Opposite Effects of Androgens and Estrogens on Adipogenesis in Rat ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 2 Printed in U.S.A.

Opposite Effects of Androgens and Estrogens on Adipogenesis in Rat Preadipocytes: Evidence for Sex and Site-Related Specificities and Possible Involvement of Insulin-Like Growth Factor 1 Receptor and Peroxisome Proliferator-Activated Receptor ␥2* M. N. DIEUDONNE, R. PECQUERY, M. C. LENEVEU,

AND

Y. GIUDICELLI

Service de Biochimie, INSERM CJF 94 – 02, Faculte´ de Me´decine Paris-Ouest, Universite´ Rene´ Descartes (Paris V) Centre Hospitalier de POISSY, 78303 Poissy Cedex, France mized female rats, 17␤-estradiol significantly increased (⫻1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor ␥2 was up-regulated by 17␤estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 ␤-estradiol (0.01 ␮M) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 ␮M) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor ␥2 expression (estrogens). (Endocrinology 141: 649 – 656, 2000)

ABSTRACT To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17␤-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (⫻2) of 17␤-estradiol (0.01 ␮M) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 ␮M) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17␤-estradiol (0.01 ␮M), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariecto-

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EGIONAL fat distribution differs between men and women and is thus considered as a secondary sex character. Fat distribution is also modified during various physiopathological situations (pregnancy, postmenopause, transsexualism), suggesting a potential role for sex steroid hormones in determining the site specificities of fat deposition (1– 4). Development of white adipose tissue (adipogenesis) is characterized by a sequence of events during which adipose precursor cells proliferate until confluence and then differentiate into mature adipocytes. The preadipocyte-adipocyte conversion process is induced by the transcriptional activation of adipose specific genes such as adipsine, aP2, LPL. (5–7). Master regulatory transcription factors [peroxisome proliferator-activated receptor ␥2 (PPAR␥2), C/EBP␣, C/EBP␤, C/EBP␦] are involved in activating or derepressing transcription of these genes and thus seem to be the key in

the commitment of the differentiation program (8 –10). Adipogenesis is markedly influenced by a variety of hormones and nutritional signals. Insulin, insulin-like growth factor 1 (IGF1), GH, and glucocorticoids are important positive signals for adipocyte differentiation in vivo and in vitro (11–13). IGF1, like insulin, stimulates both preadipocyte growth and differentiation. The role played by sex hormones in adipogenesis is still poorly understood. In human adipose tissue, estrogens have been reported to modulate adipogenesis in vitro by increasing preadipocyte replication (14) without altering the differentiation process (15). In rat preadipocytes, progesterone stimulates the terminal differentiation (16) whereas, in 3T3-L1 and 3T3-F442A preadipocyte cell lines and in pig preadipocytes, high concentrations of dehydroepiandrosterone (DHEA) and other androgen-related steroids were shown to block the adipose conversion process, as followed by measurement of glycerol-3 phosphate dehydrogenase (GPDH) activity, a late marker of differentiation (17–19). Biological effects of steroid hormones are primarily mediated by their specific receptors. Estrogen and androgen receptors are expressed in rat (20 –22) and human (23, 24) preadipocytes and adipocytes. In these cells, the number of estrogen and androgen receptors are also variable, according

Received June 11, 1999. Address all correspondence and requests for reprints to: Y. Giudicelli, Service de Biochimie, Centre Hospitalier, 78303 Poissy Cedex, France. E-mail: [email protected]. * This work was supported by INSERM (CJF 94 – 02), the University Rene´ Descartes Paris V, the Ligue Nationale contre le Cancer (Comite´ de´partemental des Yvelines), and Biome´rieux, SA.

