Dual specificity mitogen-activated protein (MAP) kinase phosphatase-4 plays a potential role in insulin resistance.

Insulin is the key hormone that controls glucose homeostasis. Dysregulation of insulin function causes diabetes mellitus. Among the two major forms of diabetes, type 2 diabetes accounts for over 90% of the affected population. The incidence of type 2 diabetes is highly related to obesity. To find novel proteins potentially involved in obesity-related insulin resistance and type 2 diabetes, a functional expression screen was performed to search for genes that negatively regulate insulin signaling. Specifically, a reporter system comprised of the PEPCK promoter upstream of alkaline phosphatase was used in a hepatocyte cell-based assay to screen an expression cDNA library for genes that reverse insulin-induced repression of PEPCK transcription. The cDNA library used in this study was derived from the white adipose tissue of ob/ob mice, which are highly insulin-resistant. The mitogen-activated dual specificity protein kinase phosphatase 4 (MKP-4) was identified as a candidate gene in this screen. Here we show that MKP-4 is expressed in insulin-responsive tissues and that the expression levels are up-regulated in obese insulin-resistant rodent models. Heterologous expression of MKP-4 in preadipocytes significantly blocked insulin-induced adipogenesis, and overexpression of MKP-4 in adipocytes inhibited insulin-stimulated glucose uptake. Our data suggest that MKP-4 negatively regulates insulin signaling and, consequently, may contribute to the pathogenesis of insulin resistance.

Type 2 diabetes mellitus is growing as a major public health problem in the United States and other industrialized countries (1). The rapid increase in the number of people diagnosed with the disease has triggered more interest in finding novel and better therapeutic agents (2). Type 2 diabetes is a polygenic disease with complicated etiology. Although significant progress has been made in the identification of genes that contribute to the disease, many pieces of the puzzle are still missing. Insulin resistance, characterized by elevated circulating insulin levels and impaired glucose tolerance, usually precedes Type 2 diabetes. Whether an individual with insulin resistance ultimately develops diabetes depends on the secretion capacity of pancreatic ␤ cells (3). Drugs aiming to stimulate insulin secretion can improve hyperglycemia but only work on patients with relatively mild dysfunction (4). Improving insulin sensitivity promises a preferable approach to treat diabetes.
Insulin is an anabolic hormone that controls many aspects of metabolism. The MAP 1 kinase cascade and the PI3-kinase pathway are two of the signaling networks activated by insulin upon binding to its receptor. The activation of ERK by insulin seems to play a major role in insulin-mediated mitogenesis, whereas PI3-kinase has been implicated as a major key player of the metabolic arm. Defects in the insulin-stimulated metabolic cascade lead to insulin resistance. Therefore, to identify additional genes involved in negatively regulating insulinstimulated metabolic pathway(s), we employed an expression cloning approach using the promoter of the phosphoenol pyruvate carboxykinase gene (PEPCK) placed upstream of secreted alkaline phosphatase as a surrogate reporter system for the metabolic effects of insulin.
The action of insulin modifies the activity of a multitude of proteins within minutes and regulates expression of about 100 genes in a matter of hours (5). Among the genes regulated by insulin, the expression of PEPCK, a key enzyme in hepatic gluconeogenesis, has been studied most extensively (6,7). There has been no evidence to support post-translational modification or allosteric activation of PEPCK by insulin, and transcriptional regulation is thought to be the only way by which insulin regulates PEPCK levels (5). Therefore, the PEPCK promoter is an ideal tool in a functional screening to reflect transcriptional changes in response to insulin stimulation. Furthermore, it has been shown that insulin-mediated repression of PEPCK transcription can be completely blocked by wortmannin (a PI3-kinase inhibitor), indicating the essential role of PI3-kinase in this pathway (8). On the other hand, kinase inhibitors and dominant-negative mutants of ERK pathway, which block insulin mitogenic signaling, had no effect on insulin-mediated PEPCK promoter repression (9,10). Therefore, regulation of PEPCK transcription by insulin seems to use the same signaling pathways involved in insulin-mediated metabolic effects. Our hypothesis is that genes blocking the inhibitory effect of insulin on PEPCK transcription are also likely to attenuate the metabolic signaling pathway(s) elicited by insulin. Using this approach, we identified mitogen-activated protein kinase phosphatase 4 (MKP-4), a dual specificity protein phosphatase (11), as a candidate gene involved in the negative regulation of insulin signaling. Further experiments * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY316312.

