help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2003-0213
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
18/7/1697    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bazuine, M.
Right arrow Articles by Maassen, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bazuine, M.
Right arrow Articles by Maassen, J. A.
Molecular Endocrinology 18 (7): 1697-1707
Copyright © 2004 by The Endocrine Society

Mitogen-Activated Protein Kinase (MAPK) Phosphatase-1 and -4 Attenuate p38 MAPK during Dexamethasone-Induced Insulin Resistance in 3T3-L1 Adipocytes

Merlijn Bazuine, Françoise Carlotti, Roos S. Jahangir Tafrechi, Rob C. Hoeben and J. Antonie Maassen

Department of Molecular Cell Biology (M.B., F.C., R.S.J.T., R.C.H., J.A.M.), Leiden University Medical Center, 2333 AL Leiden, The Netherlands; and Unité Mixte de Recherche 6548 (F.C.), Université de Nice, 06108 Nice, France

Address all correspondence and requests for reprints to: Dr. J. A. Maassen, Signal Transduction Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, PO Box 9503, 2333 AL Leiden, The Netherlands. E-mail: J.A.Maassen{at}LUMC.NL.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-{lambda} remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulininduced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per se.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A MAJOR PATHOGENIC factor in the genesis of type II diabetes is the occurrence of cellular resistance of muscle, liver, and/or fat cells to the actions of insulin (for a review see Refs. 1, 2, 3). In most cells insulin induces a complex pattern of signal transduction pathways, initiated by insulin binding and activation of the insulin receptor tyrosine kinase. In muscle and in adipose tissues the pathways are linked to translocation of the insulin-responsive glucose transporter 4 (GLUT4) from an intracellular vesicular storage compartment to the plasma membrane (PM), leading to enhanced glucose uptake by these tissues (4). The activation of phosphatidylinositol 3-kinase (PI-3'kinase), by binding to phosphotyrosine residues of the insulin receptor substrate (IRS)-1,2, and tyrosine phosphorylation of Cbl are essential components for insulin-induced glucose uptake (5). Apart from translocation, the amount of glucose taken up by the cell is further regulated by a subsequent intrinsic activation of GLUT4 in the PM mediated by p38 MAPK (6, 7).

The clinical use of corticosteroids is associated with the development of marked insulin resistance (8, 9, 10). These hormones act through binding to specific nuclear receptors, thereby activating or repressing gene transcription. One of the proteins induced by dexamethasone treatment is the dual-specificity phosphatase MKP-1 (MAPK phosphatase 1). Lasa et al. (11) demonstrated recently that dexamethasone treatment of HeLa cells resulted in the attenuation of p38 MAPK signaling through the actions of this dual-specificity phosphatase.

Another family member of these MAPK phosphatases is MKP-4, which was recently observed to be up-regulated in tissues of insulin-resistant ob/ob– and db/db– mice (12). These observations suggest that these dual-specificity phosphatases may play a role in the occurrence of insulin resistance.

Using 3T3-L1 adipocytes we have analyzed the insulin-receptor signaling steps that become attenuated by dexamethasone treatment in relation to their contribution to insulin-induced glucose uptake. Our results suggest that dexamethasone treatment of 3T3-L1 adipocytes induces the up-regulation of MKP-1 and MKP-4 and a concomitant block of p38 MAPK activation, rather than an effect on the process of GLUT4 translocation itself. Although fully differentiated 3T3-L1 adipocytes have long been refractory to ectopic expression, we recently described lentivirus-mediated ectopic expression as a novel tool with which to efficiently transduce these cells (13). Employing lentivirus constructs expressing either MKP-1 or MKP-4, we mimicked the effects of dexamethasone on 3T3-L1 adipocytes. Our combined data illustrate that interference with p38 MAPK signaling results in a reduction of insulin- and arsenite-induced glucose uptake. To our knowledge, this is the first physiologically relevant model of insulin resistance in 3T3-L1 adipocytes in which the p38 MAPK pathway is identified as the main culprit, rather than a defect in the PI-3'kinase pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Effect of Dexamethasone Treatment on Insulin- and Arsenite-Induced Glucose Uptake in 3T3-L1 Adipocytes
Treatment with 100 nM dexamethasone induced a time-dependent decrease in insulin-induced glucose uptake, becoming apparent after 4 h of treatment and reaching maximal levels after 48 h (see Fig. 1AGo). The level of glucose uptake did not alter further over a time period of up to 72 h (data not shown). The cellular stress-inducing agent arsenite is a strong inducer of glucose uptake in these cells in a PI-3'kinaseindependent manner (14). As can be seen in Fig. 1AGo, treatment with dexamethasone also inhibited arseniteinduced glucose uptake in 3T3-L1 adipocytes with similar time dependencies. In all subsequent experiments cells were treated for 48 h with 100 nM dexamethasone unless indicated otherwise.



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1. The Effect of Dexamethasone on Insulin- and Arsenite-Induced Glucose Uptake

3T3-L1 adipocytes were stimulated as indicated and assayed for 2-deoxy-D[14C]-glucose (2-DOG) uptake. A, 3T3-L1 adipocytes were treated with 100 nM dexamethasone for the indicated times and stimulated with 100 nM insulin for 15 min or with 0.5 mM arsenite for 30 min. B, After treatment with 100 nM dexamethasone for 48 h, 3T3-L1 adipocytes were stimulated with the indicated concentration of insulin for 15 min and assayed for 2-DOG uptake. C, After treatment with 100 nM dexamethasone for 48 h, 3T3-L1 adipocytes were stimulated with the indicated concentration of arsenite for 30 min and assayed for 2-DOG uptake. D, 3T3-L1 adipocytes were treated with 100 nM dexamethasone, 100 nM dexamethasone in the presence of 1 µM RU486 or 1 µM RU486 for 48 h and subsequently stimulated with 100 nM insulin for 15 min or with 0.5 mM arsenite for 30 min as indicated. Subsequently, 2-DOG uptake was assayed. All values are mean ± SEM of at least three independent experiments. *, P < 0.05 compared with agonist-stimulated cells.

 
Next we determined the effect of dexamethasone pretreatment on the dose-response relation for insulin- and arsenite-stimulated hexose uptake in 3T3-L1 adipocytes. As is demonstrated in Fig. 1BGo, dexamethasone reduced maximal insulin-stimulated glucose uptake by approximately 50%. Also the ED50 for insulin shifted from about 10 nM to 50 nM insulin after incubation with dexamethasone. Importantly, basal glucose uptake was not affected by dexamethasone treatment. As can be see in Fig. 1CGo, dexamethasone had a similar effect on arsenite-induced glucose uptake.