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EFFECTS OF ANDROGENS AND ESTROGENS ON ADIPOGENESIS

to their anatomical origin (22, 25, 26), suggesting that preadipocytes and adipocytes are target cells for sex hormones. The aim of the present study was to get a better understanding of the role of sex hormones in adipogenesis and its relationship to anatomical origin of the preadipocytes. For this purpose, we have compared adipogenesis in primary cultured male and female rat preadipocytes removed from deep and superficial fat depots and exposed or not to sex steroid hormones during either the growth phase or the differentiation process. Materials and Methods Materials DMEM and DMEM-F12 (phenol red free), HEPES, porcine insulin, transferrine, T3, NADH, H⫹, dihydroxyacetone phosphate, testosterone, 5␣-dihydrotestosterone (DHT), 17␤-estradiol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate, estrone (1,3,5 [10]-estratrien 3 ol 17 one), androstanediol (5␣- androstane 3␤, 17␤, diol), androstenediol (5androstene 3␤, 17␤, diol), and BSA were from Sigma (St. Louis, MO); ICI182780 was from Tocris (Bristol, UK); RU23908 was from Roussel-Uclaf (Romainville, France); collagenase was from Roche Molecular Biochemicals (Mannheim, Germany); FCS was from Life Technologies (Grand Island, NY); polyclonal rabbit anti-PPAR␥2 antiserum was from Affinity BioReagents, Inc. (Golden, CO); polyclonal rabbit anti-IGF1 receptor (anti-IGF1R) antiserum and polyclonal goat anti IGF1 antiserum were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and horseradish peroxidase-labeled rabbit anti-IgG was from Sanofi Pharmaceuticals, Inc. Pasteur (Marne la Coquette, France).

Animals Adult male and female Sprague Dawley rats (150 –200 g) were kept under controlled lighting conditions (light, 0600 h; dark, 2000 h) and constant temperature (21 C). Females were ovariectomized as previously described (27). Animals were killed by decapitation. Epididymal, parametrial, and femoral sc adipose tissues were immediately removed under sterile conditions.

Cell culture Cell preparation and culture were performed as described (22). Briefly, preadipocytes were obtained by collagenase digestion. The floating adipocytes were discarded, and the infranatant containing the stromal vascular fraction was successively filtered through 150- and 25-␮m nylon screens. The filtrate was centrifugated at 600 ⫻ g for 10 min. After two washes, cells were plated into cell culture dishes at a density of 2– 4 ⫻ 104 cells/cm2 with 8% FCS-DMEM and were maintained at 37 C under 5% CO2 atmosphere. After plating, cells were extensively washed and maintained: 1) for the cell growth experiments, in DMEM with 2% FCS; or 2) for the differentiation studies, in DMEM with 8% FCS during 2 days and then in DMEM-F12 (1:1) supplemented with insulin (5 ␮g/ ml), transferrin (10 ␮g/ml), T3 (2 nm) (ITT medium), as described (28), until the full differentiation state was reached (day 9 or 10 after plating). When added to the medium, steroid hormones were dissolved in ethanol. The same ethanol concentrations were added to control medium (final ethanol concentration never exceeding 0.01%, vol/vol). Cell viability was assessed by the trypan blue exclusion test (29) and by measurement of lactate dehydrogenase activity in the culture medium (30). The polyclonal anti IGF1 antiserum was used, at 4 ␮g/ml, to block endogenous and exogenous IGF1 in the culture medium during 3 days post plating.

Growth assays Cell number was determined at day 3 post plating. Cells were trypsinized and counted in a hemocytometer.

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GPDH assay Cell differentiation was followed using GPDH activity as a marker of differentiation. After 9 –10 days post plating, ITT medium was removed, and cells were scraped in cold buffer containing 50 mm Tris-HCl (pH 7.4), 0.25 m sucrose, 1 mm EDTA, and 1 mm dithiothreitol. Cells were sonicated in the same buffer and centrifuged at 100,000 ⫻ g for 20 min at 4 C. GPDH activity was measured in the supernatant according to Wise and Green (31) and expressed in mU (nmol NAD⫹/min) per mg protein.

