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Service de Biochimie, INSERM CJF 94–02, Faculté de Médecine Paris-Ouest, Université René Descartes (Paris V), Centre Hospitalier, 78303 Poissy, France
Address all correspondence and requests for reprints to: Dr. D. Lacasa, Service de Biochimie, Centre Hospitalier, 78303 Poissy Cedex, France.
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Abstract |
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Ovariectomy induced increased proliferation and differentiation as well as high mitogen-activated protein (MAP) kinase activity and c-fos protein induction in both confluent and differentiated preadipocytes from perirenal fat depots. In parametrial preadipocytes, ovariectomy also increased proliferation and c-fos protein induction, but failed to alter the capacities of these cells to differentiate.
Treatment of ovariectomized rats with estradiol and progesterone reversed the promoting effect of ovariectomy on proliferation, differentiation, and c-fos induction in perirenal preadipocytes, but not the MAP kinase activation observed during the proliferative phase. This treatment also reversed the promoting effect of ovariectomy on proliferation and c-fos induction seen in confluent parametrial preadipocytes.
In contrast, sc preadipocytes were totally insensitive to ovarian status in terms of proliferation and differentiation capacities, MAP kinase activity, and c-fos induction.
This study demonstrates that adipogenesis is site-specifically controlled by the ovarian status in the rat. It also suggests that ovariectomy-induced obesity (mainly abdominal) could be related to changes in some of the signaling pathways controlling adipogenesis in intraabdominal preadipocytes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adipose tissue is an important site of estrogen biosynthesis and steroid hormone storage (9). The presence of specific ovarian hormone receptors in precursor and mature fat cells of rodents (4, 10, 11, 12) suggests that these cells might be targets for estrogen and progesterone. In addition, estradiol (E2) receptor numbers vary with the anatomical origin of the fat cells (13, 14).
The development of adipose tissue (adipogenesis) proceeds from proliferation and differentiation of preadipocytes. In vivo, adipose tissue growth depends on fat localization (15). In vitro studies on cultured preadipocytes (16, 17) have also revealed important differences between the proliferating and differentiating capacities of preadipocytes from superficial and deep intraabdominal fat depots. Epididymal preadipocytes exhibit faster, but less extensive, maturation than those from sc fat depots (18). Moreover, comparison between two deep intraabdominal fat depots have revealed that proliferating and differentiating preadipocytes are more numerous in perirenal than in epididymal fat depots (16, 17).
Proliferation and differentiation are two processes controlled by numerous factors, including steroid hormones such as glucocorticoids (for reviews, see Refs. 19, 20). Ovarian hormones also seem to be involved in adipogenesis, as in female rats, changes in ovarian status have been shown to modify the in vivo growth potential of adipose tissue from various locations (21). Moreover, in vitro studies have demonstrated enhancement of human preadipocyte replication by E2 (22). However, progesterone and E2 were reported to have no effect on differentiation of cultured human preadipocytes (23), whereas in another study (24), progesterone was found to stimulate adipogenesis in the 3T3-L1 adipose cell line.
The influence of both fat localization (16, 17) and hormonal status (25) on the proliferating and differentiating capacities of preadipocytes has been assessed with primary cultured preadipocytes. Such studies have demonstrated a reduction of the replicative capacity of preadipocytes from perirenal and epididymal fat depots in hypophysectomized rats (25).
The mitogen-activated protein kinase (MAP kinase) cascade is activated
by several growth factors and thus plays an important role in cell
growth and differentiation (26). MAP kinase is activated upstream by
MEK (MAP-ERK kinase) (27), which can itself be activated by PKC
in
response to mitogenic factors (28). Activation of the MAP kinase
cascade in response to growth factors induces rapid transcription of
the c-fos gene and protein synthesis (29). This
protooncogene plays a pivotal role in mitogenesis and differentiation
of preadipocytes (30).
Ovariectomy is known to increase deep intraabdominal fatness in rodents (7). To determine the part played by adipogenesis in the pathogenesis of this particular type of obesity, we compared the influence of ovarian status on adipose conversion, MAP kinase activity, and c-fos expression of primary cultured rat preadipocytes isolated from sc and deep intraabdominal fat depots.
