p42/p44 Mitogen-Activated Protein Kinases Activation Is Required for the Insulin-Like Growth Factor-I/Insulin Induced Proliferation, but Inhibits Differentiation, in Rat Fetal Brown Adipocytes
Almudena Porras,
Alberto M. Álvarez,
Amparo Valladares and
Manuel Benito
Departamento de Bioquímica y Biología
Molecular II (A.P., A.V., M.B.) Instituto de Bioquímica
(Centro Mixto del Consejo Superior de Investigaciones
Científicas y de la Universidad Complutense) and Centro de
Citometría de Flujo y Microscopía Confocal (A.M.A.)
U.C.M.; Facultad de Farmacia Universidad Complutense Ciudad
Universitaria, 28040 Madrid, Spain
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ABSTRACT
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Insulin-like growth factor I (IGF-I)/insulin
induced cytosolic p42/p44 mitogen-activated protein kinase (MAPK)
activation in a time-dependent manner in fetal brown adipocytes,
reaching a maximum at 5 min. Concurrently, nuclear p42/p44 MAPKs were
also activated by IGF-I and insulin. This cytosolic and nuclear MAPK
activation was totally prevented by pretreatment with the MAPK kinase
(MEK1) inhibitor, PD98059. These results indicate that MEK mediates the
IGF-I/insulin-induced p42/p44 MAPK activation. IGF-I and insulin also
increased the number of cells in the S+G2/M
phases of the cell cycle, PCNA levels, and DNA synthesis at 24 h.
This IGF-I/insulin-induced proliferation was completely blunted by the
presence of MEK1 inhibitor. In contrast, inhibition of MEK1 potentiated
the IGF-I-induced uncoupling protein (UCP-1) and the insulin-induced
fatty acid synthase mRNAs expression after short and long-term
treatments. Moreover, transient expression of a transfected active MEK
construct (R4F) decreased IGF-I-induced UCP-1 and insulin-induced fatty
acid synthase mRNA expression. These results demonstrate that p42/p44
MAPKs are essential intermediates for the IGF-I/insulin-induced
mitogenesis, but may have a negative role in the regulation of
adipocytic and thermogenic differentiation in brown adipocytes.
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INTRODUCTION
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Mitogen-activated protein kinases (MAPKs) or extracellular
signal-regulated kinases (ERKs) are a family of serine/threonine
kinases activated by receptor tyrosine-kinases such as the
insulin-like growth factor I (IGF-I) and insulin receptors, cytokine
receptors, and seven spanning heterotrimeric G proteins (1, 2). Two
members of this family, p42 and p44 MAPKs, have been widely studied,
and a role for these kinases in the proliferation (3, 4, 5) or in the
differentiation signal transduction pathways (4, 6) has been proposed.
These MAPKs are activated by tyrosine and threonine phosphorylation
(7, 8). Upon activation, they can be translocated to the nucleus (9, 10) where they can phosphorylate transcription factors (10, 11).
IGF-I and insulin as well as other growth factors activate MAPKs in
different cell types (12, 13, 14, 15, 16) through MAPK kinase (MEK) activation
(17, 18), but the possible role for MAPKs in the regulation of
proliferation/differentiation balance is poorly understood. A role for
MAPKs in the regulation of fibroblast cell growth (3, 4, 5) has been
demonstrated. However, its role in the differentiation processes is not
so well defined, and although it has been proposed that MAPKs play a
role in the neuronal differentiation of PC12 cells (4, 6, 19) and in
the insulin-induced 3T3 L1 adipocyte differentiation (20), other data
indicate that sustained MAPK activation is not sufficient to determine
neuronal differentiation (21) and that MAPK cascade has a negative role
in the insulin- induced 3T3 L1 adipocyte differentiation (22).
