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Service de Biochimie, INSERM CJF 94–02, Faculté de Médecine Paris-Ouest, Université René Descartes (Paris V) and Centre Hospitalier de Poissy 78 303 Cédex, France
Address all correspondence and requests for reprints to: D. Lacasa, Centre Hospitalier, Service de Biochimie Medicale, 10, rue du Champ-Gaillard, Poissy Cedex, 78303 France.
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Abstract |
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Therefore, the influence of androgenic status on the Fos protein induction and the Raf/mitogen-activated protein (MAP) kinase kinase (MEK)/MAP cascade, which are all required for adipose conversion of preadipocytes, was compared in proliferating and differentiated preadipocytes from femoral sc and deep intraabdominal (epididymal and perirenal) fat depots.
In epididymal and perirenal proliferating preadipocytes, increased proliferation due to castration is associated with increased MAP kinase activity. However, higher immunoreactive levels of the upstream activators of MAP kinase, Raf-1 and MEK, were observed only in epididymal cells. Moreover, in vivo testosterone treatment corrected the effects of castration on Raf-1 but not on MEK and MAP kinase.
MAP kinase activity was decreased during the course of adipogenesis. In differentiated cells, MAP kinase activity showed variations according to the anatomical origin of preadipocytes but not to the androgenic status. In contrast, MEK and Raf-1 immunoreactive levels were both sensitive to androgenic status but were differently affected depending on cell origin. Finally, the defective adipogenesis seen in epididymal preadipocytes from castrated rats was associated with reduced Fos protein induction in these cells, an alteration which was partly corrected by testosterone-treatment.
Taken together, these results suggest that androgenic status affects adipogenesis from deep intraabdominal preadipocytes through alterations of some components of the MAP kinase cascade/Fos signaling pathways.
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Introduction |
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The molecular basis of the regulation of adipose tissue distribution by sex hormones is still poorly understood. Direct effects of androgens on adipose tissue are suggested by the presence of androgen receptors in precursor and mature fat cells (2, 3).
Increased adipogenesis (preadipocyte proliferation and differentiation) is one important mechanism contributing to the development of obesity. The influence of both the anatomical origin (4, 5) and the hormonal status (6) on adipogenesis can be studied in vitro by means of primary culture of preadipocytes. By using this experimental approach, we have recently shown that castration exerts site-specific effects on adipogenesis. Namely, an increased proliferation capacity and a loss of differentiation capacity were observed in epididymal preadipocytes from castrated rats (7). In contrast, adipose conversion of preadipocytes from sc territories was insensitive to castration (7).
The mitogen-activated protein kinase (MAP kinase) cascade is activated by growth factors such as EGF and insulin and plays a crucial role in cell proliferation and differentiation (for a review, see 8 . Two isoforms of MAP kinase (p42 and p44 MAP kinases) are activated by phosphorylation of their tyrosine 185 and threonine 183 residues by MAP kinase kinases (MEKs). MEKs are themselves activated by several kinases such as Raf-1 (9).
MAP kinase activation by growth factors results in phosphorylation of transcriptional factors which in turn, induces a rapid increase in c-fos expression (10). This proto-oncogene plays a pivotal role in the control of preadipocyte proliferation and differentiation (11).
Altered MAP kinase cascade/Fos protein signaling pathways can thus be considered as one possible mechanism explaining the increased proliferation and defective differentiation capacities of rat epididymal preadipocytes after castration. This hypothesis was presently tested by comparing the effects of the androgenic status on Raf/MEK/MAP kinase and Fos protein induction in confluent and differentiated preadipocytes from rat sc, epididymal, and perirenal fat depots.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PKC were obtained from Life
Technologies (Grand Island, NY). The antisera and synthetic peptides
specific for Raf-1 (C12) and MEK1 (C18) were from Santa-Cruz
Biotechnology (Santa-Cruz, CA). The antibody and synthetic peptide
specific for c-fos (OP17) were from Oncogene Science (Cambridge, MA).
Western blotting protocols and p42/p44 MAP kinase enzyme assay system
were from the Radiochemical Centre (Amersham, Buckinghamshire, UK).
Prestained molecular weight markers were from Sigma Chemical Co. (St.
Louis, MO). Testosterone RIA tests were provided by Biomérieux
(Marcy l’Étoile, France). All other chemicals were of
reagent grade.
Methods
Animals.
