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Insulin Response Sequence-Dependent and -Independent Mechanisms Mediate Effects of Insulin on Glucocorticoid-Stimulated Insulin-Like Growth Factor Binding Protein-1 Promoter Activity

Departments of Medicine and Physiology and Biophysics, University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Terry G. Unterman, M.D., Room 6229, Medical Research Unit (MP 151), Veterans Affairs Chicago Health Care System (West Side), 820 South Damen Avenue, Chicago, Illinois 60612. E-mail: unterman{at}uic.edu.

	Abstract

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IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor- coactivator-1, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in which insulin regulates IGFBP-1 expression through both IRS-dependent and IRS-independent mechanisms.

	Introduction

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IGF BINDING PROTEINS (IGFBPs) are a family of proteins that bind IGFs with high affinity and exert both IGF-dependent and -independent effects (1). IGFBP-1 is an approximately 30-kDa protein that is produced largely in the liver in which its expression is rapidly regulated at the level of gene transcription (2, 3). Circulating levels of IGFBP-1 are rapidly regulated in response to fasting and feeding, and circulating IGFBP-1 is thought to be the major short-term modulator of IGF bioavailability (4). IGFBP-1 also exerts local effects in the liver, in which it is thought to contribute to the capacity for regeneration in response to injury (5), and in the decidualized endometrium, in which it is a major secretory product and a marker of differentiation (5, 6).

Early studies regarding the regulation of IGFBP-1 revealed that its expression is stimulated by glucocorticoids and that this effect is suppressed by insulin in liver-derived cells (7, 8). Glucocorticoids regulate the abundance of IGFBP-1 mRNA in the liver through effects on both gene transcription (9) and mRNA stability (10, 11). Whereas the mechanisms mediating effects of glucocorticoids on IGFBP-1 mRNA stability are poorly defined, the effects of glucocorticoids on IGFBP-1 gene transcription are better understood. We previously identified a glucocorticoid response element (GRE) in the proximal IGFBP-1 promoter located approximately 100 bp upstream from the transcription start site based on DNase footprinting and reporter gene studies (12). This GRE is contiguous to two insulin response sequences [insulin response sequence (IRS)A and IRSB], which are essential for the ability of insulin to suppress basal IGFBP-1 promoter activity (13). Interestingly, mutation of the IRSA also reduces the ability of glucocorticoids to stimulate promoter activity (14, 15), suggesting that factors that interact with this IRS function cooperatively with glucocorticoids to stimulate promoter activity. This observation also suggested that disrupting the function of these factors might contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter activity (14). In the present study, we used reporter gene constructs to further define the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression.

Studies with the IGFBP-1 promoter have been important in defining the mechanisms by which insulin can suppress gene expression through an IRS. We reported that members of the Forkhead transcription factor family binding to this insulin response region (14) and that signaling through the phosphatidylinositol-3' kinase (PI3K)/protein kinase B (PKB) pathway is necessary and sufficient to mediate effects of insulin on basal promoter activity through an IRS (13). These results suggested that signaling by PI3K/PKB to Forkhead proteins may be important in mediating effects of insulin on gene expression. Subsequently, studies in Caenorhabditis elegans provided genetic evidence that members of the FoxO subfamily of Forkhead proteins are major targets of insulin-like signaling downstream from PKB (16, 17), and studies in this and other laboratories confirmed that FoxO proteins associate with IRSs in the IGFBP-1 promoter (18, 19, 20) and that phosphorylation by PKB promotes nuclear exclusion and suppresses transactivation by FoxO proteins (20, 21). The phosphorylation of FoxO proteins, and possibly other Forkhead proteins (22), is now thought to contribute to the effects of insulin on a number of genes involved in gluconeogenesis (23, 24, 25) and effects of growth factors on cell growth and survival (26, 27). At the same time, it is important to note that other factors interacting with IRS-like sequences also may contribute to the effects of insulin and growth factors on gene expression (28, 29, 30).

FoxO proteins also function cooperatively with glucocorticoids to stimulate the expression of IGFBP-1 (31) and several other genes, including pyruvate dehydrogenase kinase 4 (32) and atrogin (33), possibly by helping to recruit cAMP response element binding protein/p300 and/or other coactivator proteins (31). Disrupting FoxO functions, or possibly other factors that interact with the IRS, might help to mediate the effects of insulin on both basal and glucocorticoid-stimulated IGFBP-1 promoter activity.

To better understand the mechanisms mediating effects of insulin on gene expression, we examined the role of IRS-associated factors in mediating effects of insulin on the glucocorticoid-stimulated promoter. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal but not glucocorticoid-stimulated IGFBP-1 promoter activity, indicating that IRS-independent effects also contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter. Cotransfection studies indicate that this effect of insulin is not due to suppression of peroxisomal proliferator-activated receptor- coactivator (PGC)-1, an insulin-regulated coactivator that contributes to transcriptional activity of the glucocorticoid receptor (25, 34). Studies with kinase inhibitors indicate that multiple signaling pathways contribute to the effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. Together these results indicate that the regulation of IGFBP-1 gene expression by insulin and glucocorticoids reflects an integrated response that is mediated through both IRS-dependent and -independent mechanisms.