MATERIALS AND METHODS
Cells and Reagents-The rat hepatoma cell line H4IIE was cultured in DMEM supplemented with 5% fetal bovine serum (Invitrogen). For the PEPCK promoter assay, H4IIE cells were cultured in DMEM supplemented with 0.1% fetal bovine serum as well as a mixture of 100 M cAMP, 1 M dexamethasone, and/or 10 nM insulin, as indicated in the figures. The 3T3-F442A cell line was cultured in DMEM supplemented with 10% bovine calf serum (Hyclone). For differentiation, 3T3-F442A cells were switched to DMEM supplemented with 10% cosmic calf serum (Hyclone). Cells were first induced with a mixture of 5 g/ml insulin, 1 M dexamethasone, and 0.5 mM isobutyl methylxanthine for 3 days and then maintained in 5 g/ml insulin until they fully differentiated into adipocytes.
Construction of the Reporter Construct and cDNA Library-The PEPCK promoter (Ϫ548 to ϩ73) (12) was amplified from rat genomic DNA and cloned upstream of the placental alkaline phosphatase coding sequence (13) in the pMet7 vector. For the cDNA library construction, RNA was extracted from the white adipose tissue of ob/ob mice and reverse-transcribed using oligo(dT). The cDNAs were then cloned into the pMet7 expression vector.
Transfection of H4IIE Cells-Cells were seeded into 96-well plates (1:5 dilution from a confluent 10-cm dish) 1 day prior to transfection. Fresh medium (DMEM/5% fetal calf serum) was added 5 h prior to transfection. The following day, the PEPCK reporter construct (0.156 g/well) was co-transfected with dominant-negative p85 (DNp85) or single cDNA clones from the white adipose library (0.125 g/well) into H4IIE cells using FuGene 6 (0.38 l/well) (Roche Applied Science). Medium was then exchanged, the following morning, to DMEM/0.5% fetal calf serum. Stimulants and/or inhibitors were added 5 h later, as indicated in the figures, and incubated for 16 h. Then, medium was collected on the following day and assayed for alkaline phosphatase activity.
Alkaline Phosphatase Assay-The medium was collected from transfected H4IIE cells and spun down to remove floating cells. The samples were heat-inactivated at 65°C for 30 min. The Great EscAPE kit (Clontech) was used to detect the alkaline phosphatase activity in conditioned medium. The samples were read on the Wallac Luminometer (Dynex Tech).

Construction of Expression Vectors and Establishment of Cell Lines Stably Expressing MKP-4 -
The full-length cDNA of mouse MKP-4 was cloned into a retroviral vector expressing the puromycin resistance gene. The retroviral expression vectors were transfected into a packaging cell line Bosc23 (14) by the LipofectAMINE method (Invitrogen). Forty-eight h after transfection, the conditioned medium containing retrovirus was collected and filtered through a 0.45-M syringe filter. The viral supernatant was then used to infect the recipient 3T3-F442A cells, which were seeded 24 h before infection (15). After an overnight incubation, cells were fed with fresh medium, and puromycin was added at the concentration of 5 g/ml for selection and subsequent maintenance.
RNA Extraction and Northern Blot Analysis-RNA samples were extracted using the TRIzol reagent (Invitrogen). Following denaturation, RNAs were loaded on a 1% agarose gel containing 3% formaldehyde. After electrophoresis, RNAs were transferred to a Biotran membrane (ICN), UV cross-linked, and baked at 80°C for 1 h. Hybridization with [␣ 32 P[dCTP (PerkinElmer Life Sciences)-labeled cDNA probes and subsequent washings were done as described previously (16). Photographs of ethidium bromide gels were presented for loading adjustment.
