Elevated production and metabolic clearance rates of androgens in morbidly obese women

Blood production rates of testosterone, dihydrotestosterone (DHT), and 3 alpha-androstanediol (3 alpha-diol) were found to be approximately 2-fold elevated in morbidly obese, nonhirsute, normally menstruating women. Values were intermediate between those found in normal women and those in a group of nonobese normally menstruating women with idiopathic hirsutism. Elevated androgen production rates in obese women were associated with 2- to 3-fold increases in MCRs, presumably due to decreased levels of sex hormone-binding globulin. Thus, increased production rates were offset by increased MCRs, resulting in plasma testosterone, DHT, and 3 alpha-diol concentrations that were similar in the obese and normal women. By contrast, women with hirsutism had increased production rates associated with elevated plasma androgens as well as increased MCRs. Urinary excretion of testosterone glucuronide and 3 alpha-diol glucuronide (3 alpha-diol G) were elevated in both obese and hirsute women, paralleling the increased androgen production rates. Despite increased production rates and excretion of androgens, obese women exhibited no menstrual abnormalities, hirsutism, or other signs of virilism. To explore the apparent ineffectiveness of increased androgen production to produce virilizing symptoms, we measured plasma 3 alpha-diol G levels as a measure of peripheral androgen action. The mean +/- SE plasma 3 alpha-diol G was 53 +/- 8 ng/dl in obese women and 36 +/- 6 in normal women; by contrast, women with idiopathic hirsutism had levels of 440 +/- 99, a 12-fold elevation. Plasma testosterone glucuronide in obese and hirsute women were only 2- to 3-fold elevated, while plasma DHT glucuronide was not increased in obese women and was only 2-fold elevated in hirsute women. Thus, obesity is a state of increased androgen production and accelerated clearance. 3 alpha-diol G levels in obese women were only minimally elevated, in contrast to values in the hirsute women, perhaps reflecting the apparent androgen ineffectiveness.     

Identification of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of rat Leydig cells as type II retinol dehydrogenase

Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5alpha-reductase, 2) conversion of DHT to 5alpha-androstane-3alpha, 17beta-diol (3alpha-DIOL) by the reductive activity of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), and 3) conversion of 3alpha-DIOL by an oxidative 3alpha-HSD activity. While the type I 3alpha-HSD enzyme (3alpha-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3alpha-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3alpha-HSD1, and a microsomal NAD(H)-dependent activity. The two activities were separable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3alpha-HSD in rat Leydig cells, degenerate primers were used to amplify a number of short-chain alcohol dehydrogenases. Sequence analysis of cloned PCR products identified retinol dehydrogenase type II (RoDH2) as the prevalent species in purified Leydig cells. RoDH2 cDNA was subcloned into expression vectors and transiently transfected into CHOP and COS-1 cells. Its properties were compared with transiently transfected 3alpha-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3alpha-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3alpha-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3alpha-HSD cDNA produced a protein that was exclusively a 3alpha-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 3alpha-HSD oxidation and reduction reactions that were NAD(H) dependent, and the enzyme activities were located in the microsomes. Type I 3alpha-HSD also catalyzed both oxidation and reduction, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3alpha-HSD and RoDH2 have distinct 3alpha-HSD activities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells.     

The use of human skin fibroblasts to obtain potency estimates of drug binding to androgen receptors

