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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0583
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 264-270
Copyright © 2005 by The Endocrine Society

Expression and Activity of 20{alpha}-Hydroxysteroid Dehydrogenase (AKR1C1) in Abdominal Subcutaneous and Omental Adipose Tissue in Women

Sophie Blanchette, Karine Blouin, Christian Richard, Pierre Dupont, Van Luu-The and André Tchernof

Molecular Endocrinology and Oncology Research Center (S.B., K.B., C.R., V.L.-T., A.T.), Department of Nutrition (K.B., A.T.), and Gynecology Unit (P.D.), Laval University Medical Center and Laval University, Québec City, Canada G1V 4G2

Address all correspondence and requests for reprints to: André Tchernof, Ph.D., Molecular Endocrinology and Oncology Research Center, Department of Nutrition, Laval University Medical Center, 2705 Laurier Boulevard, Room T3–67, Québec City, PQ, Canada G1V 4G2. E-mail: andre.tchernof{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We examined the expression and activity of 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD) in abdominal adipose tissue in women. This recently characterized enzyme from the aldoketoreductase 1C family is responsible for the conversion of progesterone into 20{alpha}-hydroxyprogesterone. Abdominal sc (SC) and omental (OM) adipose tissue biopsies were obtained from a sample of 32 women aged 47.7 ± 5.9 yr (body mass index 27.6 ± 5.0 kg/m2) undergoing abdominal hysterectomies. Body composition and body fat distribution measurements were performed before the surgery by dual-energy x-ray absorptiometry and computed tomography, respectively. The expression of 20{alpha}-HSD was determined by real-time RT-PCR, and its activity was measured in whole-tissue homogenates. mRNA and activity of the enzyme were detected in both the SC and OM fat depots, the two measures being significantly higher in the SC compartment. Women characterized by a visceral adipose tissue area of 100 cm2 or greater had an increased 20{alpha}-HSD conversion rate in their OM adipose tissue, compared with women without visceral obesity (13.99 ± 2.07 vs. 7.92 ± 0.83 fmol/µg protein per 24 h, P < 0.05). Accordingly, a positive correlation was found between OM adipose tissue 20{alpha}-HSD activity and computed tomography-measured visceral adipose tissue area (r = 0.36, P < 0.05). Significant positive correlations were also found between OM 20{alpha}-HSD activity and OM adipocyte diameter (r = 0.49, P < 0.05) and OM adipose tissue LPL activity (r = 0.36, P = 0.06). In conclusion, 20{alpha}-HSD activity and mRNA were detected in SC and OM adipose tissue in women, and OM 20{alpha}-hydroxylation of progesterone was highest in women with visceral obesity. Additional studies are required to establish whether local conversion of progesterone may impact on the metabolism and function of adipocytes located within the abdominal cavity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
EXCESSIVE ACCUMULATION OF adipose tissue in the abdominal region (abdominal obesity) has been demonstrated to be a strong correlate of obesity-related metabolic alterations leading to type 2 diabetes and cardiovascular disease (1, 2). More specifically, fat accumulation located within the abdominal cavity (visceral obesity) has been closely related to the cluster of metabolic abnormalities now defined as the metabolic syndrome, which includes insulin resistance, hyperinsulinemia, elevated triglyceride levels, low high-density lipoprotein-cholesterol concentrations, and hypertension (1, 2, 3).

The well-known sex difference in body fat distribution suggests that depot-specific regulation of adipose tissue mass is achieved at least partly through hormonal regulation of many cellular processes in each depot. Sex steroids are thought to regulate adipose tissue deposition by acting on the proliferation and/or differentiation of preadipocytes as well as lipogenesis and/or lipolysis in mature adipocytes (4, 5, 6). The specific role of progesterone on fat accumulation is not yet fully understood. Some studies found that progesterone stimulated fat accumulation, lipoprotein lipase (LPL) activity, lipogenesis, and steroid- mediated differentiation of preadipocytes from various sources (7, 8, 9, 10, 11, 12). On the other hand, Björntorp (13) suggested that progesterone could be responsible for the female fat distribution pattern via an antiglucocorticoid action in abdominal fat, a hypothesis that was supported by in vivo and in vitro experiments reporting an inhibition of glucocorticoid-induced fat cell differentiation, lipogenesis, or body fat accumulation by progesterone (14, 15).