Glucose Uptake-For glucose uptake experiments, fully differentiated 3T3-L1 cells were infected with adenovirus expressing MKP-4 or vector control on 24-well plates. Cells were serum-starved for 24 h prior to insulin stimulation. Cells were then washed three times with Krebs Ringers Henseleit buffer (120 mM NaCl, 5 mM KCl, 1 mM KH 2 PO 4 , 0.6 mM MgSO 4 , 1 mM CaCl 2 , 20 mM Hepes, pH 7.5, and 0.5% bovine serum albumin) and incubated in 1 ml of Krebs Ringers Henseleit Ϯ insulin for 10 min at 37°C. Glucose uptake was initiated by the addition of 2-deoxy-D-[1,2-3 H] glucose (1 ci/ml) for 10 min. The reaction was terminated by immediately removing the medium and washing cell monolayers four times with cold Krebs Ringers Henseleit. Cells were then removed in 0.15 ml of 0.1% SDS, and the radioactivity was determined by liquid scintillation counting.

Identification of MKP-4 -As a Negative Regulator of Insulin
Signaling-Individual clones of a cDNA expression library derived from the white adipose tissue of ob/ob mice were cotransfected with a PEPCK promoter (Ϫ600 bp) construct into H4IIE cells, an insulin-responsive rat hepatoma cell line (17). The PEPCK promoter was placed upstream of the secreted alkaline phosphatase (PEPCKp600-SEAP). It is well known that dexamethasone and cAMP strongly stimulate transcription of the PEPCK gene, whereas insulin shuts down the PEPCK gene expression exclusively at transcription level (6, 7). The 600-bp PEPCK promoter used in this study contains an insulin response unit, a glucocorticoid response unit, and a cAMP response unit (7). Using the secreted placental alkaline phosphatase (AP) as a reporter gene, the rate of transcription is reflected by the activity of AP in the conditioned media. Complementary DNA clones that resulted in high AP activity even in the presence of insulin were selected and confirmed in secondary assays. As a positive control, a dominant-negative form of the regulatory subunit of PI3-kinase, p85 (DNp85) (18), was cotransfected with PEPCKp600-SEAP. The mutant p85 rescued AP activity to ϳ50% of the control level in the presence of insulin (Fig. 1). The control level was defined as AP activity stimulated by dexamethasone plus cAMP in the absence of insulin. About 10,000 clones were screened using this system. This study describes the identification of one of the cDNA clones, which reproducibly restored AP activity to a similar extent when compared with the DNp85 (Fig. 1). Follow-up sequence analysis revealed that this clone is the mouse orthologue of human MKP-4 (11). MKP-4 belongs to the dual specificity protein phosphatase family and was originally identified as a phosphatase that blocks activation of the MAP kinase signaling cascade. Human and mouse MKP-4 sequences have been previously reported (11,19), and we identified the rat orthologue in this study by sequence homology search. The sequence identity is 94% between human and rat MKP-4 at the amino acid level and 83% between human and mouse MKP-4 (Fig. 2). The relative lower homology between human and mouse MKP-4 is due to the presence of a 67-residue repetitive sequence, which is present in the N terminus of the mouse enzyme (the insert is 63 residues in the literature (19)), absent in the human orthologue, but partially present in the rat orthologue. Since the 67-residue repetitive sequence is also pres-  lanes 1 and 2), the dominant-negative 85-kDa subunit of PI3-kinase (DNp85, lane 3), or MKP-4 (lane 4). All samples were stimulated with a mixture of cAMP and dexamethasone, and samples from lanes 2-4 were also stimulated with insulin. AP activities were measured by using conditioned media. ent in genomic DNA, it is unlikely to be an artifact of cloning. It remains to be determined why the repetitive sequence exists in the mouse and rat genome.