Although several drugs with antiandrogenic properties have been used to treat such conditions as prostatic carcinoma, precocious puberty, acne, and hirsutism, their relative strengths in human tissues are not known. Most of the compounds that are effective clinically in opposing androgen action interact with the androgen receptor in various assay systems. To determine in human cells the relative potencies of these agents as well as others with androgenic properties, we measured the abilities of various compounds to compete with [3H]dihydrotestosterone [( 3H]DHT) for androgen-binding sites in dispersed human genital skin fibroblasts at 22 degrees C. The concentrations of unlabeled DHT, methyltrienolone (a synthetic non- metabolizeable androgen), and testosterone required for 50% inhibition of [3H]DHT binding were similar, approximately 1 nM [0.87 +/- 0.12 (+/- SE), 1.18 +/- 0.18, and 1.01 +/- 0.20 nM, respectively]. The relative binding activities, defined by the ratio of the concentration of methyltrienolone to the concentration of competitor required for 50% displacement of [3H]DHT, were as follows: spironolactone greater than R2956 (a synthetic antiandrogen) greater than megestrol acetate greater than cyproterone acetate greater than estradiol greater than flutamide much greater than testolactone greater than cimetidine. Danazol, an androgen agonist that causes hirsutism, was nearly as effective as spironolactone in its ability to compete for the fibroblast androgen receptor, 50% inhibition of fibroblast [3H]DHT binding was achieved by 1.76 +/- 0.31 nM spironolactone and 2.85 +/- 0.50 nM danazol. Two other compounds that induce hirsutism, diphenylhydantoin and diazoxide, did not displace [3H]DHT. We conclude that 1) of the compounds tested, spironolactone, which is rapidly metabolized in vivo to a much less potent competitor, is the most potent antiandrogen in its ability to interact in vitro with human skin fibroblast androgen receptors; 2) estradiol is a relatively potent androgen receptor binder; and 3) this receptor assay, combined with metabolic clearance and pharmacokinetic considerations, should be useful in selecting drugs for androgen and antiandrogen therapy.     

THE EFFECT OF ENDOGENOUS PROGESTERONE ON SERUM LEVELS OF 5α-REDUCED ANDROGENS IN HIRSUTE WOMEN

This study was to examine indirectly the effect of endogenous progesterone, a known competitor for 5 alpha-reductase, on androgen metabolism in target organs in hirsute women. Serum levels of progesterone, testosterone (T), androstenedione (A), dihydrotestosterone (DHT) and 5 alpha-androstane 3 alpha 17 beta-diol (3 alpha-diol) and sex hormone binding globulin (SHBG) were assessed serially over a four week period in normal women, six hirsute women with regular menstrual cycles, eight hirsute women with oligomenorrhoea (and presumptive polycystic ovaries) and seven non-hirsute women with oligomenorrhoea. Serum T and A levels were significantly higher than normal in both hirsute and non-hirsute women with oligomenorrhoea, while serum SHBG was significantly lower than normal in the two groups of hirsute women. The calculated free T level was higher than normal in all three groups of patients. DHT levels were not significantly different from normal in any of the three groups of patients. The 3 alpha-diol level showed considerable overlap with normal in all groups of patients and was only significantly higher than normal in hirsute women with oligomenorrhoea (P less than 0.05). There was a small fall in DHT in the late luteal phase of the cycle of those women with a sustained rise in serum progesterone in the second half of the cycle, but no change in serum 3 alpha-diol. These studies suggest that serum 3 alpha-diol may not be as good an indicator of peripheral androgen metabolism in hirsute women as previously reported and that a rise in serum progesterone has only a minimal effect on circulating levels of the active 5 alpha-reduced androgen metabolites. Although in vitro 3 alpha-diol has been shown to be a potent inhibitor of 5 alpha-reductase this casts doubt on its role in this regard in vivo.     