The results presented herein reveal that the ovarian status influences adipogenesis in deep intraabdominal preadipocytes specifically and, therefore, suggest an important role for adipogenesis in the pathogenesis of the obesity caused by ovariectomy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and FBS were obtained from Life Technologies (Eragny,
France). p42/p44 MAP kinase enzyme assay and chemiluminescence Western
blot protocols were obtained from Amersham (Aylesbury, UK). Anti-MEK-1
antibodies (C-18) were purchased from Santa Cruz Biotechnologies (Santa
Cruz, CA), and antiserum and synthetic peptide specific for
c-fos (OP-17) were obtained from Oncogene Science (San
Diego, CA). All other chemicals were of reagent grade.
Animals
Female Sprague-Dawley rats (125–150 g) were ovariectomized
(OVX) and treated as previously described (7). Two weeks after the
operation, half of the OVX rats received one sc injection of
E2 benzoate (5 µg/animal) and oxyprogesterone caproate
(P; 5 mg/animal) every other day for 2 weeks, and the other half of the
OVX group and the sham-operated rats (SHAM) received the vehicle only.
The rats were killed by decapitation 1 day after the last injection.
Femoral sc, parametrial, and perirenal fat pads were removed
aseptically. Characteristics of the adipose tissues of these animals
were previously described (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31). Serum steroid levels of the animals
were: E2, 10 ± 5, undetectable, and 15.6 ± 2.3
ng/ml; and P, 4.6 ± 0.3, 2.50 ± 0.05, and 3.2 ± 0.2
pg/ml in SHAM, OVX, and OVX, E2, and P rats,
respectively.
Cell culture
Cell preparation and cell culture were performed as described
previously (32). After washing, cells were plated into cell culture
dishes at a density of 1–2 104 cells/cm2 in
8% FBS-DMEM. After 12 h, cultures were washed with DMEM and fed
8% FBS-DMEM. At confluence (2–3 days postplating), cells were
harvested or allowed to differentiate in DMEM-Ham’s F-12 containing 5
µg/ml insulin, 10 µg/ml transferrin, and 200 pM T3 (ITT
medium) (32).
Preadipocytes were routinely cultured in DMEM with phenol red. However, as a phenol red contaminant was reported to have estrogenic activity (33), parallel experiments were performed to study adipogenesis in the absence of phenol red. The following data show no influence of such a contaminant on either the proliferation (cell number per well on day 4 of culture, 1.15 ± 0.02 with vs. 1.20 ± 0.02 x 104 without phenol red) or differentiation capacity [glycerol 3-phosphate dehydrogenase (GPDH) activity on day 8 postconfluence, 3760 ± 54 with vs. 3744 ± 58 mU/mg protein without phenol red] of preadipocytes.
Cell counting
Preadipocytes from femoral sc, parametrial, and perirenal fat
depots removed from the three animal groups were isolated and initially
seeded at 1 105 cells/well. Cell number was determined 1,
2, 3, and 4 days postplating. Cell cultures were washed three times
with saline, then trypsinized with calcium- and magnesium-free Hanks’
solution containing 0.2% trypsin, and finally counted in a
hemocytometer.
As FBS may contain endogenous steroids, preliminary experiments were performed comparing growth rates of cells cultured in the presence of 8% FBS or 8% charcoal-treated FBS. On day 4 postplating, cell densities per well were 2.8 ± 0.2 vs. 0.37 ± 0.09 104 cells for FBS and charcoal-treated FBS, respectively, indicating a probable removal of serum mitogenic factors by the charcoal treatment. Moreover, we found undetectable (RIA) levels of E2 and P in FBS (i.e. <10 pg/ml and <0.1 ng/ml, respectively). Therefore, charcoal-treated FBS was no longer used to study preadipocyte proliferation.
