of P450c17 activity and 5-20% of 3beta HSDII activity. Although intracellular troglitazone concentrations have not been reported, we found similar IC50 values in both the microsome assay and in whole-cell experiments, suggesting that intracellular troglitazone concentrations may be equivalent to circulating concentrations. Our kinetic analyses showed that troglitazone exerted competitive inhibition of P450c17 and a mainly noncompetitive inhibition of 3beta HSDII. Thus, the interaction of troglitazone with each enzyme is distinct.

Rosiglitazone and pioglitazone also inhibited P450c17 and 3beta HSDII activities, however, with IC50 values clearly beyond their usual therapeutic serum concentrations of 0.2-1 µM (37, 38). Thus, although the direct inhibition of both P450c17 and 3beta HSDII seems to be a general property of thiazolidinedione drugs, only troglitazone does so at concentrations achieved in clinical use. Although the potential actions of rosiglitazone and pioglitazone in PCOS have not been reported, it is possible that all thiazolidinediones can decrease androgen production by indirect mechanisms involving decreased circulating insulin concentrations.

The observed differential inhibitory effects of thiazolidinedione drugs on the key androgen biosynthetic enzymes P450c17 and 3beta HSDII do not correlate with their differential binding affinity to PPARgamma . Troglitazone shows the weakest binding to PPARgamma and is only a partial agonist, whereas rosiglitazone and pioglitazone bind to PPARgamma with much higher affinity and concurrently exhibit more potent insulin-sensitizing effects (39-42). Thus, distinct and different regions of the thiazolidinedione drugs might be involved in PPARgamma binding and inhibition of P450c17 and 3beta HSDII, respectively. Further understanding of structure-function relationships in thiazolidinedione drugs may facilitate more specific drug design, aiming at inhibitory effects on steroidogenesis as well as at more potent insulin-sensitizing effects.

    ACKNOWLEDGEMENTS

We thank Drs. John W. M. Martens and Amit V. Pandey for helpful discussions and Dr. Wolfgang Jacobson for assistance with the liquid chromatography/mass spectrometry analysis.

    FOOTNOTES

* This work was supported by National Cooperative Program for Infertility Research Grant U54-HD 34449 (to W. L. M.), National Institutes of Health Grants K08-DK 02387 (to R. J. A.) and R01-DK 37922 (to W. L. M.), and research fellowship Grant Ar 310/2-1 from the Deutsche Forschungsgemeinschaft (to W. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pediatrics, Bldg. MR-IV, Rm. 209, University of California, San Francisco, CA 94143-0978. Tel.: 415-476-2598; Fax: 415-476-6286; E-mail: wlmlab@itsa.ucsf.edu.