Procedures with experimental animals are authorized
and follow the guidelines of the Ministère of Agriculture
(France) (authorization 006614). Male Sprague-Dawley rats (125–150 g)
were castrated under pentobarbital anesthesia (40 mg/kg ip) and treated
as previously described (7, 12). Briefly, 5 days after the operation,
half of the castrated rats received one sc injection of testosterone
propionate (0.5 mg/100 g BW) every other day for 10 days (CAST + T)
while the other half (CAST) and the sham-operated rats (SHAM) received
the vehicle (polyethylene glycol) only. One day after the last
injection, rats were killed by decapitation. Serum testosterone levels
were measured as described in (7) and were on the day of sacrifice:
1.5 ± 0.2, less than 0.1 and 13.5 ± 3.7 nmol/liter in SHAM,
CAST and CAST + T rats. The characteristics of the adipose tissues of
the different experimental groups were given in (12).
Cell culture
Cell preparation and culture were performed as described in Ref.
13. Briefly, preadipocytes obtained from the stroma-vascular fraction
of adipose tissue by collagenase digestion were plated at a density of
1–2 104 cells/cm2 in cell culture dishes.
After 12 h, cells were washed and fed with DMEM-8% FBS. Medium
was changed every other day. At confluence, (2–3 days postplating),
cells were harvested or allowed to differentiate in DMEM-Ham’s F12
medium (50:50) containing 5 µg/ml insulin, 10 µg/ml transferrin,
and 200 pM T3 (ITT medium) (13). Whatever the anatomical
origin, 80% at least of the cultured control cells were fully
differentiated at day 8–10 postconfluence.
Cellular extract preparation
Preadipocyte cytosolic and membrane fractions were prepared as
described in (12). For c-fos assay, preadipocytes were
serum-starved for 18 h and then exposed to 10% serum for 90 min.
Then, cells were scraped and sonicated in cold buffer containing 50
mM Tris, pH 8.0, 120 mM NaCl, 1% NP40, 0.5%
deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulfonyl
fluoride, 25 µg/ml aprotinin and 20 µg/ml leupeptin. After
centrifugation at 100,000 x g for 15 min at 4 C, the
resulting supernatant was denaturated with Laemmli buffer and stored at
-20 C.
Western blot analysis
Equal amounts (10–50 µg) of cellular fractions and prestained
molecular weight markers were subjected to SDS-PAGE (10–12.5%).
Proteins were transferred to polyvinylidenedifluoride membranes. The
filters were subsequently stained to verify equal protein loading and
transfer. After blocking with TBS containing 1% Tween-20 (TTBS) and
2.5% gelatin for 2 h, membranes were incubated overnight with the
antibodies diluted in TTBS-2.5% gelatin (anti-Raf-1,
anti-c-fos and anti- PKC at 0.5 µg/ml and anti-MEK at
0.1 µg/ml). Membranes were washed and incubated with the secondary
antiserum coupled to horse radish peroxydase (1:2000 dilution in TTBS).
Membranes were extensively washed, incubated with the ECL detection
solution and then exposed to x-ray films. Signals were quantified by
densitometry. Reprobing of the membranes gave identical results.
Specificity of the immunoreactive proteins was verified by loss of the
immunoreactivity of samples when incubated with the antiserum
neutralized by the corresponding specific peptide. Bands presented in
the figures are the only ones visible on the films. Control experiments
with various amounts of protein (10–75 µg) were performed to ensure
that the densitometric signal intensity was proportional to the loaded
amount of protein.
MAP kinase assay
Proliferating and differentiated preadipocytes were scraped and
sonicated in cold buffer containing 10 mM Tris, pH 7.4, 150
mM NaCl, 2 mM EGTA, 2 mM DTT, 1
mM sodium orthovanadate, 30 mM
ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride,
20 µg/ml leupeptin, and 25 µg/ml aprotinin. After centrifugation at
100,000 x g for 30 min at 4 C, cytosolic extracts were
kept at -80 C. MAP kinase activity was measured in the extracts using
p42/p44 MAP kinase enzyme assay system as described in Ref. 14.
Cell counting
Perirenal preadipocytes from SHAM, CAST, and CAST + T rats were
isolated and seeded at 1 105 cells per well. Cell number
was determined at days 1, 2, 3, and 4 postplating. Cells were
trypsinized and counted in a hemocytometer. Cell viability experiments
showed that, whatever the animal treatments, about 95% of the cells
excluded trypan blue after three washings.
Glycerol 3-phosphate deshydrogenase (GPDH) assay
Cell differentiation was followed by measuring the GPDH activity
according to Wise and Green (15).
Other determinations
Protein concentration and lactate dehydrogenase activity were
assessed as previously described (7). All results are expressed as
means ± SEM from at least four 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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compare the influence of rat
androgenic status on the in vitro proliferation and
differentiation capacities of preadipocytes from the perirenal fat
depots. As can be seen, castration increased the number of cells at day
4 by about 70% (P < 0.05) (mean of four separate
experiments). This effect was not corrected by testosterone treatment.