Immunoblotting PPAR␥ protein expression. Differentiated preadipocytes were scraped and sonicated in cold buffer containing 50 mm Tris (pH 8), 120 mm NaCl, 1% nonidet P40, 0.5% deoxycholate, 0.1% SDS, 0.5 mm phenylmethylsulfonylfluoride, 25 ␮g/ml aprotinin, and 105 ␮m leupeptin. After centrifugation at 100,000 ⫻ g for 20 min at 4 C, the resulting supernatant was denatured with Laemmli buffer (vol/vol) and stored at ⫺20 C. Cellular extracts (20 ␮g) were resolved by SDS/PAGE (12.5% acrylamide). Proteins were transferred to polyvinylidenedifluoride membranes overnight at 4 C and blocked for 2 h at room temperature in buffer A (137 mm NaCl, 20 mm Tris-HCl, 0.1% Tween 20) with 5% nonfat dry milk. Primary polyclonal PPAR␥1–2 antiserum (1:2000 dilution) was then added in buffer A and incubated overnight at 4 C. The primary antiserum was then removed and the blot washed extensively with buffer A. Secondary antiserum (horseradish peroxidase-labeled antirabbit IgG) was added (1:4000 dilution) and incubated with the blot for 1 h at room temperature. After washing, an enhanced chemiluminescence kit from Amersham Pharmacia Biotech (Aylesbury, UK) was used for signal detection. Films were quantified using a densitometer. Control experiments with various amounts of protein (5–50 ␮g) were performed to ensure that densitrometric signal intensity was proportional to the loaded amount of protein. IGF1R protein expression. Confluent preadipocytes were scraped and sonicated in cold buffer containing 10 mm Tris (pH 7.4), 0.25 m sucrose, 5 mm EDTA, 0.5 mm phenylmethylsulfonylflouride, 25 ␮g/ml aprotinin, and 105 ␮m leupeptin. After centrifugation at 21,000 ⫻ g for 20 min at 4 C, the pellet was resuspended and denatured with Laemmli buffer (vol/vol) and stored at ⫺20 C. Membrane extracts (40 ␮g) were resolved by SDS-PAGE (7.5% acrylamide). Proteins were transferred to polyvinylidenedifluoride membranes overnight at 4 C and blocked for 2 h, at room temperature, in buffer A with 2.5% gelatin. Primary polyclonal IGF1R antiserum (1:300 dilution) was then added in buffer A with 2.5% gelatin and incubated overnight at room temperature. Incubation with the secondary antiserum and signal detection were performed as described above. Control experiments with various amounts of protein (20 –100 ␮g) were performed to ensure that densitrometric signal intensity was proportional to the loaded amount of protein.

Other determinations Protein concentration was measured according to Bradford (32), with BSA as standard. All results are expressed as means ⫾ sem from at least three individual experiments. Statistical significance was established using Student’s t test or ANOVA test when multiple treatment groups were compared.

Results Cell growth

Androgens and estrogens are known to control proliferation of their respective target cells (epithelial cells in the prostate, mammary gland, endometrium. . . . ) (33, 34). In the present study, we have investigated the influence of different sex steroids on preadipocyte replication from day 1– 4 after plating, in the presence of 2% FCS. These experimental conditions are similar to those demonstrating a mitogenic effect of sex steroids in other cell types (35). Because the proliferative capacities of preadipocytes are


