Dehydroepiandrosterone attenuates preadipocyte growth in primary cultures of stromal-vascular cells

M. McIntosh1, D. Hausman2, R. Martin2, and G. Hausman3

1 Department of Food, Nutrition and Food Service Management, University of North Carolina at Greensboro, Greensboro, North Carolina 27402-6170; 2 Department of Foods and Nutrition, University of Georgia, Athens 30602; and 3 Animal Physiology Unit, Richard Russell Research Center, United States Department of Agriculture-Agricultural Research Station, Athens, Georgia 30613

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of this study was to determine whether the antiobesity actions of dehydroepiandrosterone (DHEA) are due to an influence on preadipocyte proliferation and/or differentiation in primary cultures of pig and rat stromal-vascular (SV) cells. Pig SV cells were isolated from dorsal subcutaneous adipose tissue of 7-day-old pigs. For the proliferation assays, pig SV cells were grown for 4 days in plating medium containing DHEA at 0, 15, 50, or 150 µM. For the differentiation assays, pig SV cells were grown in plating medium for 3 days and then switched to a serum-free medium containing DHEA at 0, 15, 50, or 150 µM for the next 6 days. Rat SV cells were isolated from inguinal fat pads of 5-wk-old male rats. Rat SV cells were exposed to DHEA at 0, 5, 25, or 75 µM during proliferation. For the differentiation assays, rat SV cells were grown for 8 days in a serum-free medium containing DHEA at 0, 5, 25, or 75 µM. Preadipocyte differentiation [lipid staining, glycerol-3-phosphate dehydrogenase (GPDH) activity] and proliferation (preadipocyte-specific antigen staining) decreased with increasing levels of DHEA in cultures of pig SV cells. In cultures of rat SV cells, preadipocyte differentiation (lipid staining, GPDH activity) and proliferation ([3H]thymidine incorporation) were decreased in the 25 and 75 µM DHEA groups compared with the control and 5 µM DHEA groups. The level of expression of CCAAT enhancer binding protein-alpha , a master regulator of adipogenesis, in cultures of pig SV cells treated with 150 µM DHEA was 38% of control cultures. These data support the hypothesis that DHEA directly attenuates adipogenesis via attenuation of preadipocyte proliferation and differentiation.

proliferation; differentiation; CCAAT enhancer binding protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

DEHYDROEPIANDROSTERONE (DHEA) sulfate (DHEAS) is the most abundant steroid in the plasma of humans (39, 40). Plasma levels of DHEAS and DHEA decrease steadily as adults age (39, 40). In contrast, aging is associated with an increased risk of adiposity. Given this inverse relationship between plasma levels of DHEA(S) and adiposity, it has been suggested that maintaining "youthful" or higher levels of DHEA(S) may prevent the development of obesity (3, 6, 8, 38). Alternatively, treating obese individuals with these steroids may attenuate adiposity, returning their body fat levels to "normal." Indeed, many studies have shown that administration of DHEA, the intracellular form of the steroid, to rodents blocks or retards fat accretion (2, 7, 21, 24-29, 32, 34, 46). Furthermore, recent studies by our group demonstrated that DHEAS is more effective than DHEA in reducing adipose tissue mass and cellularity in rats (19, 20). Moreover, several clinical trials have demonstrated that DHEA treatment reduces body fat (15, 37) and increases lean body mass (15) and strength (33, 56) in mature adults. However, the mechanism by which DHEA(S) treatment reduces adiposity has not yet been determined.

For example, it is still not known whether treatment of animals or humans with DHEA(S) directly attenuates the growth of preadipocytes, thereby reducing adiposity. In support of this concept, Gordon et al. (9) and Shantz et al. (43) demonstrated that DHEA inhibited the differentiation of 3T3-L1 cells [half-maximal effective dose (ED50) = 250 µM], a clonal line of preadipocytes of embryonic murine origin capable of differentiating into adipose-like cells. Furthermore, we have preliminary data suggesting that DHEA attenuates not only 3T3-L1 preadipocyte differentiation (ED50 = 25 µM) but preadipocyte proliferation as well (ED50 = 30 µM) (50). In contrast to the antiadipogenic actions of DHEA, DHEAS had no impact on 3T3-L1 preadipocyte proliferation or differentiation (50). These data suggest that DHEAS must be converted to DHEA or a downstream metabolite before entry into the preadipocyte, which then attenuates adipocyte growth and adiposity. However, it is still not known whether DHEA directly attenuates the growth of primary cultures of preadipocytes isolated from animals or humans, as it does in a cell line of preadipocytes such as the 3T3-L1 cells.