Local androgen inactivation in abdominal visceral adipose tissue

We examined the expression and activity of two enzymes from the aldoketoreductase (AKR) family 1C, namely type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD-5, AKR1C3) and type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD-3, AKR1C2) in female sc and omental adipose tissue and in preadipocyte primary cultures. 17beta-HSD-5 preferentially synthesizes testosterone from the inactive adrenal precursor androstenedione, whereas 3alpha-HSD-3 inactivates dihydrotestosterone. mRNAs of both enzymes were detected in adipose tissue from the omental and sc compartments. Real-time PCR quantification indicated a 3-fold higher 3alpha-HSD-3 expression compared with 17beta-HSD-5, and the expression of both enzymes tended to be higher in the sc vs. the omental depot. Accordingly, dose-response and time-course experiments performed in preadipocyte primary cultures indicated that 3alpha-HSD activity was higher than 17beta-HSD activity (13-fold maximum velocity difference). We measured 3alpha-HSD activity in omental and sc adipose tissue samples of 32 women for whom body composition and body fat distribution were evaluated by dual-energy x-ray absorptiometry and CT, respectively. We found that androgen inactivation in omental adipose tissue through 3alpha-HSD activity was significantly higher in women with elevated vs. low visceral adipose tissue accumulation (1.7-fold difference; P < 0.05). Moreover, omental adipose tissue 3alpha-HSD activity was positively and significantly associated with CT-measured visceral adipose tissue (r = 0.43; P < 0.02) and omental adipocyte diameter (r = 0.42; P < 0.02). These results indicate that local androgen inactivation is a predominant reaction in female abdominal adipose tissue, with the greatest conversion rates observed in the presence of abdominal visceral obesity. Increased androgen inactivation in omental adipose tissue of abdominally obese women may impact locally on the regulation of adipocyte metabolism.     

5 alpha-Reductase activity in the genital skin of hirsute women

A simplified, rapid, and highly reproducible technique is described for measuring 5 alpha-reductase activity (5 alpha RA) in small skin biopsies. Human genital skin was obtained from 23 nonhirsute and 20 hirsute premenopausal women (HW) and 5 normal men. Skin samples were minced at 4 C and incubated with RPMI-1640 in the presence of 95% O2-5% CO2 and 4.15 nmol [14C]testosterone ([14C]T) for 2 h at 37 C. Steroids were extracted with diethyl ether and separated by Celite and paper chromatography. Radioactivity in specific eluates was quantified, and the mass of each steroid was measured by RIA. The separate formation of 5 alpha-androstane-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha, 17B diol (3 alpha diol), androstenedione, and androsterone from [14C]T was measured. In separate experiments it was demonstrated that an incubation time of 2 h was optimum and that the addition of cofactors was unnecessary. Radiochemical purity was confirmed after chromatography. The mean +/- SE conversion ratio (CR) of T to DHT (in 2 h) in HW was higher than that in normal women (16.80 +/- 1.62% vs. 4.48 +/- 0.36%; P less than 0.01). In men, the CR of T to DHT averaged 31.60 +/- 3.96%. Individual values for the CR of T to DHT in HW and normal women did not overlap. The CR of T to 3 alpha diol was significantly higher in HW (9.66 +/- 0.86%) and men (15.98 +/- 2.0%) compared to that in normal women (2.96 +/- 0.32%; P less than 0.05). The CR of T to androstenedione was significantly greater in HW and men (6.18 +/- 0.42 and 7.28 +/- 1.92%) compared to that in normal women (2.64 +/- 0.64%; P less than 0.05). The CR of T to androsterone was very low and was similar in the three groups. The production of DHT in HW (4.50 +/- 1.0 pmol/mg X 2 h) was significantly greater than that in normal women (0.48 +/- 0.08; P less than 0.01) and was similar to the production in men (6.18 +/- 1.94 pmol/mg X 2 h). There was a significant correlation between the CR of T to DHT and DHT production, and the CR of T to 3 alpha diol and 3 alpha diol production as well as between the CRs of T to DHT and T to 3 alpha diol. These data suggest that measurements of DHT formation are best suited for the assessment of 5 alpha RA and that the measurement of 5 alpha RA in vitro from small skin biopsies is suitable for the clinical evaluation of hirsutism.     