variable according to fat depots (36, 37), this study was performed on male and female rat preadipocytes from femoral sc and deep intraabdominal fat depots. Under these experimental conditions, none of the steroids tested (testosterone, DHT, androstanediol, androstenediol, DHEA, DHEA-S, 17␤estradiol, estrone), at 10 nm concentration, had any effect on preadipocyte growth in male rats (data not shown). However, as recent studies have demonstrated a positive effect of estrogens on both creatine kinase B expression and leptin secretion in female rat adipose tissue specifically (38, 39), we have reexamined the effects of estrogens on the proliferation rate of sc and parametrial preadipocytes removed from nonovariectomized or ovariectomized female rats. As shown in Fig. 1, exposure to 10 nm 17␤-estradiol resulted in a significant increase in the growth rate of sc (⫻1.9) but not of parametrial preadipocytes from ovariectomized rats (Fig. 1). Moreover, this positive effect of 17␤-estradiol was abolished by the estrogen receptor antagonist ICI182780 (1 ␮m) (Fig. 1). Identical results were observed with preadipocytes from nonovariectomized females (⫻2.06 and ⫻1.17 in sc and parametrial preadipocytes, respectively; data not shown). Thus estrogens elicit in vitro a mitogenic response in sc preadipocytes from female, but not from male, rats. Cell differentiation

The direct influence of steroids on the adipose conversion process was next investigated in preadipocytes from the epididymal, parametrial, and sc fat depots of male and ovariectomized female rats. Cells were rapidly allowed to differentiate (2 days after plating) in a standard differentiation medium (ITT) and exposed at the same time to the different steroids for 7– 8 days. Absence of detectable lactate dehydrogenase levels in the culture media, with or without sex steroids, indicated that these long-term exposures had no cytotoxic effect. The modulatory effects of androgens on the adipose conversion process were studied in preadipocytes from male rats. As shown in Fig. 2, A and B, GPDH activity was either

FIG. 1. Effect of 17␤-estradiol on the cell growth of sc and parametrial preadipocytes from ovariectomized female rats. After 3 days of culture in DMEM with 2% FCS, in the presence or absence of 17␤-estradiol (0.01 ␮M) or in the presence of 17␤-estradiol (0.01 ␮M) and ICI182780 (1 ␮M) simultaneously, cells were collected and counted. Data are the means ⫾ SEM of six to eight separate experiments. Each experiment was performed in triplicate. *, P ⬍ 0.05; CONT, control; EST, 17␤estradiol; ICI, ICI182780.

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markedly decreased in epididymal (⫺60 to ⫺70%) or slightly reduced in sc (⫺20 to ⫺30%) preadipocytes after exposure to pharmacological concentrations (10 ␮m) of testosterone or DHT. Moreover, in epididymal preadipocytes, this antiadipogenic effect of androgens was dose-dependent, being already significant at 10 nm DHT concentration (30% inhibition P ⬍ 0.05) and was almost completely reversed by simultaneous exposure to the potent androgen receptor antagonist RU23908 (10 ␮m) (Fig. 2A). The influence of estrogens on the adipose differentiation process was compared in male (Fig. 2C) and ovariectomized female rats (Fig. 2D). Exposure to 10 nm 17␤-estradiol failed to alter GPDH activity in epididymal and sc preadipocytes from male rats. However, at high 17␤-estradiol concentration (10 ␮m), a weak (but significant) stimulating effect on GPDH activity was observed in epididymal, but not in sc, preadipocytes. In ovariectomized females, exposure to 10 nm 17␤estradiol concentrations resulted in a ⫹30% increase in GPDH activity in parametrial, but not in sc, preadipocytes. Higher concentrations (0.1 ␮m or 1 ␮m) of 17␤-estradiol did not further increase GPDH activity in preadipocytes from these ovariectomized rats (not shown). Moreover, the proadipogenic effect of 17␤-estradiol in parametrial preadipocytes was suppressed by simultaneous exposure to the estrogen receptor antagonist ICI 182780 (0.1 ␮m) (Fig. 2D). In various cell types, regulation of growth and differentiation by steroid hormones seems to be mediated, at least in part, through changes in the expression of some growth factors and/or their receptors (40 – 42). Among these growth factors, IGF1 is considered as an important mitogenic and adipogenic factor (43). However, the mitogenic potency of IGF1 seems to be variable according to the preadipocyte anatomical origin. As a matter of fact, experiments performed with anti IGF1 antiserum in the culture medium (4 ␮g/ml), during 3 days post plating, revealed that depletion of exogenous and endogenous IGF1 results in a significantly higher decrease of cell growth in sc than in parametrial preadipocytes (⫺58.5% ⫾ 6.5 vs. ⫺23% ⫾ 3, P ⬍ 0.05). These observations led us to compare the influence of androgens and estrogens on IGF1R expression during the early phase of differentiation (4 days culture) in preadipocytes from different anatomical origins. As shown in Fig. 3, exposure to DHT (10 nm) resulted in a significant decrease (⫺40%) in IGF1R expression in epididymal preadipocytes. This effect was prevented by simultaneous exposure to RU23908 (10 mm). In contrast, in male rat sc preadipocytes, IGF1R expression was not modified by DHT exposure (Fig. 3). Next, IGF1R expression was compared, under the same conditions as above, in preadipocytes from nonovariectomized and ovariectomized female rats. As shown in Fig. 4, ovariectomy induced a significant decrease (⫺60%) in IGF1R expression. Now when preadipocytes from ovariectomized rats were exposed in vitro to 17␤-estradiol (10 nm), a significant increase in IGF1R expression was observed in sc preadipocytes (⫹75%) but not in parametrial preadipocytes (not shown). As also depicted in Fig. 5, these effects of estrogens were abolished by the estrogen receptor antagonist ICI182780 (0.1 ␮m). The identification of a major transactivating factor, PPAR␥2, that activates the expression of adipose specific genes such as aP2, adipsine, and LPL during adipogenesis