On the basis of these data on DHEA(S) and adipocyte growth, the working hypothesis of this study is that the antiadipogenic actions of DHEA(S) in vivo are mediated via direct inhibition of preadipocyte proliferation and differentiation by DHEA or a downstream metabolite. Therefore, the purpose of this study was to investigate whether DHEA directly attenuates the growth of primary cultures of preadipocytes isolated from pigs and rats. To examine a proposed mechanism by which DHEA prevents adipogenesis, the impact of DHEA on the expression of a family of transcription factors that regulate adipogenesis, the CCAAT enhancer binding proteins (C/EBPs), was also determined. Pigs and rats were chosen as models because these two species are commonly used for studying preadipocyte growth, and protocols for these studies are firmly established.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and culture of stromal-vascular cells. Pigs from 5 to 7 days of age were obtained from a commercial producer, overdosed with thiopental sodium, and exsanguinated. Five-week-old male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with pentobarbital sodium (50 mg/kg ip), freed of hair, and disinfected with iodine and alcohol. All procedures for animal care were carried out in accordance with protocols approved by the University of Georgia's Institutional Animal Care and Use Committee. Subcutaneous adipose tissue (dorsal for pigs; inguinal for rats) was removed aseptically, and stromal-vascular (SV) cells were obtained by using a collagenase digestion procedure (12). Briefly, adipose tissue was minced with scissors and incubated with 5 ml of the digestion buffer per gram of tissue for 60-90 min in a shaking 37°C water bath (90 oscillations/min) (4). A fivefold excess of buffer containing no enzymes was then added to the digestion flasks at room temperature. Flask contents were mixed and filtered sequentially through nylon screens with 250- and 20-µm mesh openings to remove undigested tissue and large cell aggregates. The isolated cells were then centrifuged at 500 g for 10 min to separate the floating adipocytes from the pellet of SV cells. The SV cells were washed with a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F12; 1:1, vol/vol; Sigma, St. Louis, MO) containing 50 mg/l gentamicin, 50 mg/l cephalothin, and 2.5 mg/l amphotericin B antibiotics and then centrifuged at 500 g for 10 min and resuspended. Aliquots of SV cells were stained with Rappoport's stain and counted on a hemocytometer. Porcine SV cells were seeded in 35-mm tissue culture dishes containing 2 ml of plating medium at a density of 2 × 104/cm2. Rat SV cells were seeded at a density of 1 × 104/cm2 in 35-mm tissue culture dishes for the differentiation assays or at a density of 5 × 103/cm2 in 25-cm2 culture flasks for the proliferation assays. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. Media were changed every 2-3 days.

Proliferation and differentiation assays for pig SV cells. For the proliferation assays, pig SV cells were grown for 4 days in DME/F12 containing 10% fetal bovine serum (FBS), 15 mmol NaHCO3, 5 mmol glucose, the antibiotics just mentioned, 80 nmol dexamethasone (DEX), and DHEA at 0, 15, 50, or 150 µM. For the differentiation assays, pig SV cells were grown in DME/F12 medium containing 10% FBS for 3 days until confluent. Cells were then switched for the next 6 days to a serum-free DME/F12 medium that contained ITS (800 nM insulin, 5 mg/l transferrin, and 5 µg/l selenium), DEX, and DHEA at 0, 15, 50, or 150 µM. DHEA was dissolved in DMSO, which was present in all cultures including the controls, at a level of ~0.5%. For all experiments, unless otherwise indicated, each treatment was performed in triplicate and each experiment was repeated at least once.