To determine whether the classical glucocorticoid receptor is responsible for this phenomenon, the glucocorticoid-receptor antagonist RU486 was added with dexamethasone. In the presence of this glucocorticoid receptor antagonist the deleterious effects of dexamethasone on insulin- or arsenite-induced glucose uptake were fully prevented (see Fig. 1DGo). Intriguingly, whereas RU486 treatment slightly improved insulin-induced glucose uptake in unstimulated cells, it had a deleterious effect on arsenite-induced glucose uptake. Although this was observed consistently, we have no explanation for this phenomenon. At any rate, the results of combined dexamethasone/RU486 treatment support the concept that in 3T3-L1 adipocytes dexamethasone acts through the glucocorticoid receptor to reduce glucose uptake.

The Effect of Dexamethasone Treatment on the Insulin-Induced Activation of the PI-3'Kinase Signaling Pathway in 3T3-L1 Adipocytes
Incubation of 3T3-L1 adipocytes with dexamethasone had no effect on insulin-induced insulin receptor tyrosine phosphorylation, although insulin-stimulated IRS-tyrosine phosphorylation was slightly reduced in response to dexamethasone treatment. However, it must also be noted that both IRS-1 and -2 were still appreciably tyrosine phosphorylated. The amount of PI-3'kinase associating with the IRS proteins closely followed the tyrosine phosphorylation pattern (data not shown) To analyze whether these changes in IRS-tyrosine phosphorylation and the resulting reduction in the amount of associated PI-3'kinase have functional consequences for the activation of downstream signaling steps, we determined the effect of dexamethasone treatment on the in vivo phosphorylation of several downstream components of the IRS/PI-3'kinase axis. Although phosphorylation of the PI-3'kinasedependent site of protein kinase B (PKB), Thr308, was reduced (but not fully absent), phosphorylation of Ser473 of PKB was not affected by dexamethasone treatment (see Fig. 2Go). This suggests that despite a reduction in PI-3' kinase activity, PKB activity remained intact. To investigate this point further, we analyzed the phosphorylation of FKHR-L1, an intracellular downstream target of PKB kinase activity (15). Indeed, phosphorylation of Thr32 of FKHR-L1 remained intact after dexamethasone treatment (see Fig. 2Go). Another downstream target of PI-3'kinase activity involved in GLUT4 translocation is atypical protein kinase C (PKC){lambda} (16). Phosphorylation of Thr403 (in the activation loop) of PKC{lambda} was elevated in unstimulated, dexamethasone-treated adipocytes. However, upon insulin stimulation an increase in Thr403 phosphorylation was observed both in untreated and in dexamethasone-treated adipocytes (see Fig. 2Go). These data show that dexamethasone slightly affects PI-3'kinase activity, as was reported previously (17, 18). However, our data add to these previous data the observation that this reduction in PI-3'kinase activity is not sufficient to reduce the insulin-induced activation of downstream-signaling intermediates such as PKB and FKHR-L1. Another signaling pathway contributing to insulin-induced GLUT4-translocation, but independent of PI-3'kinase activity, is the tyrosine phosphorylation of c-Cbl (19). This phosphorylation is also induced by stimulation of 3T3-L1 adipocytes with arsenite (14). Dexamethasone had no effect on the level of either insulin- or arsenite-induced tyrosine phosphorylation of Cbl (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 2. The Effect of Dexamethasone Treatment on the Insulin-Induced Activation of the PI-3'Kinase Signaling Pathway in 3T3-L1 Adipocytes

Whole-cell lysate of adipocytes (10 µg) stimulated with 100 nM insulin for 5 min and/or pretreated with 100 nM dexamethasone for 48 h as indicated was analyzed by immunoblot using phospho-specific antibodies against T308 or S473 of PKB (pThr308 and pSer473 PKB), T32 of FKHR-L1 (pFKHR-L1), T403 of PKC-{lambda} (pPKC-{lambda}), or antibodies against PKB (PKB) or PKC{lambda} (PKC-{lambda}), as indicated. Data shown are representative immunoblots of at least three independent experiments.

 
The Effect of Dexamethasone on Insulin- or Arsenite-Induced GLUT4 Translocation
Insulin and arsenite induce the translocation of the insulin-responsive GLUT4 glucose transporter from an intracellular vesicular storage compartment [low density microsome (LDM)] toward the PM in 3T3-L1 adipocytes. To analyze the effects of dexamethasone on GLUT4 translocation, we performed subcellular fractionation of dexamethasone-treated adipocytes. As can be seen in Fig. 3AGo, dexamethasone treatment did not affect the translocation of GLUT4 toward the PM in response to either insulin or arsenite stimulation. A representative immunoblot used to obtain the quantified data as represented in Fig. 3AGo is shown in Fig. 3BGo.



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 3. The Effect of Dexamethasone on Either Insulin- or Arsenite-Induced GLUT4 Translocation to the PM in 3T3-L1 Adipocytes

3T3-L1 adipocytes were pretreated with 100 nM dexamethasone for 48 h and subsequently stimulated for 15 min with 100 nM insulin (insulin) or for 30 min with 0.5 mM arsenite (arsenite). Adipocytes were fractionated, and equal amounts of protein from both LDMs and PMs were analyzed by immunoblot with anti-GLUT4 antibodies (panel B). GLUT4 levels in each fraction subjected to immunoblot analysis as in panel B were quantified using a LumiImager and expressed as a fraction residing in either PM or LDM (panel A). The total amount of GLUT4 in the high density microsome fraction did not alter during treatment. Data are expressed as the mean value ± SE of at least three independent observations. *, P < 0.05 compared with the same stimulation in untreated cells.

