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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). Maximum value was reached with 20 nM to 1 µM insulin or 2.5–10 nM IGF-I.



View larger version (44K):
[in this window]
[in a new window]
 
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.

 
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. 2Go), returning to essential baseline values after 25 min to 2 h of treatment either with IGF-I or insulin.



View larger version (43K):
[in this window]
[in a new window]
 
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.

 
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. 3Go). MAPK activity detected in the nuclear fraction of untreated cells (control) was around 30–40% 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 15–25 min of treatment in both cases (Fig. 3Go), although a certain amount of phospho-MAPKs were detected after 3–5 h.



View larger version (42K):
[in this window]
[in a new window]
 
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.

 
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. 4AGo, 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. 4BGo).



View larger version (24K):
[in this window]
[in a new window]
 
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.

 
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 1Go. 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.


View this table:
[in this window]
[in a new window]
 
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

 
Data obtained from quantification of PCNA amounts detected by Western blot analysis (Fig. 5AGo) 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).



View larger version (35K):
[in this window]
[in a new window]
 
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.

 
Data from the [3H]thymidine incorporation assay (Fig. 5BGo) 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. 6Go, the presence of the MEK1 inhibitor potentiated the IGF-I effect, increasing the UCP-1 mRNA expression at 24 h (Fig. 6AGo) and 4 days (Fig. 6BGo) (**, 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.



View larger version (47K):
[in this window]
[in a new window]
 
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 (5–8 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.

 
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. 7AGo, 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. 7BGo). 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.



View larger version (54K):
[in this window]
[in a new window]
 
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.