For the proliferation assays, a monoclonal antibody (AD-3) that recognizes a cell-surface preadipocyte-specific antigen during and before porcine preadipocyte differentiation in vivo and in vitro was used (13, 14). This method for determining porcine preadipocyte proliferation has been previously validated (13, 14). AD-3 staining was conducted using an ExtrAvidin Peroxidase staining kit for monoclonal antibodies according to the manufacturer's instructions (Sigma). The number of preadipocytes (e.g., cells expressing the AD-3 antigen) per 35-mm dish was counted manually under a 484-mm2 coverslip that was placed on each 35-mm dish, with glycerol as an anchor.

For the differentiation assays, the activity of glycerol-3-phosphate dehydrogenase (GPDH; EN 1.1.1.8) in cytosolic fractions of cells was measured (51). At the time of cell harvest, each 35-mm culture dish containing cells was rinsed three times with Hanks' balanced salt solution (HBSS; pH = 7.4). Cells from each well were scraped into 1 ml of ice-cold sucrose buffer and stored at -80°C for later determination of GPDH activity. The activity of GPDH was measured in cells suspended in ice-cold sucrose buffer and homogenized in a probe sonicator for 5 s at a 20% output level. Suspensions were centrifuged at 8,000 g for 10 min at 4°C. The resulting supernatant was used for the determination of GPDH activity with dihydroxyacetone phosphate as substrate by measuring the oxidation of NADH at 340 nm over time. One unit of activity corresponds to the oxidation of 1 nmol of NADH · min-1 · mg cell protein-1. Protein concentrations of cell sonicates were determined using the Bio-Rad protein assay (Bio-Rad Labs, Hercules, CA) according to the manufacturer's instructions.

The presence of lipid within adipocytes was visualized by staining with Oil Red O. Nuclei were stained with hematoxylin to determine cell number. To stain adipocytes, cells in 35-mm dishes were fixed for 30 min at 4°C with Baker's Formalin (10 ml of 37% formaldehyde, 10 ml calcium chloride, and 80 ml water). Cells were then washed three times with HBSS and stained for 10 min with 0.3% Oil Red O in 60% isopropanol. The cells were then carefully washed with distilled water and counterstained for 2 min with Harris hematoxylin to identify nuclei. A drop of heated glycerol gel was added to the center of each dish, and a 484-mm2 coverslip was used to cover the dishes. The amount of lipid-stained material per field under the coverslip in 10-15 fields per dish was quantified by computer-assisted image analysis by use of a Dage CCD-72 camera (Dage-MTI, Michigan City, IN) and IM-3000 software (Analytical Imaging Concepts, Irving, CA). Data are expressed as the area of stained cells per field, as previously described (14).

The total number of SV cells at the end of differentiation was also measured to determine whether DHEA treatment altered cell number during preadipocyte maturation. Cultures in 35-mm dishes were washed with HBSS and then incubated in a prewarmed cell-counting solution for 45 min at 37°C in a humidified 5% CO2 atmosphere. The cell-counting solution, pH 7.4, contained 0.01 M sodium phosphate, 0.154 M sodium chloride, 25 mM glucose, 5 mM EDTA, and 2% BSA. An aliquot from each dish was diluted in saline and counted with a Z1 Coulter Counter (Coulter, Miami, FL).