Brown adipose tissue is specialized in heat production by the mechanism
called "non-shivering" thermogenesis (23) that is carried out by
the uncoupling protein (UCP-1). In particular, rat fetal brown
adipocytes already express UCP-1, and this expression continues after
birth (24). Different signals that induce the UCP-1 expression, such as
noradrenaline (25) and T3 (26, 27), seem to be involved in
the thermogenic differentiation. Insulin induces the lipogenic
differentiation in rat fetal brown adipocytes (28), while IGF-I is a
potent mitogen that also plays an important role in rat fetal brown
adipocytic differentiation (29, 30), particularly in the thermogenic
differentiation (31). However, the mechanisms mediating the IGF-I and
insulin effects are still poorly understood, although it is known that
Ras proteins play a crucial role in some IGF-I/insulin-signaling
pathways (13, 32, 33). In particular, Ras proteins mediate the
insulin/IGF-I induced adipocytic differentiation of 3T3 L1 cells (13, 32) and the IGF-induced UCP-1 expression in rat fetal brown adipocytes
(34, 35). Moreover, since Ras proteins mediate the IGF-I/insulin
induced MAPK activation in different cell types (13, 14, 15, 16, 36), this
IGF-I/insulin-Ras-MAPK cascade might have an important role in the
regulation of proliferation and/or differentiation in rat fetal brown
adipocytes. Therefore, we have characterized the IGF-I/insulin-induced
MAPK pathway and tried to establish its role on the balance between
proliferation and differentiation processes in rat fetal brown
adipocytes. We present evidence demonstrating that IGF-I and insulin
activate p42/p44 MAPKs through a MEK-dependent pathway and this
IGF-I/insulin-induced MAPK activation is a requirement for
proliferation, but inhibits differentiation in brown adipocytes.
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RESULTS
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Cytosolic p42/p44 MAPKs Are Activated by IGF-I and Insulin in Rat
Fetal Brown Adipocytes
IGF-I and insulin are known to activate MAPKs in different cell
types with a maximum activation after approximately 5 min of treatment
(12, 13); therefore, we wished to determine whether this was also true
for rat fetal brown adipocytes.
Cytosolic MAPK activity, determined by quantification of active p44/p42
MAPKs detected by Western blot analysis using an antiphospho-p44/p42
MAPKs antibody, was increased after treatment of the cells with
different concentrations of IGF-I or insulin for 5 min (Fig. 1
). Maximum value was reached with 20
nM to 1 µM insulin or 2.510 nM
IGF-I.

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Figure 1. Dose-Response of IGF-I and Insulin-Induced MAPK
Activity in Brown Adipocytes
Amount of active p44/p42 MAPKs in cytosolic lysates from rat fetal
brown adipocytes. Serum-starved cells were treated with different IGF-I
and insulin concentrations for 5 min as indicated, and cytosolic
extracts were obtained and analyzed by anti-phospho-MAPK Western blot
as described in Materials and Methods. Upper
panel, Representative Western blot analysis using 10 µg total
protein. Lower panel, Anti-phospho-MAPK Western blot
densitometric analysis. Results presented are normalized to the level
of control (C) untreated cells and are means ± SEM of
three different experiments. Autoradiograms were quantitated using
computer-assisted densitometry from Molecular Dynamics.
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Even though we measured MAPK activity after 5 min of treatment with
IGF-I or insulin, which is considered the point of maximum activation,
a complete time course study of the MAPK activity was carried out in
rat fetal brown adipocytes. MAPK stimulation by IGF-I and insulin
showed a fast kinetic of activation with a peak of activation at about
5 min of treatment (Fig. 2
), returning to
essential baseline values after 25 min to 2 h of treatment either
with IGF-I or insulin.

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Figure 2. Time Course of Induction of Cytosolic MAPK Activity
by IGF-I and Insulin in Brown Adipocytes
Amount of active p44/p42 MAPKs in cytosolic lysates from rat fetal
brown adipocytes. Serum-starved cells were treated with 2.5
nM IGF-I (A) or 20 nM insulin (B) for different
time periods as indicated, and cytosolic extracts were obtained and
analyzed by anti-phospho-MAPK Western blot as described in
Materials and Methods. Upper panels,
Representative Western blot analysis for IGF-I (A) and insulin (B)
using 10 µg of total protein. Lower panels, Western
blot densitometric analysis of anti-phospho-MAPKs. Results presented
are normalized to the level of control (C) untreated cells and are
means ± SEM of three separate experiments.
Autoradiograms were quantitated using computer-assisted densitometry
from Molecular Dynamics.