Tissue Distribution and Regulation of MKP-4 -The human orthologue of MKP-4 was only detected in kidney and placenta among more than 20 tissues examined in the original cloning study (11). However, no information was available regarding the expression of MKP-4 in adipose tissue. Therefore, the expression of MKP-4 was examined in various murine tissues including the white and brown adipose tissues. Mouse MKP-4 was only detectable in white adipose tissue, kidney, and testis (Fig. 3A). The adipose expression of this gene was also confirmed in multiple human fat samples (data not shown). To determine the source of MKP-4 signal in adipose tissue, white fat was separated into stromal-vascular cells and adipocytes. The expression of MKP-4 was predominantly in the adipocyte fraction (Fig. 3B), indicating that it is an adipocyte gene. The expression of MKP-4 was also determined in a cultured preadipocyte cell line 3T3-F442A. The expression level of this gene was low in preadipocytes but up-regulated during adipogenesis (Fig. 3C), indicating a role for MKP4 in mature adipocytes. To understand the physiological relevance of this dual specificity phosphatase in obesity and insulin resistance, the MKP-4 expression was examined in insulin-responsive tissues of several rodent obesity models. The expression level of MKP-4 was significantly increased in the white adipose tissue of ob/ob, db/db, and diet-induced rodent obesity models (Fig. 4, top panels, lanes 1-8). In the lean mice on regular diet, MKP-4 was undetectable in brown adipose tissue, liver, and muscle. In the obese state, however, MKP-4 could be detected in each of these tissues (Fig. 4, top right panel, lanes 9 -12; bottom left panel,  lanes 1-8). Although MKP-4 was also expressed in kidney and testes, no regulation was observed in the above rodent obesity models (Fig. 4, bottom panels, lanes 9 -16), suggesting that the up-regulation of MKP-4 is specific to insulin-responsive tissues. Since liver, muscle, white adipose tissue, and brown adipose tissue express high levels of insulin receptor and play important roles in insulin sensitivity, the up-regulation of MKP-4 in insulin-responsive tissues indicates a potential role for MKP-4 in insulin signaling and in obesity-related insulin resistance.
MKP-4 in Adipocyte Function-To further understand the role of MKP-4 in adipocyte biology, MKP-4 was stably expressed in 3T3-F442A preadipocytes by a retroviral approach. Then, the cells were induced to differentiate by either insulin alone or a mixture of insulin, dexamethasone, and isobutylmethylxanthine. The experiment was terminated when more than 90% of control vector-expressing cells induced by the mixture fully differentiated into adipocytes, which is 7 days after induction. Cells stably expressing MKP-4 differentiated poorly under both induction conditions as shown by oil red O staining (Fig. 5A). This experiment indicates that MKP-4 has an inhibitory effect on adipogenesis under both conditions. The differentiation was quantificated by the percentage of cells stained positive by oil red O with vector-expressing cells arbitrarily set as 100% (Fig. 5B, bottom graph). MKP-4-expressing cells treated with the full mixture did not fully differentiate when kept up to 2 weeks after induction (data not shown). To study the effect of MKP-4 on adipogenesis at the molecular level, two adipocyte differentiation marker genes were examined by Taqman analysis. As shown in Fig. 5B, the expression levels of both glut4 and peroxisome proliferator-activated receptor-␥ were significantly reduced in MKP-4-expressing cells when compared with control cells.
The fact that MKP-4 has the ability to inhibit insulin-stim-FIG. 2. Amino acid homology among orthologues of MKP-4. Amino acids are indicated using the single-letter codes. Sequence alignment was performed by using Clustal W version 1.7 software. * indicates positions that have fully conserved residues; : indicates strongly conserved residues; and . indicates weakly conserved residues. The strong and weak groups are defined as strong score Ͼ0.5 and weak score Ͻ0.5, respectively, in the Gonnet Pam250 matrix. GenBank TM accession number for mMKP-4, AY316312; GenBank TM accession number for rMKP-4, XM_2197117; GenBank TM accession number for hMKP-4, Y08302.