Proliferation and differentiation assays for rat SV cells. Proliferation of rat SV cells in the absence or presence of DHEA was determined in an established preadipocyte proliferation assay by use of the [3H]thymidine pulse-labeling technique (41). Briefly, freshly isolated SV cells were seeded in 25-cm2 flasks in 4 ml of plating medium at a density of 2.0 × 104 cells/cm2. Plating medium consisted of DME/F12, antibiotics (listed above), and 10% FBS. On day 2, cultures were incubated with plating media containing 2.5% pig serum and DHEA at 0, 5, 25, or 75 µM. On day 3, cultures were labeled for 24 h with 0.5 µCi [3H]thymidine (ICN, Irvine, CA) per flask in DME/F12 medium containing 2.5% pig serum and DHEA at 0, 5, 25, or 75 µM. DHEA was dissolved in DMSO, which was present in all cultures including the controls, at a level of ~0.25%. After the labeling period, cultures were rinsed twice with 5 ml of HBSS containing 50 mM (cold) thymidine and refed with 4 ml of fresh, unlabeled test medium. On day 5, cultures were refed a differentiation-promoting medium without DHEA. This differentiation medium contained 10% pig serum, 1.0 nM porcine insulin, and 1,000 U/l heparin in DME/F12 medium. Differentiation medium was changed every other day until day 14. Cells were harvested on day 14 in HBSS containing 0.22% trypsin, 0.02% collagenase, and 0.5% BSA. Lipid-filled adipocytes were separated from undifferentiated preadipocytes and SV cells by centrifugation on an HBSS-Percoll gradient with a density of 1.02. [3H]thymidine incorporation into these cell fractions was determined by standard liquid scintillation counting on a Beckman LS counter (Beckman Instruments, Palo Alto, CA).

For the differentiation assays, rat cells were seeded in DME/F12 medium containing 10% FBS for 1 day and switched to a serum-free differentiation medium containing ITTS (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, and 2 nM triiodothyronine) and DHEA at 0, 5, 25, or 75 µM. DHEA was dissolved in DMSO, which was present in all cultures including the controls, at a level of ~0.25%. On day 8, rat SV cells were harvested, and GPDH activity, Oil Red O staining, and cell numbers were measured as described for the pig differentiation protocols.

C/EBP expression in pig SV cells. Cultures of pig SV cells were grown in 10-cm plates, as described for the differentiation protocol. Each treatment was performed in duplicate, and the experiment was repeated once. Cells were harvested in 0.4 ml of lysis buffer (60 mM Tris · HCl, 1.0 mM phenylmethylsulfonyl fluoride, and 1.0% SDS) containing protease inhibitors. The cell lysate was then sonicated and centrifuged for 20 min at 14,000 g at 4°C, the supernatant was collected, and the protein content was determined using the DC protein assay by Bio-Rad. Each sample (30 µg protein) was separated by standard SDS-PAGE (15% SDS gels) and transferred electrophoretically to polyvinylidene fluoride membrane and hydridized with primary and secondary antibodies. Antibodies for C/EBP-delta , C/EBP-beta , and C/EBP-alpha were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG (Sigma). The membranes were incubated in enhanced chemiluminescence (ECL) reagents (Amersham, Arlington Heights, IL) and exposed to ECL hyperfilm (Amersham), and the bands on the developed film were quantified via densitometry (Bio-Rad Labs, Richmond, CA).

Statistics. Data were analyzed by one-way analysis of variance (ANOVA) using the SuperANOVA program (Abacus Concepts, Berkeley, CA). Differences between treatment means were considered statistically significant at P < 0.05 (44).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pig SV cell proliferation and differentiation. The number of preadipocytes (AD-3 stained cells) decreased as the level of DHEA increased in cultures of pig SV cells (Fig. 1). Furthermore, the amount of lipid in maturing pig SV cultures decreased as the level of DHEA in the media increased (Figs. 2 and 3). Likewise, GPDH activity decreased with increasing levels of DHEA in the media (Fig. 3). As seen in Fig. 2, the SV cells treated with DHEA appear to be larger in size than the control SV cells, especially those receiving the 150 µM level. DHEA treatment had no adverse effect on the total number of pig SV cells. In fact, cultures of pig SV cells treated with 50 µM DHEA had significantly more cells (5.0 × 105) than cultures treated with 0, 15, or 150 µM DHEA (4.0 × 105, 4.3 × 105, and 4.1 × 105, respectively; pooled SE = 0.13 × 105).