In recent years adipose tissue was increasingly perceived as a metabolically active organ displaying endocrine, paracrine, and autocrine signals (16). Several steroidogenic enzymes and steroid-converting activities have also been detected in adipose tissue or adipose cells (17), including activities and/or mRNAs of aromatase; 3ß-hydroxysteroid dehydrogenase (HSD); type 3 3{alpha}-HSD; type 1 11ß-HSD; types 2, 3, and 5 17ß-HSD; 7{alpha}-hydroxylase; 17{alpha}-hydroxylase; 5{alpha}-reductase; and UDP-glucuronosyltransferase 2B15 (18, 19, 20, 21, 22, 23, 24, 25, 26). In the present study, we report on the expression and activity of 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD), a member from the aldoketoreductase (AKR1C) 1C enzyme family involved in the conversion of progesterone to its 20{alpha}-hydroxylated metabolite, in abdominal sc (SC) and omental (OM) adipose tissue in women.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Women of this study were recruited through the elective surgery schedule of the Gynecology Unit of the Laval University Medical Research Center for studies on adipose tissue metabolism. Thirty-two healthy women aged 40–60 yr undergoing abdominal gynecological surgery were included in the present analysis. All women of the study elected for total (n = 31) or subtotal (n = 1) abdominal hysterectomies, some with salpingo-oophorectomy of one (n = 5) or two (n = 19) ovaries. Reasons for surgery included one or more of the following: menorrhagia (n = 11), myoma (n = 18), pelvic pain (n = 11), benign cyst (n = 1), endometriosis (n = 7), cystitis (n = 3), and hydrosalpinx (n = 3). Medication included thyroid hormones (n = 3); GnRH agonist and/or hormone therapy (n = 9); lipid-lowering (n = 1) or antihypertensive therapy (n = 8); nonsteroidal antiinflammatory drugs (n = 4); antidepressors (n = 9); and antivertigo (n = 1), antiseizure (n = 1), and asthma (n = 6) medication. Seven women were taking nutritional supplements (vitamins, iron, calcium, or glucosamine). Other types of medication included digoxin, acetaminophen, aspirin, allergy medicine, antibiotics, mesalamine, and antispasmodic medicine. Approbations by the medical ethics committees of Laval University and Laval University Medical Research Center were obtained. All subjects provided written informed consent before their inclusion in the study. Adipose tissue samples and biological/clinical data of the present group of women were used for a previous study on other steroidogenic enzymes (27).

Body fatness and body fat distribution measurements

These tests were performed on the morning of or a few days before the surgery. Measures of total body fat mass, fat percentage, and fat-free mass were determined by dual-energy x-ray absorptiometry, using a QDR-2000 densitometer and the enhanced array whole-body software (version 5.73A, Hologic Inc., Bedford, MA). Measurement of abdominal sc and visceral adipose tissue cross-sectional areas was performed by computed tomography as previously described (28), using a Light Speed 1.1 CT scanner (General Electric Medical Systems, Milwaukee, WI) and the Light Speed QX/I 1.0 production software. Subjects were examined in the supine position, with arms stretched above the head. The scan was performed at the L4-L5 vertebrae level using a scout image of the body to establish the precise scanning position. The quantification of visceral adipose tissue area was done by delineating the intraabdominal cavity at the internal-most aspect of the abdominal and oblique muscle walls surrounding the cavity and the posterior aspect of the vertebral body with the computer interface of the scanner. Adipose tissue was highlighted and computed using an attenuation range of –190 to –30 Hounsfield units. The coefficient of variation between two analyses from the same observer (n = 10) were 0, 0.50, and 2.14% for total, sc, and visceral adipose tissue areas, respectively.