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Nuclear p42/p44 MAPKs Are Activated by IGF-I and Insulin Treatment
in Rat Fetal Brown Adipocytes
We also analyzed whether IGF-I or insulin could induce changes in
the nuclear p42/p44 MAPK activity. A time course of MAPK activity was
carried out in nuclear extracts from cells treated with IGF-I or
insulin (Fig. 3
). MAPK activity detected
in the nuclear fraction of untreated cells (control) was around
3040% that of the cytosolic fraction. Treatment of the cells with
IGF-I or insulin rapidly increased the levels of active MAPKs with a
peak around 5 min, decreasing to baseline levels after 1525 min of
treatment in both cases (Fig. 3
), although a certain amount of
phospho-MAPKs were detected after 35 h.

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Figure 3. Time Course of Induction of Nuclear MAP Kinase
Activity by IGF-I and Insulin in Brown Adipocytes
Amount of active p44/p42 MAPKs in nuclear extracts from rat fetal brown
adipocytes. Serum-starved cells were treated with 2.5 nM
IGF-I (A) or 20 nM insulin (B) for different time periods
as indicated. Nuclear extracts were obtained and analyzed by
anti-phospho-MAPKs Western blot as described in Materials and
Methods. Upper panels, Representative Western
blot analysis for IGF-I (A), using 10 µg of total protein, and for
insulin (B), using 20 µg of total protein. Lower
panels, Anti-phospho-MAPK Western blot densitometric analysis.
Results presented are normalized to the level of control (C) untreated
cells and are means ± SEM of three separate
experiments. Autoradiograms were quantitated using computer-assisted
densitometry from Molecular Dynamics.
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MEK1 Mediates the IGF-I and Insulin-Induced Activation of
Cytosolic and Nuclear p42/p44-MAPKs in Rat Fetal Brown Adipocytes
Since it has been described that MEK mediates the
p42/44mapk activation induced by several signals in
different cell types (2), we tried to determine whether MEK was also
upstream of MAPK in the IGF-I/insulin-induced MAPK cascade in rat fetal
brown adipocytes. To accomplish that, serum-starved cells untreated or
pretreated with the MEK1 inhibitor, PD98059 (20 µM), were
triggered with IGF-I or insulin for 5 min, and phospho-MAPK levels were
measured by Western blot in cytosol and nucleus.
As is shown in Fig. 4A
, IGF-I and insulin
increased cytosolic MAPK activity in the cells maintained in the
absence of the MEK1 inhibitor, while no effect was observed in cells
pretreated with this compound. Similarly, MEK1 inhibition blocked
insulin/IGF-I-induced p42/p44-MAPKs activation in the nucleus (Fig. 4B
).

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Figure 4. MEK1 Mediates the IGF-I and Insulin-Induced MAPK
Activation in Brown Adipocytes
Amount of active p44/p42 MAPKs in cytosolic (A) and nuclear extracts
(B) from rat fetal brown adipocytes. Serum-starved cells untreated or
pretreated with 20 µM of the MEK1 inhibitor, PD98059, for
1 h were treated with 2.5 nM IGF-I or 20
nM insulin for 5 min as indicated, and nuclear extracts
were obtained and analyzed by anti-phospho-MAPKs Western blot as
described in Materials and Methods. Upper
panels, Representative Western blot analysis of cytosolic
lysates (A), using 10 µg of total protein, and of nuclear extracts
(B), using 20 µg of total protein. Lower panels,
Anti-phospho-MAPK Western blot densitometric analysis. Results
presented are normalized to the level of control (C) untreated cells
and are means ± SEM of three separate experiments.
Autoradiograms were quantitated using computer-assisted densitometry
from Molecular Dynamics.
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Based on these results, MEK1 appears to be the essential upstream
element for p42/p44-MAPK activation in cytosol and nucleus in the
IGF-I/insulin-MAPK cascade in rat fetal brown adipocytes.