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Fig. 1.   Effect of dehydroepiandrosterone (DHEA) treatment for 4 days on the number of cells immunoreactive for a preadipocyte-specific monoclonal antibody (AD-3) in cultures of pig stromal-vascular (SV) cells. Pig SV cells were grown for 4 days in a combination of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F12; 1:1, vol/vol) containing 10% fetal bovine serum (FBS), 15 mmol NaHCO3, 5 mmol glucose, 40 mg/l gentamicin sulfate, 50 mg/l cephalothin, 2.5 mg/l amphotericin B, 80 nmol dexamethasone (DEX), and DHEA at 0, 15, 50, or 150 µM. Values represent least square means ± SE. Means not sharing a common superscript (a or b) are significantly (P < 0.05) different.


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Fig. 2.   Micrographs (×10) of pig SV cell cultures treated with DHEA for 6 days and stained with Oil Red O. Pig SV cells were grown in DME/F12 medium containing 10% FBS for 3 days until confluent. Cells were then switched for the next 6 days to a serum-free DME/F12 medium containing ITS (800 nM insulin, 5 mg/l transferrin, and 5 µg/l selenium), DEX, and DHEA at 0 (A), 15 µM (B), 50 µM (C), or 150 µM (D). Arrows indicate adipocytes loaded with Oil Red O-stained lipid droplets.


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Fig. 3.   Effect of DHEA treatment for 6 days on Oil Red O-stained lipid content (A) and on specific activity of glycerol-3-phosphate dehydrogenase (GPDH) activity (B) in cultures of pig SV cells. Pig SV cells were grown in DME/F12 containing 10% FBS for 3 days until confluent. Cells were then switched for the next 6 days to a serum-free DME/F12 medium containing ITS (800 nM insulin, 5 mg/l transferrin, and 5 µg/l selenium), DEX, and DHEA at 0, 15, 50, or 150 µM. Values represent least square means ± SE. Means not sharing a common superscript (a, b, or c) are significantly (P < 0.05) different.

Rat SV cell proliferation and differentiation. [3H]thymidine incorporation into preadipocytes (e.g., SV cells that became adipocytes at the end of the culture period) decreased as the level of DHEA in the media of rat SV cultures increased above 5 µM (Fig. 4). Similarly, [3H]thymidine incorporation into nonpreadipocytes (e.g., SV cells that did not become adipocytes at the end of the culture period) decreased as the level of DHEA in the media of rat SV cultures increased above 5 µM (12,895, 14,137, 11,434, and 7,721 cpm/flask for 0, 5, 25, and 75 µM, respectively; pooled SE = 745). The amount of stainable lipid in maturing adipocytes also decreased with increasing levels of DHEA in the media (Figs. 5 and 6). Furthermore, GPDH activity decreased as the level of DHEA in the media of rat SV cells increased above 5 µM (Fig. 6). As seen in Fig. 5, the SV cells treated with DHEA appeared to be more fibroblast-like. In contrast to the pig SV cells, rat SV cells treated with 75 µM DHEA were fewer in number and appeared smaller in size than the control cells. For example, cultures of rat SV cells treated with 75 µm DHEA had significantly fewer SV cells (1.1 × 105) than cultures treated with 0, 5, or 25 µM DHEA (2.6 × 105, 2.9 × 105, and 2.5 × 105 SV cells, respectively; pooled SE = 0.20 × 105).


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Fig. 4.   Effect of DHEA treatment for 3 days on [3H]thymidine incorporation into preadipocytes in cultures of rat SV cells. Cells were seeded in DME/F12 medium containing 10% FBS for 1 day and switched to several different types of media conditions (described in detail in MATERIALS AND METHODS) over the next 13 days in the absence and presence of DHEA using an established thymidine incorporation protocol. Values represent least square means ± SE. Means not sharing a common superscript (a, b or c) are significantly (P < 0.05) different. cpm, Counts/min.


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Fig. 5.   Micrographs (×10) of rat SV cell cultures treated with DHEA for 8 days and stained with Oil Red O. Arrows indicate adipocytes loaded with Oil Red O-stained lipid droplets. Cells were seeded in DME/F12 medium containing 10% FBS for 1 day and switched to a serum-free differentiation medium containing ITTS (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, and 2 nM triiodothyronine) and DHEA at 0 µM (A), 5 µM (B), 25 µM (C), or 75 µM (D) for the next 8 days.