Plasma hormone measurements

Plasma concentrations of dehydroepiandrosterone (DHEA), androstenedione, dihydrotestosterone, estrone, and estradiol were determined in 26 subjects of the study using high-performance gas chromatography and chemical ionization mass spectrometry. Intra- and interassay precision did not exceed 5.9% for these measurements. Dihydrotestosterone, estrone, and estradiol values were below quantification level for four, five, and three subjects, respectively. DHEA sulfate and estrone sulfate concentrations were determined in 25 subjects of the study using HPLC and mass spectrometry and a PE Sciex API 300 tandem mass spectrometer (Perkin-Elmer, Foster City, CA) equipped with a Turbo ionspray source. Intra- and interassay precision did not exceed 6.4% for these measurements. Progesterone levels were determined in 27 subjects with a RIA from Diagnostic Systems Laboratories (Webster, TX).

Adipose tissue sampling

Paired OM and SC adipose tissue samples were collected during the surgical procedure and immediately carried to the laboratory in 0.9% saline preheated at 37 C. A portion of the biopsy was used for adipocyte isolation, and the remaining tissue was immediately frozen at –80 C for subsequent analyses.

Adipocyte isolation, lipolysis, and lipase activity

Tissue samples were digested with collagenase type I in Krebs-Ringer-Henseleit buffer for 45 min at 37 C according to a modified version of the Rodbell method (29). Adipocyte suspensions were filtered through nylon mesh and washed three times with Krebs-Ringer-Henseleit buffer. For cell size measurements, mature adipocyte suspensions were visualized using a contrast microscope attached to a camera and computer interface. Pictures were taken and the Scion Image software (Scion Corporation, Frederick, MA) was used to measure the size of 250 adipocytes.

Basal lipolysis was measured by incubating isolated cell suspensions for 2 h at 37 C. Glycerol release in the medium was measured by bioluminescence using the nicotinamide adenine dinucleotide hydroxide-linked bacterial luciferase assay (30, 31), a Berthold Microlumat Plus bioluminometer (LB 96 V, EG&G, Bad Wildbad, Germany), and the WinGlow software (EG&G). The average coefficient of variation for duplicate glycerol release measurements was 14.1%. Lipid weight of the cell suspension was measured by performing Dole’s extraction, and lipolysis results were expressed as a function of adipocyte surface area (nanomoles glycerol per square micrometers x 108/2 h).

LPL activity was determined in 30- to 50-mg frozen adipose tissue samples by the method of Taskinen et al. (32). Tissue eluates were obtained by incubating the sample in Krebs Ringer phosphate buffer and heparin at 28 C for 90 min. The eluates were then incubated with excess concentrations of unlabeled and 14C-labeled triolein in a Tris-albumin buffer emulsified with ultrasound. The reaction was carried out at 37 C for 60 min with agitation. The resulting free fatty acids liberated from triolein by the LPL reaction were isolated by the Belfrage extraction procedure. Porcine plasma was used as a source of Apo-CII to stimulate LPL activity and unpasteurized cow’s milk as an internal LPL activity standard for interassay variations. Activity results were expressed in nanomoles oleate per 106 cells per hour.