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M100040200

    ABBREVIATIONS

The abbreviations used are: 17-Preg, 17alpha -hydroxypregnenolone; DHEA, dehydroepiandrosterone; 3beta HSDII, 3beta -hydroxysteroid dehydrogenase type II; 17OHP, 17alpha -hydroxyprogesterone; PCOS, polycystic ovary syndrome; PPARgamma , peroxisome proliferator-activated receptor gamma ; OR, oxidoreductase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Miller, W. L. (1988) Endocr. Rev. 9, 295-318[Medline] [Order article via Infotrieve]
2. Nakajin, S., Shively, J. E., Yuan, P. M., and Hall, P. F. (1981) Biochemistry 20, 4037-4042[Medline] [Order article via Infotrieve]
3. Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1986) Science 234, 1258-1261[Medline] [Order article via Infotrieve]
4. Lin, D., Harikrishna, J. A., Moore, C. C., Jones, K. L., and Miller, W. L. (1991) J. Biol. Chem. 266, 15992-15998[Abstract/Free Full Text]
5. Penning, T. M. (1997) Endocr. Rev. 18, 281-305[Abstract/Free Full Text]
6. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., Michael, M. D., Mendelson, C. R., and Bulun, S. C. (1994) Endocr. Rev. 15, 342-355[Medline] [Order article via Infotrieve]
7. Lin, D., Black, S. M., Nagahama, Y., and Miller, W. L. (1993) Endocrinology 132, 2498-2506[Abstract]
8. Auchus, R. J., Lee, T. C., and Miller, W. L. (1998) J. Biol. Chem. 273, 3158-3165[Abstract/Free Full Text]
9. Ehrmann, D. A., Barnes, R. B., and Rosenfield, R. L. (1995) Endocr. Rev. 16, 322-353[Medline] [Order article via Infotrieve]
10. Dunaif, A. (1997) Endocr. Rev. 18, 774-800[Abstract/Free Full Text]
11. Velazquez, E. M., Mendoza, S., Hamer, T., Sosa, F., and Glueck, C. J. (1994) Metabolism 43, 647-654[Medline] [Order article via Infotrieve]
12. Nestler, J. E., and Jakubowicz, D. J. (1996) N. Engl. J. Med. 335, 617-623[Abstract/Free Full Text]
13. Dunaif, A., Scott, D., Finegood, D., Quintana, B., and Whitcomb, R. (1996) J. Clin. Endocrinol. Metab. 81, 3299-3306[Abstract]
14. Ehrmann, D. A., Schneider, D. J., Sobel, B. E., Cavaghan, M. K., Imperial, J., Rosenfield, R. L., and Polonsky, K. S. (1997) J. Clin. Endocrinol. Metab. 82, 2108-2116[Abstract/Free Full Text]
15. DeFronzo, R. A., and Goodman, A. M. (1995) N. Engl. J. Med. 333, 541-549[Abstract/Free Full Text]
16. El-Mir, M. Y., Nogueira, V., Fontaine, E., Averet, N., Rigoulet, M., and Leverve, X. (2000) J. Biol. Chem. 275, 223-228[Abstract/Free Full Text]
17. Owen, M. R., Doran, E., and Halestrap, A. P. (2000) Biochem. J. 348, 607-614[CrossRef][Medline] [Order article via Infotrieve]
18. Spiegelman, B. M. (1998) Diabetes 47, 507-514[Abstract]
19. Kersten, S., Desvergne, B., and Wahli, W. (2000) Nature 405, 421-442[CrossRef][Medline] [Order article via Infotrieve]
20. Olefsky, J. M. (2000) J. Clin. Invest. 106, 467-472[Free Full Text]
21. Lee, T. C., Miller, W. L., and Auchus, R. J. (1999) J. Clin. Endocrinol. Metab. 84, 2104-2110[Abstract/Free Full Text]
22. Pompon, D., Louerat, B., Bronine, A., and Urban, P. (1996) Methods Enzymol. 272, 51-64[CrossRef][Medline] [Order article via Infotrieve]
23. Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve]
24. Hadfield, C., Cashmore, A. M., and Meacock, P. A. (1986) Gene (Amst.) 45, 149-158[CrossRef][Medline] [Order article via Infotrieve]
25. Geller, D. H., Auchus, R. J., and Miller, W. L. (1999) Mol. Endocrinol. 13, 167-175[Abstract/Free Full Text]
26. Dixon, M., and Webb, E. C. (1979) Enzymes , 3rd Ed. , Academic Press, New York
27. Cornish-Bowden, A. (1995) Analysis of Enzyme Kinetic Data , Oxford University Press, Oxford
28. Chung, B. C., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, P. F., Shively, J. E., and Miller, W. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 407-411[Abstract]
29. Thomas, J. L., Myers, R. P., and Strickler, R. C. (1989) J. Steroid Biochem. 33, 209-217[CrossRef][Medline] [Order article via Infotrieve]
30. Rheaume, E., Lachance, Y., Zhao, H. F., Breton, N., Dumont, M., de Launoit, Y., Trudel, C., Luu-The, V., Simard, J., and Labrie, F. (1991) Mol. Endocrinol. 5, 1147-1157[Abstract]
31. Nestler, J. E., and Jakubowicz, D. J. (1997) J. Clin. Endocrinol. Metab. 82, 4075-4079[Abstract/Free Full Text]
32. la Marca, A., Egbe, T. O., Morgante, G., Paglia, T., Cianci, A., De Leo, V., and Ciani, A. (2000) Hum. Reprod. 15, 21-23[Abstract/Free Full Text]
33. Sambol, N. C., Chiang, J., O'Conner, M., Liu, C. Y., Lin, E. T., Goodman, A. M., Benet, L. Z., and Karam, J. H. (1996) J. Clin. Pharmacol. 36, 1012-1021[Abstract]
34. Kiddy, D. S., Hamilton-Fairley, D., Bush, A., Short, F., Anyaoku, V., Reed, M. J., and Franks, S. (1992) Clin. Endocrinol. 36, 105-111[Medline] [Order article via Infotrieve]
35. Crave, J. C., Fimbel, S., Lejeune, H., Cugnardey, N., Dechaud, H., and Pugeat, M. (1995) J. Clin. Endocrinol. Metab. 80, 2057-2062[Abstract]
36. Loi, C. M., Alvey, C. W., Vassos, A. B., Randinitis, E. J., Sedman, A. J., and Koup, J. R. (1999) J. Clin. Pharmacol. 39, 920-926[Abstract/Free Full Text]
37. Maeshiba, Y., Kiyota, Y., Yamashita, K., Yoshimura, Y., Motohashi, M., and Tanayama, S. (1997) Arzneim. Forsch. 47, 29-35[Medline] [Order article via Infotrieve]
38. Cox, P. J., Ryan, D. A., Hollis, F. J., Harris, A. M., Miller, A. K., Vousden, M., and Cowley, H. (2000) Drug Metab. Dispos. 28, 772-780[Abstract/Free Full Text]
39. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A. (1995) J. Biol. Chem. 270, 12953-12956[Abstract/Free Full Text]
40. Berger, J., Bailey, P., Biswas, C., Cullinan, C. A., Doebber, T. W., Hayes, N. S., Saperstein, R., Smith, R. G., and Leibowitz, M. D. (1996) Endocrinology 137, 4189-4195[Abstract]
41. Willson, T. M., Cobb, J. E., Cowan, D. J., Wiethe, R. W., Correa, I. D., Prakash, S. R., Beck, K. D., Moore, L. B., Kliewer, S. A., and Lehmann, J. M. (1996) J. Med. Chem. 39, 665-668[CrossRef][Medline] [Order article via Infotrieve]
42. Camp, H. S., Li, O., Wise, S. C., Hong, Y. H., Frankowski, C. L., Shen, X., Vanbogelen, R., and Leff, T. (2000) Diabetes 49, 539-547[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.