 
The Effect of Dexamethasone Treatment on ERK and p38 MAPK Activation
Apart from translocation of the insulin-responsive GLUT4 glucose transporter, the amount of glucose taken up by the cell is additionally regulated by the activity of p38 MAPK (6, 7). In response to either insulin or arsenite, p38 MAPK is activated leading to an intrinsic activation of the GLUT4 transporter and thus, resulting in a maximal uptake of glucose. As can be seen in Fig. 4Go, treatment with dexamethasone resulted in a strong reduction (~80%) of p38 MAPK phosphorylation in response to insulin and arsenite. Dexamethasone treatment had no effect on the amount of p38 MAPK. Importantly, insulin-induced phosphorylation of the MAPK family members ERK-1 and -2 was not altered by dexamethasone treatment (see Fig. 4Go). To determine whether the reduction of p38 activation has functional consequences for the in situ action of this kinase, we also monitored the phosphorylation of activating transcription factor 2 (ATF-2), a downstream target of p38 activity (20, 21). Dexamethasone treatment completely inhibited insulin- and arsenite-induced phosphorylation of ATF-2 in 3T3-L1 adipocytes (see Fig. 4Go).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4. The Effect of Dexamethasone on Insulin- or Arsenite-Induced p38-MAPK and ERK-1 and -2 Pathways

3T3-L1 adipocytes were pretreated with 100 nM dexamethasone for 48 h and subsequently stimulated with either 100 nM insulin for 15 min or 0.5 mM arsenite for 30 min as indicated. Total cell lysate (10 µg) was subjected to immunoblot analysis using phospho-specific antibodies against Thr202/Tyr204 of ERK-1/-2(pT202/Y204 ERK), Thr180/Tyr182 of p38 (pT180/Y182 p38), Thr71 of ATF-2 (pT71-ATF2), or antibodies against either ERK-1/-2 (ERK) or p38 MAPK (p38) as indicated. Demonstrated are representative immunoblots of at least three independent observations.

 
The Effect of Dexamethasone Treatment on the Levels of MKP-1 and MKP-4
MKP-1 is a dual-specificity, MAPK phosphatase (22, 23). Lasa et al. (11) demonstrated recently that MKP-1 is up-regulated by dexamethasone acting through the glucocorticoid receptor in HeLa cells. Consequently, in these cells, p38 MAPK activation and stabilization of Cox-2 mRNA (a p38-dependent event) is lost, without an effect on ERK signaling. Reasoning that a similar mechanism could be central to the observed effect of dexamethasone on glucose uptake in 3T3-L1 adipocytes as well, we analyzed the effects of dexamethasone treatment on MKP-1 levels in 3T3-L1 adipocytes. As can be seen in Fig. 5AGo, by applying real-time quantitative RT-PCR, dexamethasone treatment was observed to increase MKP-1 mRNA 4-fold after 2 h. The level of MKP-1 mRNA was still increased at the 4-h time point and stabilized at a level 3-fold higher than in untreated adipocytes up until 48 h.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 5. The Effect of Dexamethasone on Intracellular MKP-1 and MKP-4 mRNA and Protein Levels

3T3-L1 adipocytes were treated with 100 nM dexamethasone as indicated. A, First strand cDNA was prepared from mRNA isolated at the indicated time points from dexametha sone-treated adipocytes. When indicated, 1 µM RU486 was added concomitant with the dexamethasone treatment. Equal amounts of cDNA were subjected to real-time quantitative RT-PCR. Data shown are representative of four independent experiments, each performed in triplicate. Data are expressed as relative amount of mRNA compared with untreated adipocytes ± SEM. Representative experiments are depicted in panels B and C for MKP-1 and MKP-4, respectively. D, Equal amounts of protein (10 µg) of whole-cell lysate isolated at the indicated time points of dexamethasone treatment were subjected to immunoblot analysis and probed with an antibody against MKP-1 or MKP-4 as indicated. Equal loading was reconfirmed by immunoblot analysis using antibodies against p38 MAPK and PKB. Immunoblots were subjected to LumniImager analyses, and quantified levels of Boehringer light units were expressed as fold relative to untreated adipocytes ± SEM of four independent observations. For comparison, at the same time points insulin- and arsenite-induced 2-deoxy-D[14C]-glucose (2-DOG) uptake was measured and expressed as fold relative to untreated adipocytes ± SEM. Insulin-induced levels of glucose uptake in the untreated adipocytes were 30.0 (pmol/min·mg) and 10.8 (pmol/min·mg) for arsenite; basal levels of glucose were 3.5 (pmol/min·mg). Data are expressed as the mean relative value ± SEM of three independent observations. Panels E and F depict a representative immunoblot for MKP-1 and -4, respectively, used to obtain the data presented in panel D.

 
MKP-4 is a homolog of MKP-1 (24, 25); this phosphatase has recently been implicated in insulin resistance and was found to be up-regulated in ob/ob– and db/db– mice (12). Thus we also analyzed the effects of dexamethasone on MKP-4. As can be observed in Fig. 5AGo, MKP-4 mRNA was also up-regulated after dexamethasone treatment, albeit at lower levels (3-fold) and with slower kinetics. In Fig. 5Go, B and C, representative experiments used to obtain the data shown in Fig. 5AGo have been included. Importantly, cotreatment with RU486 blocked the effects of 48-h dexamethasone on both MKP-1 and -4 (Fig. 5AGo). Furthermore, neither MKP-5 and MKP-7 [two other MKP-family members (26, 27)] nor eEF-1{alpha} (a house-hold gene) mRNA changed during dexamethasone treatment.

As can be seen in Fig. 5DGo, MKP-1 protein levels followed the increase of mRNA levels with a significant increase over basal levels from the 8-h time point onward, leading up to a 2-fold increase in the amount of MKP-1 protein. Remarkably, although the increase of MKP-4 mRNA was delayed, the induction of MKP-4 protein levels matched the kinetics of MKP-1 (Fig. 5DGo). Representative immunoblots used to obtain the data presented in Fig. 5DGo are included as Fig. 5Go, E and F. When quantified and expressed as fold/basal vs. fold/maximal response, respectively, the increase in MKP protein levels in response to dexamethasone treatment correlates closely to the reduction in insulin- or arsenite-induced glucose uptake over time (Fig. 5DGo).