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Fig. 6.   Effect of DHEA treatment for 8 days on Oil Red O-stained lipid content (A) and on specific activity of GPDH activity (B) in rat SV cell cultures. Cells were seeded in DME/F12 medium containing 10% FBS for 1 day and switched to a serum-free differentiation medium containing ITTS (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, and 2 nM triiodothyronine) and DHEA at 0, 5, 25, or 75 µM for the next 8 days. Values represent least square means ± SE. Means not sharing a common superscript (a, b, c, or d) are significantly (P < 0.05) different.

C/EBP expression in pig SV cells. The level of expression of C/EBP-alpha in cultures treated with 150 µM DHEA was 38% of control cultures (Fig. 7). In contrast, the expression of C/EBP-delta and C/EBP-beta was not affected by DHEA treatment (data not shown).


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Fig. 7.   Effect of DHEA treatment on expression of CCAAT enhancer binding protein-alpha (C/EBP-alpha ) in pig SV cell cultures. Pig SV cells were grown in DME/F12 medium containing 10% FBS for 3 days until confluent. Cells were then switched for the next 6 days to a serum-free DME/F12 medium containing ITS (800 nM insulin, 5 mg/l transferrin, and 5 µg/l selenium), DEX, and DHEA at 0, 15, 50, or 150 µM. Values represent least square means ± SE. Means not sharing a common superscript (a or b) are significantly (P < 0.05) different.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study provides direct evidence that DHEA attenuates preadipocyte proliferation and differentiation in primary SV cultures. To our knowledge, this is the first time the antiadipogenic actions of DHEA have been demonstrated in primary cultures of preadipocytes isolated from pigs and rats. These data suggest that the antiobesity effects of DHEA observed in rats (2, 7, 21, 24-29, 32, 34, 46, 47) and pigs (A. Tagliaferro, unpublished data) may indeed be due to the direct actions of DHEA or downstream metabolites on adipose tissue mass and cellularity. In support of this concept, we have demonstrated that DHEA, but not DHEAS, attenuates the proliferation and differentiation of 3T3-L1 preadipocytes (50). It is not known whether the antiadipogenic effects of DHEA are due to DHEA itself or to a metabolite of DHEA such as androstenedione, testosterone, or estrogens. Three major downstream metabolites of DHEA in human SV cells, 7alpha -hydroxy-DHEA, androstenedione, and estrone (17, 18), have been identified. However, the impact of these metabolites on adipogenesis is not known. Although treatment of rats with testosterone has been shown to reduce adipose tissue mass and adipocyte number (16), the direct influence of testosterone on preadipocyte growth is not known. We do, however, have unpublished data demonstrating that DHEA and 17alpha -estradiol attenuate 3T3-L1 preadipocyte proliferation to a greater extent than testosterone, estrone, pregnenolone, and DHEAS.

Several studies have demonstrated that treatment of nonpreadipocytes with DHEA(S) stimulates glucose and lipid metabolism. For example, treatment of primary cultures of rat myoblasts (36) and human skin fibroblasts (35) with 10 to 100 µM levels of DHEA increased glucose uptake and insulin sensitivity to a greater extent than other downstream metabolites of DHEA. As we found in 3T3-L1 cells (50), DHEAS had no impact on glucose uptake or insulin sensitivity in these two cell culture systems (35, 36). On the other hand, DHEAS enhanced peroxisomal fatty acid oxidation and carnitine acetyltransferase activity in isolated hepatocytes to a much greater extent than DHEA (55). These cell-specific differences in the actions of DHEAS are most likely due to the ability of DHEAS to be transported into hepatocytes by an energy-dependent, carrier-mediated process (42). The lack of an effect of DHEAS on rat myoblasts, human skin fibroblasts, and 3T3-L1 preadipocytes may be due to the absence of a transporter for DHEAS in these cells.