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FIG. 2. Effect of various sex steroids on GPDH activity of epididymal, parametrial, and femoral sc preadipocytes from male and ovariectomized female rats. Precursor cells from epididymal, parametrial, and sc fat deposits were cultured as described in Materials and Methods. Testosterone, DHT, and RU23908 (Fig. 2, A and B), 17␤-estradiol, and ICI182780 (Fig. 2, C and D) at 0.1 ␮M, 10 ␮M, or 100 ␮M were added during all the differentiation process in ITT medium. After 7 to 8 days in ITT medium, cells were collected, and GPDH activity was measured. Values are expressed as percentage of the mean values obtained for cells maintained in culture without steroids (1886 ⫾ 220 mU/mg protein and 928 ⫾ 106 mU/mg protein in epididymal and sc cells from male rats, respectively, and 1175 ⫾ 275 mU/mg protein and 2545 ⫾ 750 mU/mg protein in parametrial and sc cells from ovariectomized female rats, respectively). Each value represents the mean ⫾ SEM of four to seven separate experiments. *, P ⬍ 0.05; **, P ⬍ 0.01; ns, not significant; (a), vs. control; (b), vs. RU23908 in Fig. 2A; (b), vs. ICI182780 in Fig. 2D.

(for review, see Ref. 6) led us to investigate the influence of sex hormones on PPAR␥2 expression. By immunoblotting, we have first established that PPAR␥2 is present in confluent preadipocytes; and, in agreement with previous reports on preadipocyte cell lines, we have confirmed that the PPAR␥2 expression is maintained at a constant level throughout the differentiation process in control cells. As shown in Fig. 6, PPAR␥2 protein expression in differentiated cells was much lower (⫺60%) in sc than in epididymal preadipocytes from male rats. Moreover, 2-bromo palmitate, an adipogenic factor (44), increased PPAR␥2 expression in both epididymal and sc preadipocytes (⫻ 1.74 ⫾ 0.22 and ⫻ 2.11 ⫾ 0.33) and the GPDH activity in epididymal

specifically (⫻ 5.5 ⫾ 1.56 vs. ⫻ 1.13 ⫾ 1.05). Testosterone and DHT had no effect on PPAR␥2 expression, but 17␤-estradiol increased PPAR␥2 expression in epididymal preadipocytes and in parametrial preadipocytes from ovariectomized rats.