Androgen binding in nuclear matrix of human genital skin fibroblasts from patients with androgen insensitivity syndrome

Specific sex steroid-binding sites are associated with the salt-insoluble nuclear matrix from which lipids, histones, and chromatin have been extracted. In intact cultured normal human genital skin fibroblasts incubated for 1 h at 37 C with a saturating concentration (2 nM) of [3H]dihydrotestosterone [( 3H]DHT), approximately 50% of the total intracellular androgen receptor-steroid complexes were found in the nucleus. Within isolated nuclei from such cells, 28-49% of the specific androgen receptor binding was associated with the nuclear matrix. The antiandrogen cyproterone acetate inhibited DHT binding within the nuclear matrix. Cultured genital skin fibroblasts from two unrelated patients with receptor-positive complete androgen insensitivity (CAIS, AR+), had normal (approximately 50%) nuclear binding of DHT, and 35% and 45% of it was localized to the nuclear matrix. Genital skin fibroblasts from a patient with receptor-negative complete androgen insensitivity (CAIS, AR-) had no specific DHT binding in isolated nuclei or nuclear matrix. Scatchard analysis of specific DHT binding in the nuclear matrix isolated from cells of normal subjects after an in vitro exchange assay (0 C; 24 h) revealed the presence of saturable (maximum binding, approximately equal to 200 fmol/mg nuclear DNA), high affinity (Kd approximately equal to 1.0 nM) binding sites. By contrast, in the nuclear matrix isolated from cells of a patient with CAIS, AR+, the binding affinity for DHT was 3-fold lower (Kd approximately equal to 3.0 nM). When cytosolic androgen receptor-DHT complexes prepared from cells preincubated at 37 C for 1 h with [3H]DHT were incubated at 0 C for 1 h with isolated nuclei and nuclear matrix in the presence of 0.15 M KCl, 40-60% of specific nuclear binding was associated with the nuclear matrix. In these cell-free in vitro experiments, radiolabeled DHT-receptor complexes prepared from normal or mutant cells were mixed with isolated nuclei and nuclear matrix prepared from cells of normal subjects or patients with CAIS, AR+ or CAIS, AR-. Under these conditions, specific DHT binding in nuclei and nuclear matrix was quantitatively similar in the presence of a mutant (CAIS, AR+) receptor-steroid complex or in the presence of nuclei or nuclear matrix from the mutant cells (CAIS, AR- or AR+) when compared simultaneously with the same subcellular fractions prepared from the cells of normal subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of 3 alpha-androstanediol glucuronide in human genital skin

3 alpha-Androstanediol glucuronide (3 alpha diol-G) is produced extrasplanchnically and is a good clinical marker of androgen action in peripheral tissues. However, the direct formation of androgen glucuronides in peripheral sites such as skin has not been determined in man. Genital skin from 21 premenopausal women and 8 men and foreskin from 6 neonates were incubated with either [14C]testosterone [14C]dihydrotestosterone (DHT) to determine the production of DHT glucuronide and 3 alpha diol-G in skin. After hydrolysis of incubation medium with glucuronidase, followed by extraction and sequential chromatography, constant 3H to 14C ratios of 3 alpha diol confirmed the production of DHT glucuronide and 3 alpha diol-g. The conversion of DHT to 3 alpha diol-G was higher than the conversion from testosterone (P less than 0.05), and conversion was higher in men than in women. These data provide evidence for the direct formation of C19 steroid glucuronides by human skin.     

Evidence for the importance of peripheral tissue events in the development of hirsutism in polycystic ovary syndrome

Hirsutism can occur in the presence of normal or near normal levels of serum testosterone, unbound testosterone (uT), dehydroepiandrostene sulfate, androstenedione, and dihydrotestosterone. However, we have found that serum androstanediol glucuronide (3 alpha-diol G) is markedly increased in idiopathic hirsutism and it serves as an excellent marker of peripheral androgen metabolism and action. In the present work, we studied 12 hirsute (H) and 12 nonhirsute (NH) patients with polycystic ovary syndrome (PCO) and 13 age and weight matched controls in order to determine if differences in sex steroid levels or peripheral tissue androgen events were associated with hirsutism. Serum unbound estradiol levels and LH-FSH ratios were similar in both groups of women with PCO, and both were significantly higher than levels in controls (P less than 0.05). Whereas serum testosterone, uT, and androstenedione were elevated in both H-PCO and NH-PCO patients compared to controls, the levels in these two groups were similar. Serum dehydroepiandrostene sulfate was higher in PCO patients compared to controls, but H-PCO patients had slightly higher levels than NH-PCO patients. Serum delta 5-androstenediol was also slightly higher in H-PCO compared to NH-PCO patients. Dihydrotestosterone was normal and unconjugated; 3 alpha-diol was higher than normal in both groups of patients with PCO, although H-PCO patients had higher levels than NH-PCO patients. Compared to these relatively minor changes between the PCO patient groups, serum 3 alpha-diol G was markedly elevated in H-PCO patients (approximately 10-fold), yet normal in NH-PCO patients (P less than 0.01). The ratios of serum 3 alpha-diol G-uT were similar in NH-PCO patients and controls, but were elevated in H-PCO patients (P less than 0.01). These data indicate that: 1) women with PCO have increased circulating androgen levels regardless of the presence or absence of hirsutism; and 2) the presence of hirsutism is not only a function of circulating androgen levels, but may also be determined by events in peripheral tissues.     