MAPK Cascade Is Responsible for IGF-I and Insulin-Induced
Proliferation in Rat Fetal Brown Adipocytes
IGF-I and insulin are involved in the regulation of proliferation
and differentiation in rat fetal brown adipocytes (29). Since an
increase in nuclear p42/44mapk activity was induced by
IGF-I and insulin, we tried to establish whether it might have a role
in the regulation of the cell cycle. To do that, the effect of the MEK1
inhibitor, PD 98059, on the IGF-I/insulin-induced proliferation was
assessed at 24 h, since this is the optimum time point for
measuring mitogenic effects in these cells. We measured cell cycle by
flow cytometry, PCNA levels by Western blot analysis, and DNA synthesis
by [3H]thymidine incorporation.
Results from flow cytometric analysis of the cell cycle under different
conditions are represented in Table 1
. Cells treated
with IGF-I for 24 h presented a statistically significant increase
(**, P < 0.01) in the percentage of cells in the
S+G2/M phases of the cell cycle. Similar results were obtained for
insulin (*, P < 0.05). However, the IGF-I/insulin
effect on mitogenesis was blunted by MEK inhibition.
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Table 1. Inhibition of the MAPK Cascade Inhibits the IGF-I and
Insulin-Induced Increase in the Percentage of Cells in S +
G2/M Phases of the Cell Cycle in Brown
Adipocytes
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Data obtained from quantification of PCNA amounts detected by Western
blot analysis (Fig. 5A
) corroborated
those from flow cytometric cell cycle analysis. PCNA levels were
significatively increased by IGF-I and insulin after 24 h of
treatment (**, P < 0.01). However, MEK 1 inhibition
abolished this effect (**, P < 0.01).

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Figure 5. Effect of the MEK1 Inhibitor PD98059 on PCNA
Expression and [3H]Thymidine Incorporation in Brown
Adipocytes
Rat fetal brown adipocytes were serum starved overnight and treated or
not with 20 µM of the MEK1 inhibitor PD98059 for 1 h
before the treatment with either 2.5 nM IGF-I or 20
nM insulin for 24 h. A, PCNA levels quantification by
Western blot analysis. Upper panel, Representative
anti-PCNA Western blot carried out in cell lysates using 50 µg of
total protein as described in Materials and Methods.
Lower panel, Anti-PCNA Western blot densitometric
analysis. Results presented are normalized to the level of control (C)
untreated cells and are means ± SEM of three separate
experiments. Autoradiograms were quantitated using computer-assisted
densitometry from Molecular Dynamics. B, [3H]Thymidine
incorporation into DNA. Assays were carried out as described in
Materials and Methods. Results presented are normalized
to the level of control (C) untreated cells and are means ±
SEM of three separate experiments. Mean control value was
480 cpm.
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Data from the [3H]thymidine incorporation assay (Fig. 5B
)
confirmed those from cell cycle analysis and PCNA quantification. IGF-I
and insulin significantly increased [3H]thymidine
incorporation (**, P < 0.01) when compared with
control value. However, this increase in DNA synthesis induced by
IGF-I/insulin was completely prevented by the presence of the MEK1
inhibitor (**, P < 0.01). Control value was also
decreased by pretreatment with this inhibitor. These results
demonstrate that p42/p44 MAPKs are required for the
IGF-I/insulin-induced proliferation in rat fetal brown adipocytes.
MAPK Cascade Plays a Negative Role in Adipocytic and Thermogenic
Differentiation in Rat Fetal Brown Adipocytes
IGF-I/insulin induces differentiation-related gene
expression in rat fetal brown adipocytes (28), IGF-I being specially
involved in the thermogenic differentiation of these cells (31). Since
IGF-I and insulin activated p44/42MAPKs in these cells, we wished to
determine the function of these kinases in the differentiation
processes. To assess the role of MAPKs on the thermogenic and
adipocytic differentiation, we studied the effect of the MEK1 inhibitor
PD98059 on the IGF-I-induced UCP-1 mRNA expression and on the
insulin-induced fatty acid synthase (FAS) mRNA expression after short-
(24 h) and long-term treatments (4 days).
As is shown in Fig. 6
, the presence of
the MEK1 inhibitor potentiated the IGF-I effect, increasing the UCP-1
mRNA expression at 24 h (Fig. 6A
) and 4 days (Fig. 6B
) (**,
P < 0.01). Moreover, MEK1 inhibition in control cells
induced a similar increase in the UCP-1 mRNA levels as the one induced
by IGF-I at 24 h and 4 days (*, P < 0.05).