GPDH assay
Cell differentiation was followed by measuring cell GPDH
activity. After 10–12 days, ITT media were removed, and cells were
scraped and sonicated in buffer containing 50 mM Tris-HCl
(pH 7.4), 0.25 M sucrose, 1 mM EDTA, and 1
mM dithiothreitol. After centrifugation at 100,000 x
g for 20 min at 4 C, GPDH activity was measured in the
supernatant (34) and expressed in milliunits (nanomoles of NAD per min)
per 105 cells.
Cellular extract preparation
For MEK and PKC assays, cytosolic extracts were prepared as
follows. After washing, confluent and differentiated preadipocytes were
scraped in 50 mM Tris buffer, pH 7.5, containing 0.25
M sucrose, 5 mM EDTA, 10 mM EGTA,
20 mM 2-mercaptoethanol, 50 µg/ml
phenylmethylsulfonylfluoride (PMSF), 25 µg/ml aprotinin, 20 µg/ml
leupeptin, and 10 mM benzamidine. Cells were sonicated and
centrifuged at 100,000 x g for 45 min at 4 C to obtain
cytosolic fractions.
For c-fos protein detection, preadipocytes were serum deprived for 18 h and then exposed to 10% serum for 90 min. After washing, cells were scraped in cold buffer containing 50 mM Tris (pH 8.0), 120 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.5 mM PMSF, 25 µg/ml aprotinin, and 20 µg/ml leupeptin as described previously (35). Cells were sonicated and centrifuged at 100,000 x g for 15 min at 4 C. The resulting supernatant was denatured with Laemmli buffer (vol/vol) and stored at -20 C.
Total and cytosolic protein contents per cell were determined and were identical whatever the ovarian status and anatomical origin of the preadipocytes.
Western blot analysis
Equal amounts of protein (25–50 µg) corresponding to the same
amount of cells were subjected to SDS-PAGE (12.5% acrylamide).
Proteins were transferred to polyvinlidene difluoride filters.
Nonspecific sites were blocked by incubation with TBS-T buffer [137
mM NaCl, 10 mM Tris (pH 7.5), and 0.1%
Tween-20] containing 2.5% gelatin for 2 h. The filters were
incubated overnight with anti-MEK antibody (0.1 µg/ml in TBS-T and
2.5% gelatin), anti c-fos antibody (1 µg/ml in TBS-T and
2.5% gelatin), or anti-PKC antibody (0.5 µg/ml in TBS-T). After
washing, filters were incubated with the secondary antiserum coupled to
peroxidase (1:2000 dilution in TBS-T) for 1 h and washed. Filters
were next incubated with the enhanced chemiluminescence detection
system and exposed to x-ray films.
MAP kinase assay
Cytosolic extracts were prepared from proliferating and
differentiated preadipocytes exposed to 10% serum or 10 nM
insulin for 10 min. After washing, cells were scraped and sonicated in
10 mM Tris buffer, pH 7.4, containing 150 mM
NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1
mM sodium orthovanadate, 30 mM sodium
ß-glycerophosphate, 1 mM PMSF, 20 µg/ml leupeptin, and
25 µg/ml aprotinin and centrifuged at 100,000 x g
for 30 min at 4 C. Cytosolic extracts were kept at -80 C. MAP kinase
activity was measured using the p42/p44 MAP kinase enzyme assay
described previously (36). Reactions were linear up to 10 µg
protein/assay and 30-min incubation at 30 C.
Other determinations
Protein concentrations were measured according to the method of
Bradford (37) with BSA as standard. Lactate dehydrogenase activity was
measured as described previously (38). Cell viability was verified by
trypan blue exclusion. E2 and P RIA in rat serum and FBS
were performed after ether extraction using Orion Diagnostic and Ciba
Corning kits, respectively. All results are expressed as the mean
± SEM from at least three individual experiments.