Regulation of androgen receptor and 5 alpha-reductase in the skin of normal and hirsute women

The hormonal activity of androgens is mediated in target cells, particularly in human skin, by two kinds of proteins: the androgen receptor and the enzyme 5 alpha-reductase. In well differentiated androgen target cells, 5 alpha-reductase achieves the transformation of testosterone (T) into dihydrotestosterone (DHT), a more active androgen than T, because of its higher affinity for the receptor. In other words, 5 alpha-reductase acts as an amplifier of the androgen signal but is not absolutely required for androgen action. Regarding the regulation of the androgen receptor, minimal information is available. However, in genital skin, the receptor seems to be predominantly localized in the cytosolic compartment before puberty in males and in the nuclear compartment after puberty. In hirsute patients, recent data on genital skin fibroblasts do not show significant differences between the binding capacity of fibroblasts from normal and hirsute women whereas there is no difference between normal men and women. 5 alpha-Reductase activity seems to be a very important step in the processes involved in androgen action. While 5 alpha-reductase activity present in the skin of external genitalia does not seem to be androgen dependent, this is not the case for the enzyme located in pubic skin. In this area, a sex difference between males and females may be observed both in skin homogenates and in cultured fibroblasts. In addition DHT added to a medium of pubic skin fibroblasts is capable of increasing 5 alpha-reductase activity. This increase is not observed when cyproterone acetate is added to the medium and in patients with testicular feminization syndrome without receptors. Pubic 5 alpha-reductase activity is an androgen receptor mediated phenomenon. In patients with hirsutism, and particularly idiopathic hirsutism, 5 alpha-reductase activity is high without an increase in circulating androgens. This may be observed both in pubic skin homogenates and in cultured fibroblasts. Thus, an excess of skin 5 alpha-reductase activity may be considered as a cause of hirsutism but both the exact level of the abnormality in the regulation of the enzyme and its genetic control remain to be elucidated.     

Increase in H-Y antigen-positive lymphocytes in hirsute women: effects of cyproterone acetate and estradiol treatment

To investigate a possible relationship between lymphocyte H-Y antigen expression and plasma androgen concentrations in hirsute women, 27 hirsute women were studied. A significant increase in the percentage of H-Y-positive lymphocytes was found in both hirsute women with idiopathic hirsutism [13.4 +/- 2.9% (+/- SD); n = 15] and hirsute women with the polycystic ovary syndrome (13.0 +/- 2.8%; n = 12) compared to that in normal women (10.0 +/- 1.9%; n = 30; P less than 0.0005). Plasma testosterone and androstenedione concentrations, % H-Y+ lymphocytes, and hirsutism scores diminished during oral cyproterone acetate (50 mg/day) and percutaneous estradiol (3 mg/day) treatment. Significant correlations between % H-Y+ lymphocytes and hirsutism scores (P less than 0.001), % H-Y+ lymphocytes and plasma T concentrations (P less than 0.01) were found. We conclude that 1) women can produce H-Y antigen in the same way as men; 2) hirsutism is associated with an increase in H-Y antigen; and 3) the antiandrogen cyproterone acetate reduces H-Y antigen expression on lymphocytes.     