In support of DHEA's specificity for attenuating adipogenesis, we demonstrated that in cultures of differentiating pig SV cells (Fig. 2), DHEA treatment reduced only the number of maturing adipocytes, not the total number of SV cells in culture. In fact, cultures of pig SV cells treated with 50 µM DHEA had significantly more cells (5.0 × 105) than the control cultures (4.0 × 105). In contrast, differentiating rat SV cells (Fig. 5) treated with the highest level of DHEA (75 µM) had fewer mature adipocytes and SV cells. This difference in response to DHEA treatment in SV cells between pigs and rats could be due to differences in culture conditions. For example, the pig SV cells were cultured in serum-free media containing DEX during differentiation, whereas cultures of rat SV cells were cultured without DEX. DEX supplementation optimizes the differentiation of certain types of preadipocytes, including cells from pigs (13), rats (54), humans (11), and the 3T3-L1 (9) and TA1 (5) cell lines. Glucocorticoids promote differentiation by several known mechanisms, including attenuation of mitogenic activity (10). Interestingly, DHEA has been shown to antagonize the actions of glucocorticoids in vivo (24, 25, 52). Furthermore, we have unpublished data indicating that the lower the concentration of DEX in differentiating 3T3-L1 preadipocytes, the greater the antiadipogenic effects of DHEA. For example, cultures of 3T3-L1 preadipocytes treated with 250 µM DHEA and supplemented with 0.1 µM DEX had only 2% marker enzyme activity relative to control cultures. In contrast, cultures treated with 250 µM DHEA and supplemented with 1.0 µM DEX had 30% marker enzyme activity compared with the controls. If DHEA indeed antagonizes DEX's antimitogenic activity (10), this could explain why there were more nonpreadipocytes and fewer preadipocytes in the cultures of pig SV cells treated with 15 and 50 µM DHEA compared with the controls. In the absence of DEX, nonpreadipocyte growth in rat cultures was attenuated by DHEA at higher concentrations (i.e., 75 µM). Therefore, rat preadipocytes may be more susceptible to DHEA's antiadipogenic effects when grown in serum-free media devoid of DEX. The reason for this inhibitory effect of DHEA on nonpreadipocyte growth of rat cells in the absence of DEX is not known.

The mechanism by which DHEA attenuates adipogenesis is not known. DHEA or one of its metabolites could be altering preadipocyte proliferation and differentiation by a number of different mechanisms. DHEA or a metabolite could be arresting cell growth by 1) blocking the actions of a gene the activation of which is essential for proliferation or differentiation, 2) limiting the availability of substrates, 3) inducing growth arrest or apoptosis, or 4) being cytotoxic. In response to the first two potential mechanisms, the proliferation of preadipocytes and their differentiation into mature adipocytes are regulated by several "master" genes of the adipogenic program (23, 53). These master regulators include three C/EBPs, C/EBP-beta , C/EBP-delta , and C/EBP-alpha . C/EBP-beta regulates early differentiation, C/EBP-delta mid-differentiation, and C/EBP-alpha late differentiation. Without C/EBP-alpha expression, terminal differentiation does not occur because of a lack of induction of lipogenic genes involved in glucose (GLUT-4) and fatty acid (fatty acid binding protein) uptake and lipid synthesis (stearoyl CoA desaturase) and esterification (GPDH) (23, 53). For example, C/EBP knockout mice fail to develop lipid in white and brown adipose tissue and die soon after birth (49). In contrast, upregulation of these genes by glucocorticoid (DEX) in 3T3-L1 preadipocytes stimulates C/EBP expression and preadipocyte differentiation (53). Several other genes essential for adipose tissue development, such as glucose-6-phosphate dehydrogenase (G-6-PD), the rate-limiting enzyme of the pentose phosphate shunt, also have response elements for glucocorticoids (45). Therefore, antagonism of DEX-induced expression of the C/EBP family or other genes the expression of which is essential for adipogenesis could attenuate preadipocyte maturation.

