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
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ABSTRACT |
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-
, 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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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-
,
C/EBP-
, and C/EBP-
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).
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RESULTS |
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.
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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.
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C/EBP expression in pig SV cells.
The level of expression of C/EBP-
in cultures treated with 150 µM
DHEA was 38% of control cultures (Fig. 7).
In contrast, the expression of C/EBP-
and C/EBP-
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- (C/EBP- ) 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.
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DISCUSSION |
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,
7
-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
17
-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-
,
C/EBP-
, and C/EBP-
. C/EBP-
regulates early differentiation,
C/EBP-
mid-differentiation, and C/EBP-
late differentiation.
Without C/EBP-
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-
, 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-
fivefold. On the basis of these observations, it is tempting to
speculate that DHEA antagonizes glucocorticoid-induced activation of
the C/EBP-
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-
, transforming growth factor-
, 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-
) 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.
 |
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