Progesterone metabolism in adipose cells

The aim of the present study was to investigate pathways of progesterone metabolism in human adipose cells. Adipose tissue samples from the omental (OM) and subcutaneous (SC) fat compartments were surgically obtained in women. In isolated mature adipocytes, progesterone was converted to 20alpha-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo-keto reductases 1C1, 2 and 3 (20alpha-HSD, 3alpha-HSD type 3 and 17beta-HSD type 5, respectively). In cultured preadipocytes, progesterone was converted to several metabolites identified using bidimensional thin layer chromatography, with or without the dual inhibitor of 5alpha-reductase type 1 and 2 (17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan-3-one (4-MA)). Major metabolites identified in OM and SC preadipocytes which were incubated for 24h with (14)C-labelled progesterone were 20alpha-hydroxyprogesterone, 5alpha-pregnane-3alpha/beta-ol-20-one, 5alpha- and 5beta-pregnanedione, 5alpha- and 5beta-pregnane-20alpha-ol-3-one, 5alpha-pregnane-3alpha/beta-ol-20-one and 5beta-pregnane-3alpha/beta-20alpha-diol. Induction of preadipocyte differentiation increased expression levels of AKR1C1 and modified the pattern of progesterone metabolism substantially, leaving 20alpha-hydroxyprogesterone as the main metabolite generated. On the other hand, progesterone itself showed no consistent effect on adipocyte differentiation. In conclusion, preadipocytes and lipid-storing, mature adipocytes efficiently generate progesterone metabolites in women, which is consistent with rather modest effects progesterone on abdominal fat cell differentiation.     

Dihydrotestosterone accumulation in genital skin fibroblasts derived from elderly men with prostatic hyperplasia.

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) by 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 15 prepubertal boys (2-10 yr), 17 young men (20-40 yr), 13 elderly men (60-78 yr) without clinically evident prostatic pathology, and 17 elderly men (61-88 yr) with benign prostatic hyperplasia (BPH). Respective DHT formations from testosterone (5 alpha-reduction) and 3 alpha-diol (3 alpha-HSOR oxidation) were not different among genital skin fibroblasts of the 4 groups. However, DHT degradation to 3 alpha-diol (3 alpha-HSOR reduction) was significantly lower in fibroblasts from elderly men with BPH than in those from the prepubertal boys (P less than 0.01), the young men (P less than 0.01), and the elderly men without BPH (P less than 0.05). 3 beta-HSOR reduction in fibroblasts of the BPH group was significantly lower (P less than 0.05) than in those of the elderly men without BPH; however, it did not differ from values for the prepubertal boys and the young men. (3 alpha + 3 beta)-HSOR reduction was also significantly lower (P less than 0.05) in the BPH group than respective values of the three other groups. These results indicate that DHT accumulation may occur in genital skin fibroblasts from elderly men with BPH, resulting from a shift in the overall balance of androgen metabolism, which favors the net formation of DHT.     

Androgen inactivation and steroid-converting enzyme expression in abdominal adipose tissue in men