Discussion

In the present work, we have studied, in vitro, the influence of androgens and estrogens on the proliferative and differentiating phases of adipogenesis in cultured preadipocytes isolated from adult rat epididymal, parametrial, and sc adipose tissues. We have observed important differences in the


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FIG. 3. Effect of androgens on IGF1R expression in rat epididymal and sc preadipocytes. Precursor cells were cultured as described in Materials and Methods. DHT and RU23908 were added during 4 days after plating. Preadipocytes were collected, and membrane extracts were prepared as described in Materials and Methods. A, Western-blot analysis from one representative experiment. Forty micrograms of protein were immunoblotted with polyclonal rabbit anti-IGF1R antiserum; B, Densitometric analysis [values are mean ⫾ SEM obtained from five to six separate experiments and are expressed as control values (without steroids)]. *, P ⬍ 0.01; (a); vs. control. IGF1R expression in epididymal preadipocytes represents 51% ⫾ 12 (n ⫽ 4) of IGF1R expression observed in sc preadipocytes (100%).

FIG. 4. Effect of ovariectomy on IGF1R expression in sc preadipocytes. sc preadipocytes from intact and ovariectomized female rats were cultured as described in Materials and Methods, 4 days after plating cells were collected, and membrane extracts were prepared. Densitometric analysis of IGF1R immunoblot was represented. Values are the mean ⫾ SEM obtained from three experiments. *, P ⬍ 0.02; SHAM, sham-operated female rat; OVX, ovariectomized female rat.

responsiveness of these cells to sex hormones, depending on the animal gender and the cell anatomical origin. After exposure to estrogens, we have found over 2-fold increase in proliferation rate in sc preadipocytes from female rats only. This effect was mediated by estrogen receptors, because, in the presence of ICI182780, the positive effect of estrogens was completely abolished. These results are consistent with previous investigations carried out in human sc preadipocytes (14). Recent studies have reported that estrogens in mammary tumors (40) and androgens in prostatic cell lines (41) modulate cell proliferation by up-regulating EGF receptor and

IGF1R expression. EGF does not significantly affect rat preadipocyte growth (45), whereas IGF1 is a potent mitogenic factor for these cells (43). These findings led us to examine the influence of estrogens on IGF1R expression in female rat preadipocytes. In confluent sc preadipocytes, a positive effect of 17␤-estradiol on IGF1R expression was, in fact, observed, which could well contribute to explaining the mitogenic action of estrogens observed in these cells. Moreover, in the presence of the antiestrogen ICI182780, this effect of 17␤-estradiol was completely blocked. This result suggests that the estrogen receptors, identified in rat confluent sc preadipocytes (46), are involved in the IGF1R up-regulation by estrogens. Further experiments are currently in progress, to determine whether estrogens (via these receptors) are modulators of the IGF1R phosphorylation/dephosphorylation balance that is crucial for the signaling activity of this receptor (43). Under our experimental conditions, the different androgens (testosterone, DHT, DHEA(S), androstanediol, and androstenediol) tested at rather low concentration (i.e. 10 nm) failed to affect preadipocyte proliferation in male rats. This contrasts with other studies showing that DHEA concentrations in the micromolar range induce antimitogenic effects in 3T3-L1 cells or in stromal adipose precursor cells from rat and pig (18 –19). The present experiments demonstrate that testosterone acts as a negative effector of terminal differentiation of rat preadipocytes. Identical results were obtained with DHT (the nonaromatizable testosterone metabolite), suggesting the androgen specificity of these testosterone effects. Moreover, the potent androgen antagonist RU23908 (47) reversed (almost completely) the negative action of DHT on GPDH activity. This result suggests that the antiadipogenic effects of androgens seem, at least in part, to be androgen receptor dependent. Adding further weight to this suggestion is our recent finding that androgen receptors are expressed at high level