The Effect of Lentivirus-Mediated Ectopic Expression of MKP-1 or MKP-4 in 3T3-L1 Adipocytes
Recently we described lentivirus vectors as a new tool with which to transduce fully differentiated 3T3-L1 adipocytes. In contrast to previously employed methods on these cells, treatment with lentiviruses results in a high percentage of transduced cells, without the need to detach the cells from the plates and without any apparent cytotoxicity (13). Employing lentivirus-mediated ectopic expression of MKP-1, MKP-4, or their respective inactive C/S-mutants, we attempted to mimic the effects of dexamethasone in 3T3-L1 adipocytes. MKP-1 and -4 have different subcellular localizations (28, 29). To ensure that lentivirus-mediated ectopic overexpression does not alter this basic behavior of these isoforms, we employed a crude subcellular fractionation technique. As can be seen in Fig. 6AGo, when nuclei are separated from the cytosol, p38 MAPK is present in both fractions [as has been described (30, 31)], whereas the nuclear envelope protein Lamin-A/C is detected only in the nuclear fractions and ERK-1/2 is cytosolic. Indeed, MKP-1 (as well as the inactive C/S-mutant) is observed in the nuclear fraction, whereas MKP-4 (as well as the inactive C/S-mutant) resides in the cytosol.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 6. The Effect of Lentivirus-Mediated Ectopic Expression of MKP-1 or MKP-4 in 3T3-L1 Adipocytes

A, 3T3-L1 adipocytes transduced with the lentivirus constructs indicated were subjected to crude subcellular fractionation and subjected to immunoblot analysis using antibodies recognizing Lamin-A/C, p38 MAPK, ERK-1/2, MKP-1, or MKP-4 as indicated. B, 3T3-L1 adipocytes were transduced with the indicated lentivirus constructs. After stimulation with 100 nM insulin or 0.5 mM arsenite, 2-deoxy-D[14C]-glucose (2-DOG) uptake was assayed. All values are mean ± SEM of at least three independent experiments. *, P < 0.05 compared with untransduced cells. C, 3T3-L1 adipocytes were transduced with the indicated lentivirus constructs and subsequently stimulated with either 100 nM insulin for 15 min or 0.5 mM arsenite for 30 min as indicated. Whole-cell lysate (10 µg) was subjected to immunoblot analysis using phospho-specific antibodies against Thr202/Tyr204 of ERK-1/-2 (pT202/Y204 ERK), Thr180/Tyr182 of p38 (pT180/Y182 p38), or antibodies against MKP-1, MKP-4, and ERK-1/-2 (ERK) as indicated. Demonstrated are representative immunoblots of at least two independent observations.

 
Ectopic overexpression of MKP-1 or MKP-4 resulted in a reduction of insulin- and arsenite-induced glucose uptake, whereas ectopic overexpression of either C/S-mutant had no effect. Intriguingly, MKP-1 consistently induced a more pronounced effect on insulin-induced glucose uptake (~30% reduction), whereas it barely (~10%) affected arsenite-induced glucose uptake. The opposite was observed with MKP-4, which was a more potent inhibitor of arsenite-induced glucose uptake (~30%), but had only a small (but significant) effect on insulin-induced glucose uptake (~10%) (Fig. 6BGo). When p38 MAPK and ERK signaling were analyzed, a similar phenomenon was observed. Whereas insulin-induced phosphorylation of ERK remained intact with either isoform, insulin-induced p38 MAPK phosphorylation was attenuated with ectopically expressed MKP-1, and arsenite-induced p38 MAPK phosphorylation remained largely unaffected (Fig. 6CGo). Conversely, when MKP-4 was overexpressed, little or no effect was observed on insulin-induced p38 MAPK phosphorylation, but the arsenite-induced p38 MAPK phosphorylation was markedly attenuated (Fig. 6CGo). Thus, combined ectopic overexpression of MKP-1 and MKP-4 reiterates the effects observed with dexamethasone treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We analyzed the effects of dexamethasone on signal-transduction pathways leading to GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Dexamethasone reduces both insulin- and arsenite-induced glucose uptake with similar time dependencies. Cotreatment with RU486, a glucocorticoid receptor antagonist, prevents the deleterious effects of dexamethasone on glucose uptake in 3T3-L1 adipocytes, demonstrating the effects of dexamethasone are mediated through the glucocorticoid receptor. It has been suggested previously that attenuation of IRS-dependent steps is involved in most, if not all, types of insulin resistance in 3T3-L1 adipocytes (32, 33, 34). In response to dexamethasone treatment, IRS supershifting (indicative of elevated Ser/Thr-phosphorylation) and reduced PI-3'kinase association after insulin-stimulation were also observed (data not shown).

PKB is activated by growth factors in a PI-3'kinase-dependent manner by a two-step mechanism, phosphorylation of Thr308 by the PI-3'kinase effector phosphoinositide-dependent protein kinase 1 and phosphorylation of Ser473 by a distinct kinase, leading to full activation of this protein kinase (35, 36). However, after dexamethasone treatment phosphorylation of the PI-3'kinase-dependent Thr308 was reduced (but not fully lost), and phosphorylation of Ser473 of PKB in response to insulin remained intact; this suggests that the activation of PKB is unaffected. To corroborate this conclusion we analyzed PKBmediated intracellular signaling one step further. Thr32 of FKHR-L1 is a direct downstream target of activated PKB in 3T3-L1 adipocytes (15). Consistent with the lack of an effect of dexamethasone on Ser473, phosphorylation of FKHR-L1 was unaffected by dexamethasone treatment. Another AGC-kinase member implicated in insulin-induced GLUT4 translocation is the atypical PKC{lambda} (16). As can be seen in Fig. 2Go, basal levels of PKC{lambda} phosphorylation are elevated after dexamethasone treatment. However, the induction of activation loop phosphorylation by insulin remains intact after dexamethasone treatment.

Furthermore, arsenite induces GLUT4-mediated glucose uptake in 3T3-L1 adipocytes independent of PI-3'kinase activity. Given that arsenite-induced glucose uptake was similarly affected, these observations strongly suggest that PI-3'kinase signaling is not the prime target for mediating the effects of dexamethasone in these cells. These data are in agreement with a previous study by Sakoda et al. (17), who also demonstrated that dexamethasone-induced insulin resistance toward glucose uptake in 3T3-L1 adipocytes occurs independently of an effect on PI-3'kinase activity.

A PI-3'kinase-independent pathway contributing to insulin-induced GLUT4 translocation in 3T3-L1 adipocytes is initiated by tyrosine phosphorylation of c-Cbl (19, 37). Arsenite also induces tyrosine phosphorylation of Cbl (14). However, both insulin- and arsenite-induced tyrosine phosphorylation of Cbl are unaffected by dexamethasone treatment (data not shown). Consistent with these observations, dexamethasone has no effect on either the insulin- or arsenite-induced translocation of GLUT4 transporters from the LDM fraction to the PM (see Fig. 3Go).