Along these lines, DHEA treatment has been reported to have many anti-glucocorticoid actions. For example, DHEA has been shown to reduce lipid synthesis in vivo (22, 24, 25, 46) and the lipid accumulation in preadipocytes (9, 43) by inhibiting G-6-PD. By limiting G-6-PD activity, adipocytes would be deficient in ribose 5-phosphate for nucleic acid synthesis. Moreover, adipocytes would lack sufficient NADPH for fatty acid synthesis. Therefore, a DHEA-mediated reduction in G-6-PD activity would limit adipocyte growth by decreasing the availability of nucleic acids and NADPH. In the present study, 50 and 150 µM DHEA reduced the expression of C/EBP-alpha , a DEX-inducible transcription factor essential for adipogenesis. Furthermore, we have preliminary data demonstrating that treatment of 3T3-L1 preadipocytes with 25 µM DHEA for 8 days reduced the expression of C/EBP-alpha fivefold. On the basis of these observations, it is tempting to speculate that DHEA antagonizes glucocorticoid-induced activation of the C/EBP-alpha in adipose tissue, thereby inhibiting growth and lipid metabolism of preadipocytes. However, DHEA may be altering cell growth by mechanisms other than those regulated by glucocorticoids. For example, some immunoenhancing properties of DHEA have been shown to be independent of its antiglucocorticoid actions (1).

It is not known whether DHEA or its downstream metabolites are capable of inducing growth arrest or apoptosis. Certain growth factors (i.e., tumor necrosis factor-alpha , transforming growth factor-beta , fibroblast growth factor, fetuin) are essential for inducing cell mitosis and inhibiting differentiation via stimulating quiescent cells to enter a G1 from a G0 phase of the cell cycle (48). Whether DHEA interferes with the action of these growth factors that induce proliferation and suppress differentiation is not known. DHEA clearly reduced the proliferation of preadipocytes in primary cultures of pig and rat SV cells in the present study. At the highest level it also reduced the proliferation of nonpreadipocytes in cultures of rat SV cells. Therefore, 75 µM DHEA in cultures of rat SV cells grown in serum-free media devoid of DEX could be reducing cell numbers by causing cell death or necrosis. Unfortunately, we did not measure lactate dehydrogenase levels in the media of cultures treated with DHEA to assess potential cytotoxicity. Indicators of apoptosis were also not measured. In contrast, DHEA did not adversely affect the proliferation of nonpreadipocytes in cultures of pig SV cells, and therefore we do not think that DHEA at the levels tested was cytotoxic to the pig SV cells.

In summary, we have demonstrated for the first time that DHEA directly attenuates the growth of primary cultures of pig and rat SV cells. However, it is not known whether these antiadipogenic actions of DHEA treatment are due to DHEA or to a downstream metabolite. Furthermore, the molecular mechanisms of DHEA's or DHEAS's antiobesity effects are not known. We hypothesize that on ingestion by mammals, a portion of DHEAS is converted by specific cells (i.e., hepatocytes, macrophages) to DHEA along with a number of downstream metabolites. Once sequestered by preadipocytes, DHEA and/or a metabolite antagonize(s) the anti-mitogenic and differentiation-promoting actions of glucocorticoids. We further speculate that this antagonism could be at the glucocorticoid receptor level, but more likely, on the basis of work by Mohan et al. (31) and our group (30), it could be at the gene level, where glucocorticoids bind to specific response elements of glucocorticoid-responsive genes. By DHEA impairing glucocorticoids' ability to activate genes (i.e., C/EBP-alpha ) that regulate adipogenesis and lipid metabolism, the growth of preadipocytes and the lipid metabolism of mature adipocytes are attenuated. This results in fewer and smaller adipocytes and, therefore, less adipose tissue. Future studies examining DHEA's influence on glucocorticoid-responsive genes that regulate adipogenesis in preadipocytes would provide evidence for this proposed antiadipogenic mechanism of action of DHEA.

    ACKNOWLEDGEMENTS

We thank Rosetta Minish, Xiaoli Chen, and Lori Lee-Jones for technical assistance with various aspects of this research.

    FOOTNOTES

Address for reprint requests: M. K. McIntosh, Dept. of Food and Nutrition, UNCG, PO Box 26170, Greensboro, NC 27402-6170.

Received 23 December 1997; accepted in final form 12 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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