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FIG. 5. Effect of estrogens on IGF1R expression in sc preadipocytes from ovariectomized female rats. sc precursor cells were cultured as described in Materials and Methods. 17␤-estradiol and ICI182780 were added during 4 days after plating. Preadipocytes were collected, and membrane extracts were prepared as described in Materials and Methods. A, Western-blot analysis from one representative experiment. Forty micrograms of protein were immunoblotted with polyclonal rabbit anti-IGF1R antiserum; B, Densitometric analysis [values are mean ⫾ SEM obtained from three to four separate experiments and are expressed as control values (without steroids)]. **, P ⬍ 0.05; (a), vs. control; (b), vs. ICI182780.

during the first days of adipogenesis (22). In contrast, sc adipose precursor cells seem less sensitive to the antiadipogenic effect of androgens, in comparison with epididymal cells. This difference seems also to be explained by the sitespecificity of androgen receptor expression, because androgen receptor density is about two times lower in sc than in epididymal preadipocytes (22). One mechanism that could explain the antiadipogenic effect of androgens is a reduced transcription of genes encoding adipogenic transcriptional factors. Among these factors, PPAR␥2 which is considered as one of the master regulator genes of the adipoconversion process could be a target of androgens for the following reasons: 1) the PPAR␥ gene regulatory unit includes various hormone responsive elements (48) including a GRE which is a DNA binding domain for glucocorticoid receptors but also for androgen receptors (49); and 2) two recent studies reported that the glucocorticoid, dexamethasone, strongly increases the expression of PPAR␥2 in 3T3 fibroblasts (50) and of another member of the PPAR family, PPAR ␣, in hepatic cells (48). In the present study, however, we were unable to observe a negative regulation of PPAR␥2 expression by androgens. Therefore, other mechanisms have to be considered to explain the antiadipogenic action of androgens. IGF1 is an essential regulator of the adipose conversion process; although, in 3T3-L1 preadipocytes, IGF1R expression remains constant throughout the differentiation phase (51). In this study, exposure of epididymal precursor cells to androgens was found to decrease the IGF1R expression apparently through an androgen receptor-dependent mechanism as well. This limitation in the IGF1 signaling pathway, caused by androgens at the receptor level, may partly explain how androgens reduce adipose tissue development in deep fat deposits. High blood levels of the androgen precursors DHEA and DHEA-S have been reported in abdominal obesity (52), suggesting a possible role of DHEA and DHEA-S in adipose tissue development. DHEA (but not DHEA-S) was reported to clearly inhibit adipose conversion in 3T3-L1 and F442A cell

lines (17, 19) and in pig or rat preadipocytes (18). One mechanism put forward to explain how DHEA reduces the differentiation of 3T3 fibroblasts into adipocytes is: a reduced fatty acid synthesis caused by DHEA inhibition of glucose6-phosphate dehydrogenase activity (17). However, this mechanism seems to be excluded from explaining the negative effect of androgens on rat preadipocytes, because, in 3T3 fibroblasts, testosterone and DHT block adipose conversion without changing glucose-6-phosphate dehydrogenase activity (17). Another mechanism that has been proposed to explain the antiadipogenic effect of DHEA is: a reduced C/EBP␣ expression (18). Our preliminary experiments, however, failed to reveal any significant variations in C/EBP␣ and C/EBP␤ expressions after exposure to DHT and testosterone in preadipocytes undergoing differentiation. Thus, the precise molecular mechanism of the antiadipogenic effect of androgens remains to be determined. Involvement of estrogens in the regulation of adipogenesis is poorly described in the literature (16). Therefore, we have also investigated the possible influence of estrogens on the adipoconversion process. In contrast to androgens, 17␤-estradiol increased GPDH activity, an effect apparently gender dependent, because it was restricted to parametrial and, only at pharmacological concentrations, to epididymal preadipocytes. Although unestablished, the mechanisms explaining this gender-specific action of estrogens could involve differences in the expression of estrogen receptor subtypes (␣ and ␤) and of nuclear receptor cofactors between preadipocytes from male and female. In fact, these nuclear receptors regulate the transcriptional activity of specific genes by recruiting an array of coactivator proteins, including SRC1 (steroid receptor coactivator 1), whose expression was recently reported to be sex specific (53). Whether this site specificity and the gender dependency of the proadipogenic action of estrogens reported in this study may explain the changes in adipose tissue distribution and development occurring in females at puberty during pregnancy (1) and after chronic hormonal therapy (54) remains to be established.