Recently p38 MAPK has been implicated in enhancing the magnitude of GLUT4-mediated glucose transport (6, 7). Treatment of 3T3-L1 adipocytes with the p38 MAPK inhibitor SB203580 results in a reduction of glucose uptake without an effect on GLUT4 translocation toward the PM. Apart from insulin, this pathway also contributes to arsenite-induced glucose uptake in 3T3-L1 adipocytes (14). We found that dexamethasone attenuated p38 MAPK phosphorylation. Insulin-induced p38 phosphorylation after dexamethasone treatment became undetectable, and the much stronger arsenite-induced phosphorylation was markedly reduced (~80% less). The amount of p38 MAPK protein itself was unaltered by dexamethasone treatment. Both insulin- and arsenite-induced ATF-2 phosphorylation, a downstream target of p38 activity (20, 21), were abrogated by dexamethasone treatment, indicative of reduced signaling capacity of p38 MAPK. The loss in p38 activity results in the accumulation of less active GLUT4 glucose transporters in the PM, which could explain the partial reduction of glucose uptake in response to dexamethasone.

Recently Lasa et al. (11) described an up-regulation of MKP-1, and concomitant loss of p38 MAPK activity, in response to dexamethasone treatment. Other papers have since corroborated this effect of dexamethasone in several other cell types (38). Reasoning that a similar mechanism could be responsible for the reduced p38 MAPK phosphorylation observed in the 3T3-L1 adipocyte, we analyzed the induction of MKP-1 mRNA and protein levels in response to dexamethasone treatment. Indeed we observed that dexamethasone induces an up-regulation of MKP-1 mRNA, followed by the up-regulation of MKP-1 protein levels. The onset of the adverse effects of dexamethasone on both insulin- and arsenite-induced glucose uptake closely matched the rise of MKP-1 protein levels. Up-regulation of another MKP-family member, MKP-4, has recently also been implicated in insulin resistance in ob/ob– and db/db– mice (12). Reasoning that MKP-4 could play a similar role in 3T3-L1 adipocytes, we also analyzed the expression profile of this isoform. MKP-4 mRNA levels did rise after dexamethasone treatment, but only at later time points. Intriguingly, MKP-4 protein levels rose with identical time dependencies as MKP-1. Thus, in the case of MKP-4 it is likely that dexamethasone also controls transcriptional regulation of the mRNA, although we have no further data on that intriguing possibility. Importantly, mRNA levels of two other MKP family members, MKP-5 and -7 (26, 27), were unaltered by dexamethasone treatment. Cotreatment with RU486 blocked the rise in MKP-1 and MKP-4 mRNA, suggesting that both phosphatases are targets of glucocorticoid receptor signaling, although especially in the case of MKP-4 this can be a secondary effect.

To further substantiate the data on the involvement of MKP-1 and -4, we attempted to express these isoforms in 3T3-L1 adipocytes to analyze their respective effects on insulin- and arsenite-induced glucose uptake. To this end we employed the novel technique of lentivirus-mediated ectopic expression. Using these vectors to transduce fully differentiated 3T3-L1 adipocytes, high levels of transduction efficiency can be routinely and conveniently reached without any cytotoxic effects on the adipocytes (13). Importantly, ectopically expressed MKP-1 (as well as the inactive C/S-mutant) resides in the nucleus, whereas MKP-4 is cytosolic, consistent with the subcellular localization reported for these respective isoforms (28, 29).

Ectopic expression of these MKPs resulted in a reduction of glucose uptake concomitant with a loss of p38 MAPK phosphorylation, whereas ectopic expression of inactive MKP-C/S mutants remained without effect. Furthermore, neither MKP-1 nor MKP-4 expression resulted in ERK dephosphorylation. Thus, ectopic expression of these dual-specificity phosphatases mimics the effects observed with dexamethasone. An intriguing additional observation was made regarding isoform specificity. Whereas MKP-1 efficiently attenuated insulin-induced p38 MAPK phosphorylation, it had little impact on the potent arsenite-induced p38 MAPK phosphorylation. Strikingly, ectopic overexpression of MKP-1 profoundly affected insulin-induced glucose uptake, but had only a small effect on arsenite-induced glucose uptake. The converse situation was observed with MKP-4, which attenuated arsenite-induced p38 MAPK phosphorylation and glucose uptake, but had less effect on insulin-induced p38 MAPK phosphorylation and glucose uptake.

Clearly, although insulin and arsenite project toward a similar cellular outcome (i.e. GLUT4 translocation, p38 MAPK phosphorylation, and glucose uptake) in 3T3-L1 adipocytes, the two stimuli are mechanistically profoundly different. The attenuation of these pathways by specific MKP isoforms differs dramatically. Several explanations can contribute to this phenomenon: first and foremost, the insulin- and arseniteinduced pathways are different, resulting in a much higher level of p38 MAPK activation and conferring a partial sensitivity to SB203580 (a p38 MAPK inhibitor) on p38 MAPK activation itself upon stimulation of 3T3-L1 adipocytes with arsenite (14). Second, different scaffolding proteins can contribute by clustering the active components in different subcellular localizations. Thus, the nuclear localized MKP-1 might be more aptly localized for disruption of insulin-induced p38 MAPK activation, and vice versa, with the cytoplasmic MKP-4 and arsenite. Currently, we have some very preliminary data supporting a contribution of differential subcellular localization. A third option, which must be considered, is that arsenite and insulin induce different pathways, which could induce a differential response to a simultaneous up-regulation of MKP-1 and -4 through cross-talk of downstream effectors of either insulin and arsenite pathways (e.g. a potent activation of JNK by arsenite). However, at present we have no detailed explanation for these differential effects of MKP-1 and -4. Research into this phenomenon is hampered by the fact that these MKPs display only a partial preference (albeit less efficient, ectopically overexpressed MKP-4 does target insulin signaling) and by a general lack of knowledge on the upstream components of both arsenite and insulin signaling toward p38 MAPK in insulin-responsive cell types. Further research into this relatively recently uncovered pathway in regulating glucose uptake (and importantly, identification of the upstream activators), may well provide additional clues to the effects of these MAPK phosphatases.

In conclusion, our data demonstrate that dexamethasone induces the up-regulation of two MAPK phosphatases, MKP-1 and MKP-4, which, combined, are capable of attenuating p38 MAPK signaling in response to either insulin or arsenite in 3T3-L1 adipocytes. Consequently, dexamethasone reduces the magnitude of insulin- and arsenite-stimulated glucose transport in these cells without affecting the translocation of the GLUT4 glucose transporter from the LDM to the PM. To our knowledge, this is the first physiologically relevant model of insulin resistance in which the novel p38 MAPK pathway is implicated. Furthermore, interfering with these kinase-phosphatase interactions may represent an attractive target for the treatment of glucocorticoid-induced insulin resistance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DMEM was purchased from Life Technologies, Inc. (Gaithersburg, MD); fetal calf serum (lot no. A01127–318) was from Brunschwig (Amsterdam, The Netherlands); bovine insulin, 1-methyl-3-isobutylxanthine (IBMX), dexamethasone, 2deoxyglucose, and RU486 were obtained from Sigma (St. Louis, MO). 2-deoxy-D-[14C]glucose was purchased from NEN-Dupont (Boston, MA).