Similarly, pretreatment of the cells with the MEK1 inhibitor
potentiated the insulin effect, increasing the FAS mRNA expression at
24 h (**, P < 0.01) and 4 days (*,P < 0.05). However, no significant effect of this inhibitor
was detected on control FAS mRNA expression. These results suggest that
the IGF-I/insulin-induced MAPK activation mediated by MEK attenuates
the signaling involved in differentiation in brown adipocytes.

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Figure 6. Effect of the MEK1 Inhibitor PD98059 on
IGF-I-Induced UCP-1 and Insulin-Induced-FAS Expression in Rat Fetal
Brown Adipocytes
Northern blot analysis of total RNA from rat fetal brown
adipocytes untreated or treated with 20 µM of the MEK1
inhibitor PD98059 for 1 h before treatment either with 2.5
nM IGF-I or with 20 nM insulin for 24 h
(A) or 4 days (B). Total RNA was extracted and submitted (58 µg) to
Northern blot analysis by sequential hybridization with specific probes
for UCP-1 (UCP) or FAS and 18S ribosomal probe as indicated in
Materials and Methods. Left panels,
Representative Northern blot analysis. Right panels,
Relative UCP-1 (UCP) or FAS expression normalized with the 18S
ribosomal probe. Results presented are means ± SEM of
three separate experiments. Autoradiograms were quantitated using
computer-assisted densitometry from Molecular Dynamics.
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To confirm that MAPK cascade activation plays a negative role in the
IGF-I/insulin-induced differentiation in brown adipocytes, transient
transfections of an active MEK mutant construct (R4F) were carried out,
and UCP-1 and FAS mRNAs were measured. As a control, transient
transfections with the empty vector were also carried out. To do that,
a brown adipocyte cell line (MB 4.8.2) previously generated (37) by
transfection with a SV40 LTag construct and a wild-type ras construct
was used, since transfection efficiencies in primary brown adipocytes
were lower than 1% and no effects of transfected R4F could be
detected.
As is shown in Fig. 7A
, untransfected control MB 4.8.2 cells presented certain levels of active
p42/p44-MAPKs (5- to 6-fold higher than those from primary cells).
These levels were slightly increased after treatment with IGF-I and
insulin for 24 h. Similar results were obtained in cells
transfected with the empty vector. When cells were transiently
transfected with the active MEK mutant (R4F) construct, the levels of
active p42/p44-MAPKs were significantly (***, P <
0.001) increased (8- to 10-fold). This increase in MAPK activity in
transfected cells did not modify the low levels of UCP-1 and FAS mRNAs
expressed in control cells (Fig. 7B
). However, the IGF-I and insulin
effects inducing UCP-1 or FAS mRNA were reduced by 50% when compared
with untransfected cells, respectively (**, P < 0.01).
Transfection with the empty vector did not affect IGF-I-induced UCP-1
or insulin-induced FAS mRNAs. Therefore, these results indicate that
MAPK cascade plays a negative role in the regulation of the
IGF-I/insulin-induced differentiation in brown adipocytes.

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Figure 7. Effect of the Expression of a Transfected Active
MEK Mutant Construct (R4F) on IGF-I-Induced UCP-1 and
Insulin-Induced-FAS Expression in the Brown Adipocyte Cell Line, MB
4.8.2.
A, Amount of active p44/p42 MAPKs in cytosolic lysates from MB 4.8.2
cells. Serum-starved untransfected (-R4F), transfected with the empty
vector (Vector) or R4F transfected (+R4F) cells were maintained
untreated or treated with either 2.5 nM IGF-I or with 20
nM insulin for 24 h, and cytosolic extracts were
obtained and analyzed by anti-phospho-MAPKs Western blot as described
in Materials and Methods. Upper panel,
Representative Western blot analysis using 8 µg of total protein.