RT-PCR

Total RNA was isolated using Trizol (Gibco BRL, Burlington, Ontario, Canada) following the manufacturer’s recommendations. Reverse transcription was performed with the Ambion Retroscript kit (Ambion Inc., Austin, TX) using random decamer primers. PCR amplification conditions were 94 C for 30 sec, 66 C for 30 sec (63 C for control), and 72 C for 30 sec for 35 cycles. The primers used spanned the junction for a 273-bp intron: 5'-CCT-ATA-GTG-CTC-TGG-GAT-CCC-AC-3' and 5'-AGG-ACC-ACA-ACC-CCA-CGC-TGT-3' (20{alpha}-HSD). Amplified products were separated on 2% agarose gels stained with ethidium bromide. Sequencing of PCR products was performed using BigDye Terminator (version 3.1 cycle sequencing, ABI Prism, PE Applied Biosystems, Foster City, CA) and analysis on an ABI 3730 automated DNA sequencer (PE Applied Biosystems).

Real-time PCR

First-strand cDNA synthesis was accomplished using 5 µg of the isolated RNA in a reaction containing 200 U Superscript II Rnase H-reverse transcriptase (Invitrogen), 300 ng oligo dT18, 500 µM deoxynucleotide triphosphate, 10 mM dithiothreitol, and 34 U porcine RNase inhibitor (Amersham Pharmacia, Uppsala, Sweden) in a final volume of 50 µl. The resulting products were then treated with 1 µg Rnase A for 30 min at 37 C and purified thereafter with Qiaquick PCR purification kits (Qiagen, Santa Clarita, CA). For quantitative PCR analyses, a Light-Cycler PCR (Roche Diagnostics, Lewes, UK) was used to measure the expression of 20{alpha}-HSD using the primers described in the section above. The FastStart DNA Master SYBR green kit (Roche Diagnostics) was used in a final reaction volume of 20 µl containing 3 mM MgCl2, 20 ng of each primer, and 20 ng of the cDNA template. The PCR was carried out according to the following conditions: 95 C for 10 min, 50 cycles of (95 C for 10 sec, 58 C for 5 sec, 72 C for 8 sec) with a temperature transition of 3 C/sec. For all the samples tested, no amplification was detected in a control tube containing water. Amplification of 20{alpha}-HSD and the housekeeping gene, glucose-6-phosphate dehydrogenase, which was quantified using the following primers: 5'-CAG-CGC-CTC-AAC-AGC-CAC-AT-3' and 5'-AAG-GGC-TTC-TCC-ACG-ATG-ATG-C-3', generated melting curves with a single peak and negligible nonspecific amplification products. A universal standard curve was generated with ATPase from an amplification with perfect efficiency (i.e. efficiency coefficient E = 2.00) using cDNA amounts of 0, 102, 103, 104, 105, and 106 copies. The crossing points (Cp) to calculate the amount of copies in initial cDNA specimens were determined using the double-derivative method. For each sample, the Cp value of 20{alpha}-HSD was divided by that of the housekeeping gene. To minimize interassay variability, this Cp ratio was then multiplied by the average Cp generated for housekeeping gene amplifications of all samples examined in the present experiment. PCR data are expressed in normalized number of copies per microgram total RNA.

Enzymatic activities

20{alpha}-HSD activity was measured in whole-tissue homogenates. Tissue samples were homogenized with a Polytron in 50 mM sodium phosphate buffer (pH 7.4), 20% glycerol, 1 mM EDTA, and 1 mM nicotinamide adenine dinucleotide phosphate reduced (33). 3H-labeled progesterone was added and reactions were performed at 37 C in a final volume of 1 ml for 24 h. Steroids from tissue homogenates were extracted twice with 1 volume ether as described previously (27). The organic phases were pooled and evaporated to dryness. The steroids, including reference standards, were solubilized in 50 µl dichloromethane [the 20{alpha}-hydroxyprogesterone (20{alpha}-OH-Prog) standard was solubilized in ethanol] and applied to Silica Gel 60 TLC plates (Merck, Darmstadt, Germany) using 10 µl calibrated micropipets. The separation was performed by migration in toluene-acetone (4:1). Unlabeled 20{alpha}-OH-Prog was used as a standard and was detected under UV light. Radioactivity was detected using a Storm 860 phosphor imager (Amersham Pharmacia Biotech Inc), and quantification was done using the ImageQuant software version 5.1 (Amersham Pharmacia Biotech Inc., Little Chalfont, UK). Proteins in each tissue were quantified by the method of Lowry and used to normalize activity values.