Antibodies
Polyclonal antisera recognizing IRS-1 and IRS-2 were described by Telting et al. (39). Mouse monoclonal antibody recognizing PKC-{lambda} was purchased from Transduction Laboratories, Inc. (Lexington, KY). Horseradish peroxidase-conjugated mouse monoclonal antiphosphotyrosine antibody pY-20, goat polyclonal antibody recognizing GLUT4 (C-20), and rabbit polyclonal recognizing ERK were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibody recognizing insulin receptor ß-chain was purchased from Transduction Laboratories, Inc. The phospho-specific antibodies recognizing PKB (S473), PKC-{lambda} /{zeta} (T403/410), p38 (T180/Y182), ERK-1/-2 (T202/Y204), and ATF-2 (T71) were obtained from Cell Signaling Technology. Sheep polyclonal antibody recognizing PKB was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The appropriate horseradish peroxidase-conjugated secondary antibodies were obtained from Promega Corp. (Madison, WI). The MKP-1 antibody was kindly made available by Drs. P. Lenormand and J. Pouyssegur (Nice, France) and has been described elsewhere (40, 41). Sheep polyclonal recognizing MKP-4 was a kind gift of Dr. S. Keyse (Dundee, Scotland, UK) and has been described elsewhere (42, 43).

Cell Culture
3T3-L1 fibroblasts, obtained from ATCC (Manassas, VA), were cultured and differentiated to adipocytes as described previously (44). Cells were routinely used 7 d after completion of the differentiation process, with only cultures in which more than 95% of cells displayed adipocyte morphology being used.

Membrane Isolation Assay
3T3-L1 adipocytes were stimulated as indicated in the figure legends. Subsequently, cells were washed twice in ice-cold HES buffer (20 mM HEPES, pH 7.4; 1 mM EDTA; and 250 mM sucrose) on ice and scraped in HES buffer in the presence of protease inhibitors (complete protease inhibitor cocktail, Roche Clinical Laboratories, Indianapolis, IN). Samples were homogenized nine times three strokes in a glass Potter homogenizer, after which LDM and PM fractions were isolated by differential centrifugation as described by Simpson et al. (45).

Equal amounts of protein, as determined with BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) were subjected to immunoblot analysis using various antibodies.

Assay of 2-Deoxyglucose Uptake
3T3-L1 adipocytes, grown in 12-well plates (Costar, Cambridge, MA), were subjected to an assay of 2-deoxy-D-[14C]glucose (0.075 µCi per well) uptake as described previously (46).

Immunoprecipitations and Western Blotting
Dishes (9-cm) of 3T3-L1 adipocytes were stimulated with agonists. Immunoprecipitation and immunoblotting procedures were as described previously (14). Immunoblots were quantified using LumiAnalyst Software on a LumniImager (Roche Clinical Laboratories).

Real Time Quantitative RT-PCR
Briefly, 3T3-L1 adipocytes were treated with dexamethasone as indicated in the figure legends. Subsequently mRNA was isolated using RNA-B (Campro Scientific, Veenendaal, The Netherlands), and equal amounts of RNA were subjected to first strand cDNA synthesis using the SuperScript Preamplification System (Life Technologies). All procedures were performed according to the manuals provided by the manufacturers. Real-time quantitative PCR was performed on a ABI 7700 using a standard two-step procedure using SYBR Green Master Mix (ABI Advanced Biotechnologies, Inc., Columbia, MD) with the following oligonucleotides: 5'-CATCAAGGATGCTGGAGGGA (forward) and 5'-GAGGTAAGCAAGGCAGATGGTG (reverse) for MKP-1; 5'-GGAGCAAGGCAGGAACAGAGT (forward) and 5'-CCACCAGTAGGCACGTGAAAT (reverse) for MKP-4. Specificity of the PCR was confirmed after the reaction using 3% agarose gel electrophoresis and melting point analysis of the products. As an internal control on equal amounts of first strand cDNA, a real-time PCR using primers against eEF-1{alpha} was routinely included in the experiment.

MKP-Constructs and Lentivirus
cDNAs harboring MKP-1, MKP-1 C/S, and MKP-4 were a kind gift of Dr. S. Keyse (Dundee, Scotland, UK) and have been described elsewhere (22, 23, 25, 42). The MKP-4 C/S mutant described in this manuscript has been generated using QuikChange (Stratagene, La Jolla, CA), following the manufacturer’s instructions. The oligonucleotides 5'-GGGGTGCTCGTCCACGGTCTGGCAGGG (forward) and the reverse were used to introduce the Cys to Ser point mutation. Subsequently, the constructs were cloned into a pLV-PGK-driven lentivirus backbone. 3T3-L1 adipocytes were incubated with lentivirus for 24 h, after which medium was replaced. The experiments were performed 4 d after inoculation with the viral vectors. For a detailed description of lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes see Ref. 13 . Generated mutants and lentivirus constructs were all checked by sequence analysis.

Crude Subcellular Fractionation
Dishes (9 cm) of 3T3-L1 adipocytes were stimulated with agonists and scraped in cyt/nuc-lysis buffer (1 mM Na3VO4; 1 mM EGTA; 1 mM EDTA; 50 mM Tris Cl, pH 7.4; 0.5% Triton X-100; 150 mM NaCl; 5 mM NaF in the presence of protease inhibitors). Cell lysates were tumbled for 0.5 h at 4 C, and nuclei, ghosts, caveolae, and cytoskeleton were separated from the cytosol by spinning at 14,000 x g (rcf), for 10 min at 4 C in a table-top centrifuge. The fat cake was removed, and the pellet was washed three times with cyt/nuc lysis buffer. Equal amounts of protein were subjected to immunoblot analysis.

Statistical Analysis
Data were analyzed with an independent-samples t test using SPSS 10.0. Curves represent fits to data by nonlinear regression analysis using Prism 2.01 (GraphPad Software Inc., San Diego, CA).


   ACKNOWLEDGMENTS
 
We thank Drs. P. Lenormand and J. Pouyssegur for making their MKP-1 antibody available to us. We are also indebted to Dr. S. Keyse for sharing MKP-constructs, the MKP-4 antibody, and excellent scientific advice.