Lower panel, Anti-phospho-MAPK Western blot
densitometric analysis. Results presented are normalized to the level
of control (C) untreated cells and are means ± SEM of
three separate experiments. B, Northern blot analysis of total RNA from
untransfected (-R4F), transfected with the empty vector (Vector) or
R4F transfected (+R4F) MB 4.8.2 cells under the same conditions
described in panel A. Total RNA was extracted and submitted (20 µg)
to Northern blot analysis by sequential hybridization with specific
probes for UCP-1 (UCP) or FAS and 18S ribosomal probe indicated in
Materials and Methods. Left panel,
Representative Northern blot analysis. Right panel,
Relative UCP-1 (UCP) or FAS expression normalized with the 18S
ribosomal probe. Results presented are means ± SEM of
three separate experiments. Autoradiograms were quantitated using
computer-assisted densitometry from Molecular Dynamics.
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It should be also noted that, in the brown adipocyte cell line, MB
4.8.2, 4-fold more total RNA was necessary for detection of FAS and
UCP-1 mRNAs by Northern blot analysis as compared with primary cells.
This fact might be a consequence of the high basal p42/p44 MAPK
activity found in these cells, which would inhibit expression of the
mRNAs differentiation markers under control conditions.
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DISCUSSION
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It is well established that MAPK activation induces fibroblast
cell growth (3, 4, 5). However, its role in the differentiation process is
not so well defined and some controversial data have been published.
For that reason, we wanted to figure out the role played by p42/44MAPKs
in the balance between proliferation and differentiation induced by
IGF-I/insulin in fetal brown adipocytes.
IGF-I and insulin induced cytosolic MAPK activation in brown adipocytes
as was previously described for other cell types (12, 13). The analysis
of nuclear MAPK activity in our system indicated that untreated cells
present a basal activity that represents around 3040% from that
found in the cytosol. This nuclear MAPK activity was quickly increased
by insulin and IGF-I. These results are very similar to those
previously obtained in Hela cells triggered with serum (9).
The upstream MAPK element in the MAPK cascade is MEK (MEK1 or MEK2) for
many signals in different cell types (17, 18). Our results also
demonstrate that this is the case for rat fetal brown adipocytes, since
the IGF-I/insulin-induced MAPK activation in cytosol and nucleus was
completely prevented by the MEK1 inhibitor, PD 98059. In particular,
based on the inhibitor dose needed to block the pathway, MEK1 is the
mediator of MAPK activation. Therefore, MEK1 appears to be the
essential upstream element in the IGF-I/insulin-MAPK cascade in brown
adipocytes.
Cell cycle analysis revealed that IGF-I and insulin induce the entry
and progression through the cell cycle in a MAPK-dependent manner.
Moreover, DNA synthesis and quantification of PCNA levels also indicate
that MAPK activation is necessary for the IGF-I/insulin-induced
proliferation. The first peak of MAPK activation (540 min) might be
associated with the G0/G1 transition and even progression through G1
phase, similar to what has been described for synchronized Swiss mouse
3T3 cells, where an increase in MAPK activity is produced in the G0/G1
boundary (38). However, a role for MAPKs in other cell cycle phases
cannot be excluded. In fact, it has been suggested that MAPKs would be
important in cells progressing from M into G1 after release from a
metaphase block (by 2426 h) (38). Other data also support that MAPKs
can play different roles in the regulation of the different phases of
the cell cycle (39, 40).
The role for p44/p42MAPKs in the regulation of differentiation in
different cell types is not well established. In PC12 cells, some data
demonstrated that the sustained activation of the MAPK cascade induced
neuronal differentiation (4, 6, 19), while some others indicated that
this sustained MAPK activation was not sufficient to determine neuronal
differentiation in PC12 cells (41) or in H197 cells (21). Regarding
the adipocytic differentiation, some controversial data have also
emerged concerning the possible role for MAPKs in the
IGF-I/insulin-induced differentiation, particularly in 3T3 L1 cells.
These cells are able to differentiate into adipocytes after exposure to
physiological doses of IGF-I or pharmacological doses of insulin (42).
Ras proteins mediate this IGF-I/insulin-induced differentiation (13, 32), glucose transport (43), and MAPK activation (13, 17, 36).