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FIG. 6. Effect of various sex steroids on PPAR␥2 protein expression in differentiated preadipocytes from male and ovariectomized female rats. After exposure to different steroids, the differentiated preadipocytes (day 9 –10) were collected, and the cellular extracts were prepared as described in Materials and Methods. A, Western-blot analysis from one representative experiment. Twenty micrograms of protein were immunoblotted with polyclonal rabbit anti-PPAR␥2 antiserum. B, Densitometric analysis [values are mean ⫾ SEM obtained from three to four separate experiments and are expressed as control values (without steroids) for bars 2, 3, 4, and 5]; 1, without steroid; 2, ⫹ DHT; 3, ⫹ testosterone; 4, ⫹ 17␤-estradiol; 5, Br palmitate. *, P ⬍ 0.05.

In opposition to androgens, however, the promoting action of estrogens on adipose conversion in vitro does not seem related to changes in IGF1R expression, because, in parametrial preadipocytes, where estrogens enhance the adipose conversion process, estrogens do not alter IGF1R expression. Conversely, our experiments showed that, in sc preadipocytes, estrogens enhanced IGF1R expression but had no influence on the adipose conversion process. This site specificity of estrogen action on adipogenesis is in agreement with earlier reports showing that ovarian factors, in vivo, promote regional specialization in the development of adipose tissue depots (55). Interestingly, the proadipogenic action of estrogens on parametrial preadipocytes is accompanied by an increase in PPAR␥2 expression in ovariectomized rats. Finally, chronic exposure of rat preadipocytes to estrogens during the differentiation phase failed to alter the expression of C/EBP␣ and C/EBP␤, as did exposure to androgens (preliminary experiments). These negative results do not allow exclusion of the possibility that the opposite effects of androgens and estrogens on adipose differentiation process are independent of any of these transcriptional factors. In fact, the possibility that sex steroids modify the DNA binding activity of some of these factors cannot be ruled out. If so, androgens and estrogens, via their specific nuclear receptors,

would act as modulators of the adipogenic transcriptional activity of PPAR ␥2 and the members of the C/EBP family. Consistent with this hypothesis is the recent discovery that ARA70, a specific coactivator of androgen receptors, is able to enhance the transcriptional activity of PPAR␥2 in adipocytes, which strongly suggests that, in these cells, a cross-talk occurs between PPAR␥2 and the androgen receptor-mediated responses (56). Sex steroids could also modulate biosynthesis of ligands for these adipogenic transcriptional factors. Indeed, estrogens were recently reported to promote the production of a PPAR␥ ligand, PGJ2, in a PPAR␥1-expressing tissue, leading to overexpression of PPAR␥1 (57). Further investigations are currently in progress to test these different hypotheses. In conclusion, the present study shows that androgens and estrogens are able to modulate, in vitro, the adipose conversion of rat preadipocytes. On the whole, androgens elicit antiadipogenic effects contrary to estrogens that behave as proadipogenic factors; and the modulation of the differentiation process by sex steroids is more pronounced in preadipocytes from deep, rather than superficial, fat depots. The present study also suggests that estrogens and androgens exert their modulatory effects on preadipocyte terminal differentiation via their own receptors acting as regulators of the IGF1R and PPAR␥2 expression.

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