Moreover, castration increased by a factor of 2 the GPDH activity, a
late marker of adipogenesis (15). LDH activity which remained stable
throughout the differentiation process was unaffected by the androgenic
status. Thus perirenal preadipocytes differ from epididymal
preadipocytes where the differentiation process is completely inhibited
after castration (7).
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p42/p44 MAP kinase activity
First, p42/p44 MAP kinase activity was measured in cytosolic
extracts of confluent and differentiated cells.
As shown in Table 1, the MAP kinase activities found in
proliferating preadipocytes from the control groups were identical
whatever their anatomical origin. In contrast, castration induced a
significant increase in the MAP kinase activity of proliferating
preadipocytes from the two deep intraabdominal fat depots but not from
the sc region. In vivo treatment by testosterone failed,
however, to reverse these effects of castration.
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Immunoreactive MEK status
As shown in Fig. 2, the amount of MEK (determined
by Western blot) in preadipocyte cytosolic fractions was sensitive to
castration only in epididymal preadipocytes where it increased by
150 ± 3% (P < 0.05) at confluence and by
125 ± 24% (P < 0.05) at differentiation. In
these cells, however, in vivo testosterone treatment failed
to restore to normal MEK amount. In contrast, MEK amounts were not
significantly modified by the androgenic status in sc and perirenal
pre-adipocytes whatever their stage of culture.
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At confluence, cytosolic amount of Raf-1 was increased by castration in
preadipocytes from epididymal (+ 75 ± 30%, P <
0.05) but not from the other fat depots. In contrast, in differentiated
preadipocytes, castration increased Raf-1 amount only in perirenal (+
70 ± 30%, P < 0.05) and sc (+150 ± 40%,
P < 0.05) cells and testosterone treatment failed to
correct these alterations (Fig. 3).
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-PKC status
Finally, PKC status was also examined as this isoform was
recently shown to activate the MEK/MAP kinase cascade (17). -PKC
cytosolic amount was unmodified by the androgenic status whatever the
anatomical origin of the preadipocytes and their stage of culture (data
not shown). These data indicate that the androgenic status affects
specifically some but not all components of the MAP kinase signaling
pathway.
Fos protein induction
Induction of the proto-oncogene c-fos by serum was
studied by following the expression of immunoreactive Fos protein in
confluent and differentiated preadipocytes.
A Fos immunoreactive protein of about 50 kDa was detected in all
experimental groups after 1–2 h serum induction. Data presented in
Fig. 4 show that in confluent
pre-adipocytes, Fos protein induction was not significantly altered
by the androgenic status whatever the anatomical origin of the cells.
In contrast, in differentiated preadipocytes, castration reduced Fos
protein induction by almost half (-40 ± 10%, P
< 0.05) in epididymal cells, an alteration which was partly restored
by in vivo testosterone treatment. In differentiated
preadipocytes from the other anatomical origins, however, androgenic
status failed to apparently affect Fos induction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The important fact that castration can influence in two opposite
ways the adipogenic process in deep intraabdominal preadipocytes
depending on their anatomical origin is presently unexplained. However,
this demonstrates that precursor fat cells from deep intraabdominal fat
depots are not comparable at least in terms of the reactivity of their
adipogenic capacities to androgenic status. Perirenal fat depots
contain more differentiating preadipocytes than other depots (4, 5).
Moreover, neither an activator of adipogenesis like 2-bromopalmitate
(18) nor an antiadipogenic factor like TNF
(19) is able to influence
adipose conversion of perirenal preadipocytes (Dieudonné MN
et al., personal communication). These findings strongly
suggest that perirenal preadipocytes are more highly committed in the
in vivo/in vitro differentiation process than
those from the epididymal site. The opposite effects of androgen
deprivation on perirenal and epididymal preadipocyte adipose conversion
could then be explained if, as described for glucocorticoids (20), one
assumes that modulation of adipogenesis by androgens depends on a
critical time of exposure.
To get more informations on the molecular mechanisms underlying the effects of androgenic status on adipogenesis of intraabdominal preadipocytes, we have studied some elements of the MAP kinase cascade/Fos signaling pathways which are involved in preadipocyte proliferation and differentiation (11, 21).
In control rats, our results suggest that the variability of the proliferative capacity as a function of the preadipocyte anatomical origin (4, 5) is not related to differences in the MAP kinase cascade. In fact, p42/p44 MAP kinase activities and immunoblot levels of two upstream activators, MEK and Raf-1, were found to be similar in preadipocytes isolated from the three different fat depots studied.
The proliferative effect of castration specifically seen in perirenal and epididymal preadipocytes, is associated with high MAP kinase activity. It should be noted, however, that increased MEK and Raf-1 protein expression after castration were observed only in epididymal cells. The reasons explaining these site-related specificities remain to be established.