 
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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 30–40% 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 (5–40 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 24–26 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 H19–7 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 {gamma}, 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-{gamma} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (1.4–10 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 30–60% 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 manufacturer’s 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
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Blumer KJ, Johnson GL 1994 Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci 19:236–240[CrossRef][Medline]
  2. Cobb MH, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  3. Pagès G, Lenormand P, L’Allemain G, Chambard JC, Meloche S, Pouyssegur J 1993 Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319–8323[Abstract/Free Full Text]
  4. Cowley S, Paterson H, Kemp P, Marshall CJ 1994 Activation of MAP Kinase Kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77:841–852[Medline]
  5. Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ann NG 1994 Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966–970[Medline]
  6. Pang L, Sawada T, Decker SJ, Saltiel A 1995 Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem 270:13585–13588[Abstract/Free Full Text]
  7. Anderson NG, Maller JL, Tonks NK, Sturgill TW 1990 Requirement for integration of signals from two distinct pathways for activation of MAP kinase. Nature 343:651–653[CrossRef][Medline]
  8. Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, Depinho RA, Panayotatos N, Cobb MH, Yancopoulos GD 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663–675[Medline]
  9. Chen R-H, Sarnecki Ch, Blenis J 1992 Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12:915–927[Abstract]
  10. Davis RJ 1993 The Mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556[Free Full Text]
  11. Hill CS, Treisman R 1995 Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80:199–211[Medline]
  12. Ray LB, Sturgill TW 1987 Rapid stimulation by insulin of a serine/threonine kinase in 3T3–L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc Natl Acad Sci USA 84:1502–1506[Abstract]
  13. Porras A, Nebreda AR, Benito M, Santos E 1992 Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation. J Biol Chem 267:21124–21131[Abstract/Free Full Text]
  14. De Vries-Smits AMM, Burgering BMT, Leevers SJ, Marshall CJ, Bos JL 1992 Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature 357:602–604[CrossRef][Medline]
  15. Medema RH, De Vries-Smits AM, Van der Zon GCM, Maasen JA, Bos JL 1993 Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras. Mol Cell Biol 13:155–162[Abstract]
  16. Porras A, Muszynski K, Rapp UR, Santos E 1994 Dissociation between activation of Raf-1 kinase and the 42-kDa Mitogen-activated protein kinase/90-kDa S6 kinase (MAPK/RSK) cascade in the insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells. J Biol Chem 269:12741–12748[Abstract/Free Full Text]
  17. Nakielny S, Cohen P, Wu J, Sturgill T 1992 MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J 11:2123–2129[Abstract]
  18. Langer-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260:315–319[Medline]
  19. Fukuda M, Gotoh Y, Tachibana T, Dell K, Hattori S, Yoneda Y, Nishida E 1995 Induction of neurite outgrowth by MAP kinase in PC12 cells. Oncogene 11:239–244[Medline]
  20. Sale EM, Atkinson PGP, Sale GJ 1995 Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J 14:674–684[Abstract]
  21. Kuo W-L, Abe M, Rhee J, Eves EM, McArthy SA, Yan M, Templeton DJ, McHahon M, Rosner MR 1996 Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells. Mol Cell Biol 16:1458–1470[Abstract]
  22. Font de Mora J, Porras A, Ahn N, Santos E 1997 Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3–L1 adipocytic differentiation. Mol Cell Biol 17:6068–6075[Abstract]
  23. Trayhurn P, Milner RE 1989 A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis. Can J Physiol Pharmacol 67:811–819[Medline]
  24. Porras A, Peñas M, Fernández M, Benito M 1990 Development of the uncoupling protein in the rat brown adipose tissue during the perinatal period: its relationship with the mitochondrial GDP-binding and GDP-sensitive ion permeabilities and respiration. Eur J Biochem 187:671–675[Abstract]
  25. Porras A, Fernández M, Benito M 1989 Adrenergic regulation of the uncoupling protein expression in foetal rat brown adipocytes in primary culture. Biochem Biophys Res Commun 163:541–547[Medline]
  26. Guerra C, Porras A, Roncero C, Benito M, Fernández M 1994 Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression. Endocrinology 134:1067–1074[Abstract]
  27. Guerra C, Roncero C, Porras A, Fernández M, Benito M 1996 Triiodothyronine induces the transcription of the uncoupling protein gene and stabilizes its mRNA in fetal rat brown adipocyte primary cultures. J Biol Chem 271:2076–2081[Abstract/Free Full Text]
  28. Teruel T, Valverde AM, Benito M, Lorenzo M 1996 Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte primary cultures. Biochem J 319:627–632[Medline]
  29. Lorenzo M, Valverde AM, Teruel T, Benito M 1993 IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes. J Cell Biol 123:1567–1575[Abstract]
  30. Teruel T, Valverde AM, Álvarez A, Benito M, Lorenzo M 1995 Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I. Biochem J 310:771–776[Medline]
  31. Guerra C, Benito M, Fernández M 1994 IGF-I induces the uncoupling protein gene expression in fetal rat brown adipocyte primary culture: role of C/EBP transcription factors. Biochem Biophys Res Commun 201:813–819[CrossRef][Medline]
  32. Benito M, Porras A, Nebreda AR, Santos E 1991 Differentiation of 3T3–L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science 253:565–568[Medline]
  33. Lu K, Campisi J 1992 Ras proteins are essential and selective for the action of insulin-like growth factor 1 late in the G1 phase of the cell cycle in BALB/c murine fibroblasts. Proc Natl Acad Sci USA 89:3889–3893[Abstract]
  34. Lorenzo M, Valverde AM, Teruel T, Álvarez A, Benito M 1996 p21ras induced differentiation-related gene expression in fetal brown adipocyte primary cells and cell lines. Cell Growth Differ 7:1251–1259[Abstract]
  35. Porras A, Hernández ER, Benito M 1996 Ras proteins mediate the induction of uncoupling protein, IGF-I, and IGF-I receptor in rat fetal brown adipocyte cell lines. DNA Cell Biol 15:921–928[Medline]
  36. Porras A, Santos E 1996 The insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells: dissociation between Raf-1 kinase and the MAPK/RSK cascade. Int J Obesity 20:S43–S51
  37. Benito M, Porras A, Santos E 1993 Establishment of permanent brown adipocyte cell lines achieved by transfection with SV40 Large T antigen and ras genes. Exp Cell Res 209:248–254[CrossRef][Medline]
  38. Edelmann HML, Kühne C, Petritsch C, Ballou LM 1996 Cell cycle regulation of p70 S6 Kinase and p42/44 Mitogen-activated protein kinases in Swiss Mouse 3T3 fibroblasts. J Biol Chem 271:963–971[Abstract/Free Full Text]
  39. Albanese C, Johnson J, Watanabe, G, Eklund N, Vu D, Arnold A, Pestell RG 1995 Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 270:23589–23597[Abstract/Free Full Text]
  40. Chadee DN, Taylor WR, Hurta RAR, Allis CD, Wright JA, Davie JR 1995 Increased phosphorylation of histone H1 in mouse fibroblasts transformed with oncogenes or constitutively active mitogen-activated protein kinase kinase. J Biol Chem 270:20098–20105[Abstract/Free Full Text]
  41. Teng KK, Lander H, Fajardo JE, Hanafusa, Hempstead BL, Birge RB 1995 v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/Mitogen-activated protein kinase pathway. J Biol Chem 270:20677–20685[Abstract/Free Full Text]
  42. Smith P, Wise LS, Berkowitz R, Wan C, Rubin CS 1988 Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3–L1 adipocytes. J Biol Chem 263:9402–9408[Abstract/Free Full Text]
  43. Kozma L, Baltensperger K, Klarlund J, Porras A, Santos E, Czech MP 1993 The ras signaling pathway mimics insulin action on glucose transporter translocation. Proc Natl Acad Sci USA 90:4460–4464[Abstract]
  44. Tomiyama K, Nakata H, Sasa H, Arimura S, Nishio E, Watanable Y 1995 Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits adipocytic differentiation of 3T3 L1 cells. Biochem Biophys Res Comunn 212:263–265[CrossRef][Medline]
  45. Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, Gout Y, Fry MJ, Waterfield MD, Downward J 1994 Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527–532[CrossRef][Medline]
  46. Hu E, Kim JB, Sarraf P, Spiegelman BM 1996 Inhibition of adipogenesis through MAPKinase-mediated phosphorylation of PPAR{gamma}. Science 274:2100–2103[Abstract/Free Full Text]
  47. Valverde AM, Lorenzo M, Navarro P, Benito M 1997 Phosphatidylinositol 3-Kinase is a requirement for Insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. Mol Endocrinol 11:595–607[Abstract/Free Full Text]
  48. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD 098059 is a specific inhibitor of the activation of Mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 27:27489–27494[CrossRef]
  49. Andrews NC, Faller DV 1991 A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19:2499[Medline]
  50. Nebreda AR, Porras A, Santos E 1993 p21ras-induced meiotic maturation of Xenopus oocytes in the absence of protein synthesis: MPF activation is preceded by activation of MAP and S6 kinases. Oncogene 8:466–477
  51. Ridley RG, Patel HV, Gerber GE, Morton RC, Freeman KB 1986 Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targetting presequence. Nucleic Acids Res 14:4025–4035[Abstract]
  52. Amy CM, Witkowski A, Naggert J, Williams B, Randhawa Z, Smith S 1989 Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase. Proc Natl Acad Sci USA 86:3114–3118[Abstract]