Statistical analyses

Paired t tests were used to compare expression levels in SC and OM adipose tissue, and unpaired t tests were used to compare mean 20{alpha}-HSD activities between subjects with either high or low visceral fat areas. Spearman rank correlation coefficients were computed to quantify associations between progesterone-converting activity and adiposity measures. Statistical tests were performed on log10-tranformed data when the variables were nonnormally distributed as tested using the Shapiro-Wilk W test. The following variables were not normally distributed: OM 20{alpha}-HSD activity, OM LPL adipose tissue activity, and visceral adipose tissue area. A level of alpha of 0.05 or less was considered as statistically significant. Analyses were performed using the JMP statistical software (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Female SC and OM adipose tissue expressed mRNA for 20{alpha}-HSD, as shown by RT-PCR analysis (Fig. 1AGo). Expected transcripts of 160 bp were detected in tissue samples of several patients (two are shown on the figure). Sequencing confirmed that the PCR products were 20{alpha}-HSD. The minor amplicon noted for some of the samples likely corresponded to genomic DNA contamination of the sample (addition of a 273-bp intron). Using quantitative real-time PCR analysis in a subset of subjects, we found that 20{alpha}-HSD expression was 2.7-fold higher in SC than OM adipose tissue when normalizing expression levels for glucose-6-phosphate dehydrogenase expression (paired t test difference, P < 0.001, n = 8; Fig. 1BGo).



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  		FIG. 1. A, RT-PCR detection of 20{alpha}-HSD mRNA (160 bp) in SC and OM adipose tissue obtained in two women of the study (patients A and B; aged 43.8 and 48.3 yr, respectively). Results are representative of experiments performed in several patients. G6PDH, Glucose-6-phosphate dehydrogenase. B, Real-time RT-PCR quantification of 20{alpha}-HSD in SC and OM adipose tissue samples obtained in eight women of the study. Age and adiposity characteristics of the subsample (n = 8) included: age, 47.2 ± 4.5 yr; BMI, 26.9 ± 3.3 kg/m2; fat mass, 29.4 ± 7.2 kg; visceral adipose tissue area, 110.6 ± 60.2 cm2 (mean ± SD). **, P < 0.001.

 
Figure 2Go shows a thin-layer chromatogram of steroid products obtained after incubating whole SC or OM adipose tissue homogenates with radiolabeled progesterone for 24 h. The main conversion product form of progesterone was 20{alpha}-OH-Prog (Fig. 2Go, samples 1–4). As shown, enzymatic activity of 20{alpha}-HSD was detected in SC and OM adipose tissue from two patients (C and D). These results are representative of experiments performed with homogenates from other patients of the study sample (n = 32).



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  		FIG. 2. Thin-layer chromatogram showing steroid products obtained when incubating SC or OM adipose tissue homogenates with radiolabeled 3H-progesterone. The chromatogram was photographed under UV light for the identification of the 20{alpha}-OH-Prog standard, and this image was superimposed to the autoradiogram by image analysis. Results of patients C and D (aged 58.9 and 40.1 yr, respectively) were representative of experiments performed in several other homogenates.

 
Characteristics of the study sample are shown in Table 1Go. Subjects were aged 47.7 yr and had a mean weight of 71.2 kg. They covered a wide range of body mass index (BMI) values (from 19.3 to 39.4 kg/m2). The mean fat mass was 30 kg. Computed tomography measures also covered wide ranges: from 100 to 736 cm2 for SC adipose tissue area and from 34 to 229 cm2 for visceral adipose tissue area. OM and SC adipose tissue metabolism measurements were consistent with previously reported regional differences with respect to adipocyte size and LPL activity (34), the former being higher in the sc depot (P < 0.0001, test performed on log10-transformed values) and the latter being higher in the OM depot (P < 0.05, test performed on log10-transformed values).