   FOOTNOTES
 
This work was supported by Grant DFN 98.106 from the Dutch Diabetes Fund (to M.B.), a fellowship from the MENRT (Ministère Français de l’education Nationale, de la Recherche et de la Technologie) (to F.C.), and a fellowship from Eurogendis (to F.C.).

Requests for vectors should be made to: Dr. R. C. Hoeben, Virus Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, PO Box 9503, 2333 AL Leiden, The Netherlands. E-mail: R.C.Hoeben{at}LUMC.NL.

Abbreviations: ATF-2, Activation transcription factor 2; GLUT4, glucose transporter 4; IRS, insulin receptor substrate; LDM, low density microsome; MKP-1, MAPK phosphatase 1; PI-3'kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PM, plasma membrane.

Received for publication June 5, 2003. Accepted for publication April 6, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Zierath JR 2002 Exercise effects of muscle insulin signaling and action - Invited review: exercise training-induced changes in insulin signaling in skeletal muscle. J Appl Physiol 93:773–781[Abstract/Free Full Text]
  2. Mora S, Pessin JE 2002 An adipocentric view of signaling and intracellular trafficking. Diabetes Metab Res Rev 18:345–356[CrossRef][Medline]
  3. Patti ME, Kahn CR 1996 Lessons from transgenic and knockout animals about noninsulin-dependent diabetes mellitus. Trends Endocrinol Metab 7:311–319
  4. Watson RT, Pessin JE 2001 Intracellular organization of insulin signaling and GLUT4 translocation. Recent Prog Horm Res 56:175–193[Abstract]
  5. Khan AH, Pessin JE 2002 Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia 45:1475–1483[CrossRef][Medline]
  6. Somwar R, Perreault M, Kapur S, Taha C, Sweeney G, Ramlal T, Kim DY, Keen J, Cote CH, Klip A, Marette A 2000 Activation of p38 mitogen-activated protein kinase {alpha} and ß by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport. Diabetes 49:1794–1800[Abstract]
  7. Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A, Klip A 1999 An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3–L1 adipocytes and L6 myotubes. J Biol Chem 274:10071–10078[Abstract/Free Full Text]
  8. Andrew R, Gale CR, Walker BR, Seckl JR, Martyn CN 2002 Glucocorticoid metabolism and the metabolic syndrome: associations in an elderly cohort. Exp Clin Endocrinol Diabetes 110:284–290[CrossRef][Medline]
  9. Andrews RC, Walker BR 1999 Glucocorticoids and insulin resistance: old hormones, new targets. Clin Sci 96:513–523[Medline]
  10. Byrne CD 2001 Programming other hormones that affect insulin. Br Med Bull 60:153–171[Abstract/Free Full Text]
  11. Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR 2002 Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol Cell Biol 22:7802–7811[Abstract/Free Full Text]
  12. Xu H, Dembski M, Yang Q, Yang D, Moriarty A, Tayber O, Chen H, Kapeller R, Tartaglia LA 2003 Dual specificity mitogen-activated protein (MAP) kinase phosphatase-4 plays a potential role in insulin resistance. J Biol Chem 278:30187–30192[Abstract/Free Full Text]
  13. Carlotti F, Bazuine M, Kekarainen T, Seppen J, Pognonec P, Maassen JA, Hoeben RC 2004 Lentiviral vectors efficiently transduce quiescent mature 3T3–L1 adipocytes. Mol Ther 9:209–217[Medline]
  14. Bazuine M, Ouwens DM, Gomes de Mesquita DS, Maassen JA 2003 Arsenite stimulated glucose transport in 3T3–L1 adipocytes involves both Glut4 translocation and p38 MAPK activity. Eur J Biochem 270:3891–3903[Medline]
  15. Rena G, Guo SD, Cichy SC, Unterman TG, Cohen P 1999 Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem 274:17179–17183[Abstract/Free Full Text]
  16. Standaert ML, Galloway L, Karnam P, Bandyopadhyay G, Moscat J, Farese RV 1997 Protein kinase C-{zeta} as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport. J Biol Chem 272:30075–30082[Abstract/Free Full Text]
  17. Sakoda H, Ogihara T, Anai M, Funaki M, Inukai K, Katagiri H, Fukushima Y, Onishi Y, Ono H, Fujishiro M, Kikuchi M, Oka Y, Asano T 2000 Dexamethasone-induced insulin resistance in 3T3–L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction. Diabetes 49:1700–1708[Abstract]
  18. Turnbow MA, Keller SR, Rice KM, Garner CW 1994 Dexamethasone down-regulation of insulin receptor substrate-1 in 3T3–L1 adipocytes. J Biol Chem 269:2516–2520[Abstract/Free Full Text]
  19. Baumann CA, Ribon V, Kanzaki M, Thurmond DC, Mora S, Shigematsu S, Bickel PE, Pessin JE, Saltiel AR 2000 CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407:202–207[CrossRef][Medline]
  20. Goedert M, Cuenda A, Craxton M, Jakes R, Cohen P 1997 Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases. EMBO J 16:3563–3571[CrossRef][Medline]
  21. Cuenda A, Cohen P, Buee-Scherrer V, Goedert M 1997 Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J 16:295–305[CrossRef][Medline]
  22. Keyse SM, Emslie EA 1992 Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature 359:644–647[CrossRef][Medline]
  23. Alessi DR, Smythe C, Keyse SM 1993 The human CL100 gene encodes a Tyr/Thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras in Xenopus oocyte extracts. Oncogene 8:2015–2020[Medline]
  24. Muda M, Boschert U, Smith A, Antonsson B, Gillieron C, Chabert C, Camps M, Martinou I, Ashworth A, Arkinstall S 1997 Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4. J Biol Chem 272:5141–5151[Abstract/Free Full Text]
  25. Dickinson RJ, Williams DJ, Slack DN, Williamson J, Seternes OM, Keyse SM 2002 Characterization of a murine gene encoding a developmentally regulated cytoplasmic dual-specificity mitogen-activated protein kinase phosphatase. Biochem J 364:145–155[Medline]
  26. Tanoue T, Yamamoto T, Maeda R, Nishida E 2001 A Novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38 {alpha} and ß MAPKs. J Biol Chem 276:26629–26639[Abstract/Free Full Text]
  27. Tanoue T, Moriguchi T, Nishida E 1999 Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5. J Biol Chem 274:19949–19956[Abstract/Free Full Text]
  28. Keyse SM 2000 Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 12:186–192[CrossRef][Medline]
  29. Muda M, Boschert U, Dickinson R, Martinou JC, Martinou I, Camps M, Schlegel W, Arkinstall S 1996 MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J Biol Chem 271:4319–4326[Abstract/Free Full Text]
  30. Meng W, Swenson LL, Fitzgibbon MJ, Hayakawa K, ter Haar E, Behrens AE, Fulghum JR, Lippke JA 2002 Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export. J Biol Chem 277:37401–37405[Abstract/Free Full Text]
  31. Ben Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ 1998 Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol 8:1049–1057[CrossRef][Medline]
  32. Combettes-Souverain M, Issad T 1998 Molecular basis of insulin action. Diabetes Metab 24:477–489[Medline]
  33. Folli F, Saad MJA, Kahn CR 1996 Insulin receptor/IRS-1/PI 3-kinase signaling system in corticosteroid-induced insulin resistance. Acta Diabetol 33:185–192[Medline]
  34. Jiang GQ, Zhang BB 2002 PI 3-kinase and its up- and down-stream modulators as potential targets for the treatment of type II diabetes. Front Biosci 7:D904–D917
  35. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PRJ, Reese CB, Cohen P 1997 Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B {alpha}. Curr Biol 7:261–269[CrossRef][Medline]
  36. Williams MR, Arthur JSC, Balendran A, Van der KJ, Poli V, Cohen P, Alessi DR 2000 The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr Biol 10:439–448[CrossRef][Medline]
  37. Ribon V, Saltiel AR 1997 Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3–L1 adipocytes. Biochem J 324:839–845
  38. Clark AR 2003 MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids? J Endocrinol 178:5–12[Abstract]
  39. Telting D, van der Zon GC, Dorrestijn J, Maassen JA 2001 IRS-1 tyrosine phosphorylation reflects insulininduced metabolic and mitogenic responses in 3T3–L1 pre-adipocytes. Arch Physiol Biochem 109:52–62[CrossRef][Medline]
  40. Brondello JM, Mckenzie FR, Sun H, Tonks NK, Pouyssegur J 1995 Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts. Oncogene 10:1895–1904[Medline]
  41. Brondello JM, Brunet A, Pouyssegur J, Mckenzie FR 1997 The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J Biol Chem 272:1368–1376[Abstract/Free Full Text]
  42. Slack DN, Seternes OM, Gabrielsen M, Keyse SM 2001 Distinct binding determinants for ERK2/p38{alpha} and JNK MAP kinases mediate catalytic activation and substrate selectivity of MAP kinase phosphatase-1. J Biol Chem 276:16491–16500[Abstract/Free Full Text]
  43. Eblaghie MC, Lunn JS, Dickinson RJ, Munsterberg AE, Sanz-Ezquerro JJ, Farrell ER, Mathers J, Keyse SM, Storey K, Tickle C 2003 Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos. Curr Biol 13:1009–1018[CrossRef][Medline]
  44. Reed BC, Lane MD 1980 Insulin receptor synthesis and turnover in differentiating 3T3–L1 preadipocytes. Proc Natl Acad Sci USA 77:285–289[Abstract/Free Full Text]
  45. Simpson IA, Yver DR, Hissin PJ, Wardzala LJ, Karnieli E, Salans LB, Cushman SW 1983 Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim Biophys Acta 763:393–407[Medline]
  46. van den Berghe N, Ouwens DM, Maassen JA, van Mackelenbergh MG, Sips HC, Krans HM 1994 Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3- L1 adipocytes. Mol Cell Biol 14:2372–2377[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Ligands:   Dexamethasone  |  RU486