In various cell types, MAP kinase activation in response to growth factors has been found to induce rapid c-fos expression (10). As presently shown, however, the high MAP kinase activity observed in proliferating preadipocytes from epididymal and perirenal fat depots was not accompanied by a high Fos protein induction. It cannot be excluded that in these cells, like in Rat-1 fibroblasts (22), the pathways linking MAP kinase to c-fos induction are uncoupled.
On the other hand, MAP kinase is positively regulated by cAMP in preadipose cell lines (23) and in rat preadipocytes (unpublished results). However, castration was shown by us to induce defective cAMP production in rat epididymal preadipocytes (24). Therefore, the high MAP kinase activity found in epididymal preadipocytes is probably more the consequence of increased function of the tyrosine kinase/growth factor signaling pathways than of the cAMP-mediated one.
Several studies have demonstrated that high MAP kinase activity leads to increased transcription of proliferation related genes (for reviews, see Refs. 25, 26). One candidate gene could be c-myc, which plays a crucial role in preadipocyte mitogenesis (27) and whose expression is also induced after MAP kinase activation (28). Experiments are currently in progress to test this hypothesis.
In differentiated preadipocytes, MAP kinase activity was found 5- to 10-fold lower than in proliferating cells and was shown to be variable according to preadipocyte anatomical origin. The lowest MAP kinase activity was observed in perirenal cells which have a high differentiation capacity (4–5, 29, this study).
MAP kinase activity in differentiated preadipocytes was insensitive to the androgenic status whatever the anatomical origin of these cells. This could be explained by the finding that this activity sharply decreased during adipogenesis, and also by the observation, that MAP kinase is predominantly involved in the control of the proliferation and less in the differentiation process of 3T3-L1 preadipocytes (30).
Proto-oncogene c-fos has been shown to play a pivotal role
in preadipocyte proliferation by promoting the expression of
adipo-specific genes such as the lipid binding protein aP2 (11).
Therefore, the decreased Fos protein induction seen in epididymal cells
after castration could explain, at least in part, how castration
induces a defective adipogenesis in these cells. In contrast, the high
differentiation capacity elicited by perirenal cells was not correlated
with variations of Fos protein induction. Besides c-fos,
other transcriptional factors are deeply implied in the control of
adipogenesis. Among these factors, C/EBP, which is highly expressed
in adipose tissue, is important because its expression is necessary and
sufficient for adipocyte differentiation commitment (31). That
castration could influence the expression of C/EBP in preadipocytes
seems all the more likely as preliminary experiments from our
laboratory reveal that C/EBP expression is differently affected by
castration in epididymal and perirenal preadipocytes as well.
Finally, few studies have been devoted to androgen regulation of nuclear proto-oncogene transcription (32). It is thus difficult to establish whether the alterations caused by castration on the MAP kinase/c-fos signaling pathways are due or not to androgen deficiency. The question of a direct effect of testosterone on preadipocytes remains open. Androgen receptor number exhibits variations according to the anatomical origin of rat preadipocytes (2, 3). This number is decreased in castrated rats and is up-regulated both in vitro (2, 3) and in vivo (Dieudonné et al., unpublished results) by testosterone. The fact that, in vivo, testosterone treatment did not correct some of the alterations induced by castration (growth rate and MAP kinase activity) indicates that these alterations cannot be fully accounted for testosterone deficiency. These observations rather suggest that lack of some products of the testis other than testosterone (steroidal and non steroidal testicular factors) could play a significant role in these effects of castration. Additional in vitro experiments will be required to test this hypothesis.
In conclusion, this study shows that androgenic status influences site-specifically the proliferation and differentiation capacities of the deep intraabdominal preadipocytes. These influences appear to be at least in part related to alterations of some components of the signaling pathways controlling adipogenesis. Abdominal fat mass development being inversely correlated with blood testosterone levels in man (1), this report showing hyperplasia (increased proliferation and differentiation) of perirenal preadipocytes after castration could provide additional information on the regulation of deep intraabdominal fat mass deposition by sex hormones.
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Footnotes |
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Received October 21, 1996.
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. Science 256:379–382This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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), CAST (
) and CAST + T (
)
rats were prepared and plated in DMEM-8%FBS. At the indicated days,
cells were counted. Data from one representative experiment repeated
four times are presented. Each experiment was performed in duplicate.
P < 0.05 comparison between preadipocytes from
CAST and SHAM rats using the one-way ANOVA. B, Perirenal preadipocytes
from SHAM, CAST, and CAST + T rats were allowed to differentiate in ITT
medium. After 8–10 days, GPDH and LDH activities were measured in
cytosolic fractions. Data are means ± SEM of 10–12
separate experiments performed in duplicate. *, P
< 0.01 comparison between preadipocytes from CAST and SHAM rats using
the one-way ANOVA