ulated adipocyte differentiation prompted more interest in this gene. Since MKP-4 was expressed at higher levels in white adipose when compared with other peripheral insulin-responsive tissues and was also significantly up-regulated in obesity, it might have an impact on adipose glucose disposal. To explore the role of MKP-4 in insulin resistance, glucose uptake experiments were performed using fully differentiated 3T3-L1 cells overexpressing MKP-4. Mature adipocytes were infected by adenovirus expressing either MKP-4 or GFP for 48 h. Insulinstimulated glucose uptake was measured 72 h after infection. The capability of adipocytes to take up glucose was blunted about 30% by MKP-4 overexpression when compared with GFP-expressing control cells (Fig. 6), suggesting that elevated MKP-4 plays a role in reducing insulin-regulated glucose disposal. DISCUSSION A functional expression cloning approach was applied to identify genes that may play a role in insulin resistance in this specific study. Complementary DNA clones from the insulinresistant adipose tissue of ob/ob mice were screened. As a result of this screen, MKP-4, a dual specificity protein phosphatase, was found to block insulin signaling in this system. This phosphatase belongs to the MKP family, which leads to the downregulation of MAPK signaling cascades by dephosphorylating critical tyrosine and serine/threonine residues on MAPKs (20). It is interesting to note that a member of the MKP family inhibits insulin-mediated repression of the PEPCK promoter since complete blockage of ERK-mediated signaling, with kinase inhibitors and dominant negatives, has no effect on PEPCK gene expression (9,10). In addition to the ERK pathway, recent studies showed that the stress-induced P38 pathway can also be activated by insulin (21). Interestingly, P38 is involved in ATF-2-mediated induction of PEPCK transcription (22). MKP-4 has been reported to inactivate both ERK and P38, whereas its effect on JNK remains controversial (11,19). Since both P38 and MKP-4 are capable of enhancing PEPCK transcription, it is unlikely that MKP-4 blocks insulin-mediated repression on PEPCK promoter by inactivating P38. It is well known that a PI3-kinase-dependent pathway is required for repression of PEPCK transcription by insulin. These data suggest that MKP-4 is involved in dephosphorylating kinase components in the insulin-induced metabolic cascade. This piece of evidence demonstrates that the MAP kinase pathway can cross-talk with the metabolic pathway. Further studies are necessary to identify the substrates of MKP-4 in insulin-mediated metabolic responses.
To our knowledge, this is the first time that a dual specificity phosphatase has been implicated in insulin resistance. Previous work in the field was focused extensively on protein-tyrosine phosphatases. Among the protein-tyrosine phosphatases that attenuate the insulin signaling pathway, PTP-1B is the most extensively characterized and well validated, whereas the role of leukocyte antigen-related protein-tyrosine phosphatase (LAR) remains controversial (23,24). The major impact site of PTP-1B is known to be in liver, and overexpression of PTP-1B in adipocytes did not affect glucose uptake (25). Our results suggest that MKP-4 might be a major phosphatase involved in adipose insulin resistance since it is expressed and up-regulated in adipose tissue of several rodent obesity models. Furthermore, MKP-4 can blunt insulin-stimulated glucose uptake in cultured adipocytes, which supports our hypothesis. In addition, MKP-4 is undetectable in other insulin-responsive tissues under normal circumstances but becomes detectable in brown adipose tissue, liver, and muscle of obese mice, indicating that MKP-4 might also attenuate insulin signaling in other insulin-responsive tissues in the obese state.
The mitogen activated dual specificity MAP kinase phosphatase family has nine members (20). These phosphatases have distinct tissue distribution patterns and different substrate preferences. The closest homologues of MKP-4 are MKP-3 and MKP-X, sharing sequence identity of 56 and 61%, respectively, at the amino acid level. In fact, MKP-3 was also identified as a weaker candidate gene during the functional expression screen. The appearance of two dual specificity phosphatases in our screen indicates the involvement of a new class of phosphatases in insulin action and insulin resistance. When compared with tyrosine phosphatases, which only dephosphorylate tyrosine residues, dual specificity phosphatases also dephosphorylate serine/threonine residues. One feature of this class of protein phosphatases is that they contain both a regulatory domain and a catalytic domain, whereas PTP-1B is comprised only of a catalytic domain, which is constitutively active. Phosphorylation of IRS-1 on serine residues has been proposed as a mechanism for inducing IRS1 degradation as well as reducing the tyrosine kinase activity of insulin receptor (26,27). It remains to be studied whether dual specificity phosphatases such as MKP-4 have any effect on IRS1 serine/threonine phosphorylation. Future loss-of-function studies may further define the role of MKP-4 in insulin resistance in vivo. If confirmed to be an important regulator in obesity-related insulin resistance, MKP-4 presents a new molecular target for pharmaceutical intervention to improve systemic insulin sensitivity.