Comparison between groups were made using ANOVA with Bonferroni
P values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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present the growth curves of
preadipocytes from the different experimental groups. On day 4, the
number of preadipocytes from the two deep intraabdominal fat depots of
OVX rats was increased by 70 ± 17% in parametrial cells and by
105 ± 41% in perirenal cells (mean ± SEM of
four separate experiments; P < 0,05). This promoting
effect of OVX on preadipocyte proliferation was completely or partially
prevented by E2 and P treatment in parametrial and
perirenal preadipocytes, respectively. In contrast, ovarian status
failed to influence the growth rate of femoral sc preadipocytes. These
data indicate that preadipocyte growth is differently influenced by
ovarian status depending on the anatomical origin of the cells.
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p42/p44 MAP kinase activity
As the MAP kinase cascade plays an important role in cell growth
and differentiation (26), p42/p44 MAP kinase activities were first
measured in cytosolic extracts of confluent and differentiated
preadipocytes from the various experimental groups. As shown in Table 2, ovariectomy induced an increase in p42/p44 MAP kinase
activity in both proliferating and differentiated preadipocytes from
perirenal fat depots. This effect was abolished by the in
vivo E2 and P treatment only in
differentiated cells. MAP kinase activity remained insensitive to
ovarian status in femoral sc and parametrial preadipocytes whatever the
stage of culture (data not shown). It should also be noted that MAP
kinase activity markedly decreased (-70 to -87%) during the course
of adipogenesis in all experimental groups. In addition, despite
differences in the capacities of proliferation and differentiation of
preadipocytes according to their anatomical origin, MAP kinase
activities were similar at a given stage of culture in sc, parametrial,
and perirenal preadipocytes of control rats.
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status, two upstream activators of the MAP kinase
cascade, were studied by Western blotting in cytosolic fractions from
proliferating preadipocytes. Unfortunately, these experiments failed to
reveal any significant difference in MEK and PKC content whatever
the ovarian status or anatomical origin of the cells (data not
shown).
c-fos protein expression
As c-fos protooncogene induction plays an important
role in adipogenesis (30), we compared the induction of
c-fos protein by serum growth factors in confluent and
differentiated preadipocytes from the various experimental groups.
After induction by 10% serum for 1–2 h, a c-fos species of
about 50 kDa was detected. As shown in Fig. 2, c-fos protein expression was significantly increased by
ovariectomy in confluent preadipocytes from the two deep fat depots
(+100 ± 30% in parametrial cells and +50 ± 6% in
perirenal cells; P < 0.05). Conversely, in the same
cells, rat pretreatment with E2 and P resulted in
normalization of c-fos protein expression.
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). However, in perirenal cells, c-fos protein
induction was markedly increased after ovariectomy and again decreased
by the E2 and P treatment (-52 ± 8%;
P < 0.05). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present in vitro experiments have revealed that the capacity of the preadipocyte to proliferate is increased in two deep intraabdominal (perirenal and parametrial) fat depots when rats have undergone ovariectomy. However, the capacity of preadipocytes to differentiate is also increased by ovariectomy, but only in perirenal preadipocytes. This could be due to the fact that perirenal fat depots contain more differentiating preadipocytes than other fat depots (16, 17). In vivo treatment with E2 and P partly or completely corrected the effects of ovariectomy on the proliferative capacities of perirenal and parametrial preadipocytes, respectively. Alterations of adipogenesis caused by ovariectomy were also corrected in perirenal cells.
From these observations and because preadipocytes from sc fat depots are insensitive to ovariectomy in terms of cell proliferation and differentiation, it can be concluded that the ovarian status can site-specifically influence adipogenesis. Previous studies from our laboratory have shown that the ovarian status also exerts region-specific effects on the cAMP- and protein kinase C-dependent transducing pathways in mature fat cells (7, 8). The peculiar insensitivity of sc fat depots to ovarian status could be explained either by the low number of estrogen receptors in their adipose and preadipose cells (13, 14) or the low innervation and reduced blood flow of these depots compared to those of adipose tissue from other anatomical localizations.
Ovariectomy is followed by an increase in the serum concentration of several growth factors and hormones, such as epidermal growth factor, insulin-like growth factor I, and corticosterone (39, 40). Now, all of these factors are known to regulate adipogenesis (19, 20). For these reasons, it can be postulated: 1) that some, if not all, of these factors are at least in part involved in the increased intraabdominal fatness caused by ovariectomy in rats; and 2) that the effects of ovariectomy observed in vitro are probably due to modifications of the preadipocyte intrinsic properties caused by the in vivo hormonal deficiency.