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  		TABLE 1. Physical and metabolic characteristics of the sample of 32 women

 
SC and OM progesterone conversion rates were not significantly different when comparing lean and obese women based on BMI or fat mass values, with the exception of a nonsignificant trend for higher SC 20{alpha}-HSD activity in obese women (not shown). When stratifying subjects for visceral adipose tissue area using a cut-off value of 100 cm2 (35), women with visceral obesity were characterized by a 1.8-fold higher 20{alpha}-HSD activity, compared with women with low visceral adipose tissue area (Fig. 3Go). SC progesterone conversion rates were not different in these two groups. When comparing the OM-SC differences, 20{alpha}-HSD activity was higher in the SC adipose tissue compartment of women with low visceral adipose tissue areas, whereas no regional difference was observed in viscerally obese women.



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  		FIG. 3. Comparison of 20{alpha}-HSD activity measured in SC (A) and OM (B) adipose tissue homogenates in women with either low or high (<100 cm2 or ≥ 100 cm2) visceral adipose tissue (AT) areas. For the comparison of OM 20{alpha}-HSD activity, the group with adipose tissue areas less than 100 cm2 included 17 subjects, and the group with adipose tissue areas 100 cm2 or greater included 14 subjects. For the comparison of SC 20{alpha}-HSD activity, the group with adipose tissue areas less than 100 cm2 included 16 subjects, and the group with adipose tissue areas 100 cm2 or greater included 13 subjects. Statistical test performed on log10-transformed data for OM 20{alpha}-HSD activity. NS, Nonsignificant. *, P < 0.05.

 
Figure 4Go shows correlations between OM 20{alpha}-OH-Prog formation in OM fat tissue homogenates and adiposity as well as adipose tissue metabolism. We found significant positive correlations between OM 20{alpha}-HSD activity and visceral adipose tissue area (r = 0.36, P < 0.05), visceral adipocyte diameter (r = 0.49, P < 0.05), and a positive trend with OM LPL activity (r = 0.36, P = 0.06). SC 20{alpha}-HSD activity was not significantly associated with adiposity measures or adipose tissue metabolism, with the exception of a trend for a positive correlation with total fat percentage (r = 0.32, P = 0.08). Basal lipolysis in both adipose tissue depots was not significantly associated with 20{alpha}-HSD activity in either compartment (r = –0.06, P = 0.78 in SC adipose tissue; r = –0.13, P = 0.51 in OM adipose tissue).



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  		FIG. 4. Correlations between 20{alpha}-HSD activity measured in OM adipose tissue homogenates and visceral adipose tissue (AT) area (n = 31) (A), OM adipocyte diameter (n = 32) (B), and OM LPL activity (n = 29) (C). Log10-transformed values are presented when variables were not normally distributed.

 
In the present sample, nine women did not have menstrual bleeding, four of which had reached a natural menopause and five of which had received GnRH agonist therapy. No differences were noted in SC or OM adipose tissue 20{alpha}-HSD activity when comparing these groups of women (not shown). In addition, SC or OM adipose tissue 20{alpha}-HSD activity in the nine women who were on hormone/GnRH replacement therapy were not significantly different from activities measured in other subjects of the sample (not shown). Plasma levels of sex hormones including estradiol, estrone sulfate, progesterone, DHEA, DHEA sulfate, and androstenedione were not significantly associated with SC or OM adipose tissue 20{alpha}-HSD activity in the present study.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we investigated the expression and activity of 20{alpha}-HSD, a member of the AKR1C family (AKR1C1) in abdominal adipose tissue. This enzyme catalyzes the conversion of progesterone into 20{alpha}-OH-Prog and was previously found in several tissues such as the liver, ovaries, testes, adrenals, and placenta (33). We detected mRNA and activity of 20{alpha}-HSD in both SC and OM adipose tissue in women. Quantitative real-time PCR analyses showed that expression was higher in SC than OM fat. A positive association was found between visceral adipose tissue area and OM adipose tissue activity of this enzyme. Moreover, 20{alpha}-HSD in this depot correlated positively with OM adipocyte diameter and LPL activity. No association was found between SC adipose tissue 20{alpha}-HSD activity and abdominal obesity. This study is the first to describe 20{alpha}-HSD expression and activity in SC and OM adipose tissue. Our results show that OM adipose tissue 20{alpha}-OH-Prog formation is highest in women characterized by visceral obesity. Further studies are required to establish whether regional hydroxylation of progesterone may impact locally on the metabolism and function of adipocytes located within the abdominal cavity.