Therefore, the Ras/MAPK cascade or other Ras downstream pathways can be
the mediators of the adipocytic differentiation. In fact, a study done
using an antisense RNA against MAPK indicated that MAPK activation was
necessary for the insulin-induced 3T3 L1 adipocyte differentiation
(20). However, using other technical approaches, it has been
demonstrated that p42/p44MAPKs play a negative role in the regulation
of the IGF-I/insulin-induced differentiation of 3T3 L1 cells (22).
Moreover, another report (44) suggested that PI3K, another Ras
downstream parallel pathway (45), was involved in this process.
In fetal brown adipocytes, insulin plays an important role in the
adipocytic differentiation (28, 29, 30). IGF-I is also involved in the
adipocytic and thermogenic differentiation of these cells (29, 31),
with a preferential role inducing the expression of the UCP-1 gene,
which is the thermogenic differentiation marker (31). Since Ras
proteins are the mediators of the IGF-I-induced UCP-1 expression (34, 35) and Ras proteins mediate MAPK activation by IGF-I and insulin in
different cell types, the rationale would be that MAPKs might be the
downstream intermediates in the IGF-I/insulin differentiation signaling
pathway. However, results presented here show that the MEK inhibition
results in a potentiation of the IGF-I-induced UCP-1 mRNA expression
and insulin-induced FAS mRNA expression. The inhibition of the MAPK
cascade in control cells produced an increase in the expression of
these adipocytic and thermogenic differentiation markers, although it
was only statistically significant for UCP-1. Moreover, high levels of
active MAPKs, obtained by means of transfection of an active MEK mutant
(R4F) in a brown adipocyte cell line, decreased the IGF-I-induced UCP-1
expression and the insulin-induced FAS expression. Additionally, the
relatively high levels of active MAPKs present in this brown adipocyte
cell line before transfection with R4F might explain the low endogenous
expression of UCP-1 and FAS mRNAs. Therefore, these results indicate
that the activation of the MAPK pathway results in attenuation of the
adipocytic and thermogenic differentiation in brown adipocytes. These
results are in agreement with data from Hu et al. (46).
These authors found that insulin, a potent mitogen for Rat-IR cells,
induced the phosphorylation of the transcription factor, peroxisome
proliferator-activated receptor
, which resulted in a reduction of
its transcriptional activity and its ability to activate the adipocytic
differentiation. The inhibition of MEK by PD98059 completely prevented
peroxisome proliferator-activated receptor-
phosphorylation, leading
those cells to adipocytic differentiation in response to insulin. Thus,
these authors suggested that MAPKs might be very important to balance
cell growth and differentiation. In our system, fetal brown adipocytes,
which are able to proliferate and differentiate in primary culture,
p42/44MAPK activation might also balance these two processes. Thus,
depending on the extension of the activation of MAPKs and other
signaling pathways, these cells could establish a balance between
proliferation and differentiation. Upon IGF-I/insulin binding to their
receptors, an activation of MAPK cascade is produced that induces
progression of the cells through the cell cycle, while the parallel
PI3K activation is necessary for differentiation (47). The balance
between these pathways might determine whether the cells proliferate
and/or differentiate. Thus, when the MAPK cascade is inhibited by the
MEK1 inhibitor PD98059, brown adipocytes would be preferentially
committed to perform differentiation in response to IGF-I/insulin.
However, high levels of active p42/p44 MAPKs would preferentially
induce proliferation blocking the signaling that leads to
differentiation.
In conclusion, IGF-I and insulin induce mitogenesis in brown adipocytes
in a MAPK-dependent manner. Inhibition of p42/p44 MAPKs is sufficient
to arrest cells and potentiate adipocytic and thermogenic
differentiation induced by IGF-I/insulin. Conversely, a high level of
active p42/p44 MAPKs maintains cell proliferation but attenuates
IGF-I/insulin-induced differentiation.
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MATERIALS AND METHODS
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Isolation of Fetal Brown Adipocytes and Culture
Fetal brown adipocytes from 20-day-old rat fetuses were isolated
as described (24) and maintained in primary cultures. Cells were grown
to 90% confluency in MEM supplemented with 10% FCS. Cells were
starved overnight and triggered with IGF-I (1.410 nM) or
insulin (1 nM to 1 µM) when indicated, in the
presence or in the absence of the MEK1 inhibitor PD98059 from New
England Biolabs (Beverly, MA; 9900L) (6, 48). In this case, cells were
pretreated for 1 h with 20 µM of PD98059 although a
complete MEK inhibition was obtained by using a 10 µM
concentration after which the growth factors were added.