We examined 5alpha-dihydrotestosterone (5alpha-DHT) inactivation and the expression of several steroid-converting enzymes with a focus on aldoketoreductases 1C (AKR1C), especially AKR1C2, in abdominal adipose tissue in men. AKR1C2 is mainly involved in the conversion of the potent androgen 5alpha-DHT to its inactive forms 5alpha-androstane-3alpha/beta,17beta-diol (3alpha/beta-diol). Subcutaneous (s.c.) and omental (Om) adipose tissue biopsies were obtained from 21 morbidly obese men undergoing biliopancreatic derivation surgery and 11 lean to obese men undergoing general abdominal surgery. AKR1C2 mRNA and 5alpha-DHT inactivation were detected in both s.c. and Om adipose tissue. After incubation of preadipocytes with 5alpha-DHT, both 3alpha-diol and 3beta-diol were produced through 3alpha/beta-ketosteroid reductase (3alpha/beta-HSD) activity. In preadipocyte cultures, 3alpha-reductase activity was significantly predominant over 3beta-reductase activity in cells from both the s.c. and Om compartments. Expression levels of AKR1C1, AKR1C3 and of the androgen receptor were significantly higher in s.c. versus Om adipose tissue while mRNA levels of 17beta-HSD-2 (hydroxysteroid dehydrogenase type 2) and 3(alpha-->beta)-hydroxysteroid epimerase were significantly higher in Om fat. 3Alpha/beta-HSD activity was mainly detected in the cytosolic fraction, suggesting that AKR1C may be responsible for this reaction. Experiments with isoform-specific AKR1C inhibitors in preadipocytes showed that AKR1C2 inhibition significantly decreased 3alpha-HSD and 3beta-HSD activities (3alpha-HSD: 30 +/- 24% of control for s.c. and 32 +/- 9% of control for Om, 3beta-HSD: 44 +/- 12% of control for s.c.). When cells were incubated with both AKR1C2 and AKR1C3 inhibitors, no significant additional inhibition was observed. 5Alpha-DHT inactivation was significantly higher in mature adipocytes compared with preadipocyte cultures in s.c. adipose tissue, as expressed per microgram total protein (755 +/- 830 versus 245 +/- 151 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.05 n = 10 cultures). 5Alpha-DHT inactivation measured in tissue homogenates was significantly higher in the s.c. depot compared with Om fat (117 +/- 39 versus 79 +/- 38 fmol 3alpha/beta-diol per microg prot over 24 h, P < 0.0001). On the other hand, Om 3alpha/beta-HSD activity was significantly higher in obese men (body mass index (BMI) >or= 30 kg/m2) compared with lean and overweight men (84 +/- 37 versus 52 +/- 30 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.03). No difference was found in s.c. 3alpha/beta-HSD activity between these groups. Positive correlations were found between s.c. 5alpha-DHT inactivation rate and circulating levels of the androgen metabolites androsterone-glucuronide (r = 0.41, P < 0.02) and 3alpha-diol-glucuronide (r = 0.38, P < 0.03) and with the adrenal precursor androstenedione (r = 0.42, P < 0.02). In conclusion, androgen inactivation was detected in abdominal adipose tissue in men, with higher 3alpha/beta-HSD activity in the s.c. versus Om depot. Higher Om 5alpha-DHT inactivation rates were found in obese compared with lean men. Further studies are required to elucidate whether local androgen inactivation in abdominal adipose tissue is involved in the modulation of adipocyte metabolism and regional fat distribution in men.     

Clinical usefulness of plasma androstanediol glucuronide measurements in women with idiopathic hirsutism

Serum androstanediol glucuronide (3 alpha-diol G), a metabolite of the active androgens dihydrotestosterone and androstanediol, was elevated in 28 consecutive women with idiopathic hirsutism (IH). The mean 3 alpha-diol G level in the women with IH was 487 +/- 192 (+/- SD) ng/dL compared to 119 +/- 37 ng/dL in normal women (n = 50), and only 1 patient had a value overlapping with the normal range. Since 3 alpha-diol G appears to be formed entirely in target organs and has a long serum half-life, we studied its clinical usefulness by following women with IH during treatment. In 15 of 17 women with IH treated for 1-4 yr with glucocorticoids, contraceptives, or spironolactone, serum 3 alpha-diol G levels changed concordantly with clinical responses, in contrast to the poor concordance of serum testosterone (5 of 17), free testosterone (7 of 17), and androstenedione (7 of 17). Specifically, in IH patients treated with spironolactone, serum testosterone, free testosterone, and androstenedione levels changed little, yet clinical improvement frequently occurred, and this improvement was reflected by concomitantly lowered 3 alpha-diol G levels. Further, in 4 IH patients, discontinuation of effective therapy resulted in prompt increases in serum 3 alpha-diol G as harbingers of worsening hair growth. We, thus, conclude that serum 3 alpha-diol G measurements are clinically useful in evaluating hirsute women and correlate with the clinical responses to therapy.