The enzyme 20{alpha}-HSD shares structure identity and sequence homology with 20{alpha}-HSDs from several other species. In rodent models, it is believed that 20{alpha}-HSD induction and conversion of progesterone to 20{alpha}-OH-Prog in the corpus luteum may be responsible for pregnancy termination by causing the marked decrease in progesterone occurring before the initiation of parturition (36). In addition, a recent study demonstrated that an aldose reductase with 20{alpha}-HSD activity was presumably responsible for the production of prostaglandin F2{alpha} in bovine endometrium, suggesting additional roles of this enzyme in labor and luteolysis (37). Other physiological roles in animal models may also include the protection of mice thymocytes against the toxic effects of progesterone (38) and regulation of myelin formation in the brain (39). The role of 20{alpha}-HSD in humans is still poorly defined (33). The present finding of significant progesterone conversion to its 20{alpha}-hydroxylated form and detection of 20{alpha}-HSD mRNA in abdominal fat suggests that this enzyme may regulate the action of progesterone in this tissue. As opposed to the placenta, however, other progesterone metabolites may also be generated in adipose tissue through previously documented expression of 17{alpha}-hydroxylase and 5{alpha}-reductase in human adipose tissue or adipose cells (17, 21, 24).

The effects of progesterone itself on adipose tissue and adipose cell regulation have been investigated in several studies. Experiments performed in the murine 3T3-L1 cell line have shown that progesterone stimulates adipocyte differentiation as assessed by triglyceride accumulation and glyceraldehyde-3-phosphate dehydrogenase activity (10), and this lipogenic effect has been attributed to stimulation of adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c and key lipogenic genes such as fatty acid synthase (7). Progesterone has also been shown to have lipogenic properties in other models such as primary preadipocytes obtained from male and female rats (7, 40) and brown adipocytes from male mice (41). Although progesterone receptor knockout mice are not characterized by a modified adiposity phenotype (42), the presence of progesterone receptor isoforms A and B in adipose tissue may provide a direct route for the regulation of adipose tissue by this steroid in women (43).

On the other hand, Björntorp suggested that this hormone might be responsible for the female pattern of obesity by acting as a glucocorticoid antagonist in abdominal adipose tissue depots, thereby preventing abdominal fat accumulation (13). This hypothesis is supported by experiments demonstrating inhibition of glucocorticoid-induced fat cell differentiation, lipogenesis, and body fat accumulation by progesterone in rodents (14, 15). Hence, studies have shown that progesterone may exhibit both lipogenic and antilipogenic properties, depending on the model and experimental approach used. It may be speculated that the depot origin of the fat cells may be critical in the effects observed and that the hormone acts in a depot-specific manner. In this regard, modulation of progesterone concentrations through the regulation of 20{alpha}-HSD expression may serve as a tissue-specific regulator of local hormone levels and action in adipose tissue. Another possibility would be that part of the effects of progesterone in adipose tissue are mediated through a yet undefined 20{alpha}-OH-Prog action in adipose cells.