To get information on the molecular mechanisms underlying the effects of ovariectomy on adipogenesis from deep intraabdominal preadipocytes, we studied p42/44 MAP kinase activity because this kinase is absolutely required for serum and insulin stimulation of preadipocyte DNA synthesis and differentiation (41).
In sham-operated rats, our results confirm the variability of the proliferation capacities of preadipocytes as a function of their anatomical origin (16, 17, 18). This variability does not seem to be related to marked variations in MAP kinase activity because no difference could be observed between the MAP kinase activities of preadipocytes from the three different fat depots studied. In addition, MAP kinase activity did not show any site-specific variation in differentiated preadipocytes, in contrast to the situation found in male rats, in which MAP kinase activity is about three times lower in perirenal than in femoral sc and epididymal differentiated cells (our unpublished results).
The stimulatory effect of ovariectomy on preadipocyte proliferation and differentiation specifically seen in preadipocytes from the perirenal fat depots is associated with a high MAP kinase activity. MAP kinase activation in response to growth factors has been shown to phosphorylate transcriptional factors, inducing rapid c-fos gene transcription and c-fos protein synthesis (29). This protooncogene, which plays a pivotal role in mitogenesis, also leads to enhanced expression of adipose tissue-specific genes such as the lipid-binding protein aP2 gene (30). In proliferating and differentiating perirenal preadipocytes, the high MAP kinase activity was indeed accompanied by a high c-fos expression level, suggesting that the positive modulatory effect of ovariectomy on adipose conversion in these cells is secondary to c-fos expression.
In parametrial cells, enhanced proliferation induced by ovariectomy is also associated with high c-fos protein induction. However, concomitant elevation of MAP kinase activity was not observed in these cells. The reasons for such discrepancies are presently unclear. Nevertheless, it cannot be excluded that in parametrial preadipocytes, as in Rat-1 fibroblasts (42), the pathways linking MAP kinase to c-fos induction are uncoupled.
In rat uterus, estrogens were shown to induce c-fos expression, whereas PG was reported to down-regulate c-fos expression (43). It thus seems difficult to establish the part played by both estrogens and P deficiencies in the alterations caused by ovariectomy on the MAP kinase/c-fos signaling pathways described above. In vitro experiments, in which steroids are present during the course of the primary culture are currently in progress in our laboratory to answer this question.
Besides protooncogene c-fos, there are other genes
implicated in the control of adipogenesis whose expression could be
altered by ovariectomy in proliferating parametrial and perirenal
cells. For example, c-myc, a protooncogene that enhances
preadipocyte proliferation but blocks differentiation, and C/EBP
, a
transcriptional factor that inhibits proliferation but triggers
preadipocyte-adipose conversion, are pivotal controlling elements of
adipogenesis (44). As regulation by sex hormones of c-myc
expression has been recently reported in ventral prostate tissue (43),
it appears likely that ovariectomy modifies the expression of these
factors. Experiments are in progress to test this hypothesis.
In conclusion, this study shows that ovariectomy site-specifically controls the in vitro proliferation and differentiation capacities of rat preadipocytes. Our results suggest that the obesity displayed by ovariectomized rats could at least in part result from deep intraabdominal adipose hyperplasia (increased proliferation in parametrial and perirenal preadipocytes and increased differentiation of perirenal preadipocytes). The present experiments, which complete our recent studies showing site-specific control of adipogenesis by the androgenic status as well (45), provide additional information on the regulation of adiposity by sex hormones.
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Footnotes |
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Received December 4, 1996.
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), OVX (
), and OVX, E2, and P (
)
rats were prepared and plated in 8% FBS-DMEM. At the indicated days,
cells were collected and counted. Data from one representative
experiment among four are presented. Each experiment was performed in
duplicate. *, P < 0.05.