Culture of MB4.8.2 Brown Adipocyte Cell Line and Transfection
Assays
MB 4.8.2 brown adipocyte cell line was grown in DMEM
supplemented with 10% FCS. Cells were serum-starved overnight and
maintained in the absence of serum when IGF-I and insulin effects on
RNA expression or MAPK activity were studied.
Transient transfections were carried out using the MBS mammaliam
transfection kit (catalog no. 35 200388) from Stratagene (La Jolla, CA)
following the protocol supplied by the manufacturer. Cells were
transfected with an active MEK mutant construct, pMCL-MKK1 (R4F),
generously provided by Dr. N. Ahn (22).
Transfection efficiencies ranged between 3060% as determined by the
ß-galactosidase histochemical staining assay and by quantification of
active p42/p44 MAPKs levels.
Preparation of Cytosolic and Nuclear Extracts
Cytosolic and nuclear extracts were prepared essentially as
described previously (49). Nuclear extracts were examined for cytosolic
contamination by measuring the presence of malic enzyme by Western blot
analysis. Less than 1% of malic enzyme was present in these
extracts.
p44/p42-MAPK Activities Assay: Phospho-MAPK Western Blot
Analysis
p44/p42-MAPK activities in cytosolic and nuclear extracts were
determined by quantification of active MAPK levels by Western blot
analysis using an anti-phospho-MAPK antibody from Promega (V6671;
Madison, WI). Western blot analysis was carried out essentially as
described (13, 16, 50), using 10% SDS-polyacrylamide gels. Blots were
developed using the enhanced chemiluminescence system (Amersham,
Arlington Heights, IL).
Flow Cytometric Analysis
Analysis of the cell cycle was performed in a FACScan flow
cytometer (Becton-Dickinson, San José, CA). The percentages of
cells in Go/G1, S, or G2+M phases of the cell cycle were determined by
staining cellular DNA with propidium iodide using the Bio-Rad
(Richmond, CA) reagent kit (Kinesis 50, No. 470-0023), following the
manufacturers protocol. Measurements were carried out using a Double
Discriminator Module to discriminate doublets; 10,000 cells were
acquired per sample.
PCNA Western Blot Analysis
PCNA Western blot analysis was carried out using a monoclonal
anti-PCNA antibody from Boehringer Mannheim (Indianapolis, IN)
following the same procedure described for MAPKs, but running the
electrophoresis on 12% SDS-polyacrylamide gels.
[3H]Thymidine Incorporation Assay
Cells were incubated with [3H]thymidine (0.5
µCi/ml) for the last 4 h of stimulation with either IGF-I or
insulin. Radioactivity present in trichloroacetic acid-insoluble
material was quantitated in a ß-counter.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted with RNazol B (Biotecx Laboratories,
Houston, TX) following the protocol supplied by the manufacturer.
Northern blot analysis was carried out as previously described (25, 26). For hybridization with different probes, the blots were stripped
and rehybridized sequentially as needed in each case. Probes used
included UCP-1 (51), FAS (52), and 18S ribosomal to normalize.
Autoradiograms were quantitated by using a computer-assisted
densitometry from Molecular Dynamics (Sunnyvale, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Eugenio Santos for the critical reading of the
manuscript, Dr. Natalie Ahn for providing MEK constructs and for her
critical advice and Amalia Vázquez for the flow cytometry
analysis.
 |
FOOTNOTES
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Address requests for reprints to: Manuel Benito, Departamento de Bioquimica y Biologia Molecular II, Instituto de Bioquimica, Facultad de Farmacia, Universidad Compultense, Madrid, Spain 28040.
This work has been supported by a Grant (SAF96/0115) from CYCIT
(Comisión Interministerial de Ciencia y Tecnología from
Spain).
Received for publication October 23, 1997.
Revision received February 18, 1998.
Accepted for publication March 2, 1998.
 |
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