20{alpha}-HSD is highly homologous to other enzymes from the AKR1C enzyme family in humans. For example, type 3 3{alpha}-HSD (AKR1C2) shares 98% amino acid identity with 20{alpha}-HSD and the identity with type 5 17ß-HSD (AKR1C3) is 88%. However, despite being nearly identical in amino acid sequence, these enzymes have distinct substrate specificities, with AKR1C2 being mostly involved in androgen inactivation (reaction of dihydrotestosterone (DHT) to 3{alpha}-androstanediol) and AKR1C3 being involved in androgen synthesis (reaction of androstenedione to testosterone). AKR1C2 also exerts a slight 20{alpha}-HSD activity toward progesterone, whereas AKR1C3 also exerts 3{alpha} and 20{alpha} activities toward DHT and progesterone (33). We recently demonstrated in the present sample that mRNA and/or activity of these two other enzymes from the AKR1C family, namely type 3 3{alpha}-HSD (AKR1C2) and type 5 17ß-HSD (AKR1C3) are also detectable in abdominal adipose tissue and primary preadipocyte cultures (27). In addition, rates of androgen inactivation measured in OM adipose tissue (3{alpha}-HSD activity) were significantly and positively associated with visceral adiposity in this sample of women. By examining 3{alpha}-reduction of DHT and 20{alpha}-hydroxylation of progesterone activities measured in our adipose tissue samples, we found that 3{alpha}-HSD activity (androgen inactivation) was positively correlated with 20{alpha}-HSD activity (progesterone hydroxylation) in both the omental (r = 0.46, P < 0.01) and SC (r = 0.37, P < 0.05) fat compartments. The relevance of the correlations observed between both activities in their respective depot is unclear. The fact that type 3 3{alpha}-HSD and 20{alpha}-HSD are highly homologous and that their activities are both correlated positively with visceral adipose tissue accumulation may suggest similar regulation mechanisms for both enzymes.

Due to the necessity of a surgical procedure to access the OM adipose tissue depot for sampling, the present study uses a cross-sectional design. Thus, based on the significant positive correlations between OM adipose tissue 20{alpha}-HSD activity and abdominal adiposity or OM adipose tissue metabolism, one cannot reach conclusions on causality. The question as to whether elevated 20{alpha}-HSD in OM adipose tissue of viscerally obese women is a causal agent for abdominal obesity or whether it is merely a consequence or adaptation to the obese state cannot be addressed. Further studies using in vitro approaches are warranted to resolve this issue.


   Acknowledgments
 
We acknowledge the contribution of Drs. Philippe Laberge, Marleen Daris, Jacques Mailloux, Diogène Cloutier, and Caroline Rhéaume to the recruitment process of the study and the collection of surgical biopsies. The invaluable help of study coordinator Guylaine Chainé, nurses Denise Parsons, Johanne Baillargeon, Carole Boisvert, Danielle Bélanger, Carole Bérubé, and Diane Chamberland and radiology technicians Suzanne Brulotte, Lyne Bargone, Linda Marcotte, Louise Mailloux, Diane Bastien, and Monique Caron is also gratefully acknowledged. The authors thank all women who participated in the study for their excellent collaboration.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (MOP-53195). S.B. is the recipient of a fellowship from the National Science and Engineering Research Council of Canada, and K.B. is the recipient of a fellowship from Fonds de la Recherche en Santé du Québec. A.T. is the recipient of a scholarship from the Canadian Institutes of Health Research.

First Published Online October 19, 2004

Abbreviations: AKR1C, Aldoketoreductase; BMI, body mass index; Cp, crossing point; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; HSD, 3ß-hydroxysteroid dehydrogenase; 20{alpha}-HSD, 20{alpha}-hydroxysteroid dehydrogenase; LPL, lipoprotein lipase; 20{alpha}-OH-Prog, 20{alpha}-hydroxyprogesterone; OM, omental; SC, subcutaneous.

Received March 26, 2004.

Accepted October 10, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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