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. Li, I. S. Ali, and R. W. Currie
Insulin-induced myocardial protection in isolated ischemic rat hearts requires p38 MAPK phosphorylation of Hsp27
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H74 - H87.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
K. Horsch, H. de Wet, M. M. Schuurmans, F. Allie-Reid, A. C. B. Cato, J. Cunningham, J. M. Burrin, F. S. Hough, and P. A. Hulley
Mitogen-Activated Protein Kinase Phosphatase 1/Dual Specificity Phosphatase 1 Mediates Glucocorticoid Inhibition of Osteoblast Proliferation
Mol. Endocrinol., December 1, 2007; 21(12): 2929 - 2940.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
V. Gupta, N. Awasthi, and B. J. Wagner
Specific Activation of the Glucocorticoid Receptor and Modulation of Signal Transduction Pathways in Human Lens Epithelial Cells
Invest. Ophthalmol. Vis. Sci., April 1, 2007; 48(4): 1724 - 1734.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D. Qi and B. Rodrigues
Glucocorticoids produce whole body insulin resistance with changes in cardiac metabolism
Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E654 - E667.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Hiragun, Z. Peng, and M. A. Beaven
Cutting Edge: Dexamethasone Negatively Regulates Syk in Mast Cells by Up-Regulating Src-Like Adaptor Protein
J. Immunol., August 15, 2006; 177(4): 2047 - 2050.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Xu, Q. Yang, M. Shen, X. Huang, M. Dembski, R. Gimeno, L. A. Tartaglia, R. Kapeller, and Z. Wu
Dual Specificity MAPK Phosphatase 3 Activates PEPCK Gene Transcription and Increases Gluconeogenesis in Rat Hepatoma Cells
J. Biol. Chem., October 28, 2005; 280(43): 36013 - 36018.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
S. Kralisch, J. Klein, U. Lossner, M. Bluher, R. Paschke, M. Stumvoll, and M. Fasshauer
Hormonal regulation of the novel adipocytokine visfatin in 3T3-L1 adipocytes
J. Endocrinol., June 1, 2005; 185(3): R1 - R8.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Bazuine, F. Carlotti, M. J. W. E. Rabelink, J. Vellinga, R. C. Hoeben, and J. A. Maassen
The p38 Mitogen-Activated Protein Kinase Inhibitor SB203580 Reduces Glucose Turnover by the Glucose Transporter-4 of 3T3-L1 Adipocytes in the Insulin-Stimulated State
Endocrinology, April 1, 2005; 146(4): 1818 - 1824.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
T. Hiragun, Z. Peng, and M. A. Beaven
Dexamethasone Up-Regulates the Inhibitory Adaptor Protein Dok-1 and Suppresses Downstream Activation of the Mitogen-Activated Protein Kinase Pathway in Antigen-Stimulated RBL-2H3 Mast Cells
Mol. Pharmacol., March 1, 2005; 67(3): 598 - 603.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
18/7/1697    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services