	Materials and Methods

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Reporter gene constructs and expression vectors
The SauI-HgaI fragment of the IGFBP-1 promoter was inserted into the pGL2 polylinker (Promega, Madison, WI) (BP1.luc) and the mutation of the IRSs (IRSmut) reporter gene construct containing mutations of IRSA and IRSB was created by site-directed mutagenesis as reported previously (12). The IRS-GRE construct was created by mutating both halves of the GRE in the IRSmut reporter gene construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The sequence of the mutated promoter was confirmed by dideoxy sequencing in the University of Illinois at Chicago Sequence Center and is provided in Fig. 1.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Effect of dexamethasone on IGFBP-1 promoter activity. A, Role of IRS and GRE. HepG2 cells were transfected with calcium phosphate precipitates containing a total of 10 µg plasmid DNA, including 1 µg of a luciferase reporter gene containing a fragment of the IGFBP-1 promoter, which extends 320 bp 5' to the transcription start site. Mutations were introduced to disrupt the function of insulin response sequences A and B (IRSmut) or the IRSs together with the nearby glucocorticoid response element (IRS/GRE). Cells were treated with serum-free medium (SFM) with or without 10 µM dexamethasone (Dex) for 18 h before lysis and analysis of luciferase activity. For each reporter gene construct, promoter activity in cells treated with dexamethasone treated is shown relative to promoter activity in cells treated with SFM alone (mean ± SEM). Transient transfection studies were performed in triplicate and the combined results from at least three experiments are shown. B, Effect of glucocorticoid receptor is enhanced by IRS. HepG2 cells were cotransfected with IGFBP-1 promoter reporter gene constructs with or without mutation of the insulin sequences plus 0, 10, 100, or 300 ng of the human GR (hGR) expression vector. Cells were treated with SFM with or without 10 µM dexamethasone (Dex) for 18 h before lysis and analysis of luciferase activity. The effect of Dex on promoter activity is shown relative to promoter activity in cells treated with SFM alone. C, Mutations of IRSs and GRE. The sequences for IRSA, IRSB, and the GRE located nearby in the proximal IGFBP-1 promoter, and the mutations introduced into the constructs used in this study are shown.

Cell culture and transient transfection studies
HepG2 cells were plated in 65-mm dishes in Opti-MEM (Invitrogen, Carlsbad, CA) with 5% bovine calf serum (Invitrogen) and refed with DMEM with 10% bovine calf serum before transfection with calcium phosphate precipitates of plasmid DNAs, as previously reported (14). Transfected cells were rinsed twice with Hanks’ balanced salt solution (Sigma, St. Louis, MO) and then refed with DMEM with 1 mg/ml BSA with or without 10 µM dexamethasone and/or 100 nm recombinant human insulin for 18 h before lysis and analysis of luciferase activity.

	Results

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Effect of dexamethasone on IGFBP-1 promoter activity
To assess the role that IRSs play in glucocorticoid regulation of IGFBP-1 promoter activity, we performed reporter gene studies in HepG2 cells using luciferase reporter gene constructs containing the IGFBP-1 promoter with or without mutation of both the IRSs and the GRE (IRS/GRE). As shown in Fig. 1A, treatment with dexamethasone increases the activity of the intact IGFBP-1 promoter by 3-fold, whereas mutation of the IRSs reduces this effect by approximately 50%, supporting the concept that factors that interact with this region enhance the ability of glucocorticoids to stimulate promoter activity. Mutation of the GRE completely disrupts the remaining effect of glucocorticoids, indicating that the IRS-independent effect of glucocorticoids requires the presence of this GRE.

Because the expression of endogenous glucocorticoid receptors is thought to be low in HepG2 cells, we asked whether IRS-associated factors also contribute to the ability of glucocorticoids to stimulate promoter activity when the availability of glucocorticoid receptors is not limiting. As shown in Fig. 1B, we performed cotransfection studies with an expression vector for the glucocorticoid receptor (GR). Cotransfection with 30, 100, or 300 ng of the GR expression vector increases the ability of dexamethasone to stimulate the activity of both the wild-type and IRSmut IGFBP-1 promoter in a dose-dependent fashion. At each level of GR expression, the effect of dexamethasone on promoter activity is diminished by mutation of the IRSs, indicating that the effect of the IRSs on glucocorticoid function is independent of the level of GR expression.

Dexamethasone treatment enhances the effect of insulin
We also examined the effect of glucocorticoid treatment on the regulation of promoter activity by insulin using reporter gene constructs with/without mutation of the IRSs and/or GRE. As shown on the left in Fig. 2, insulin inhibits basal IGFBP-1 promoter activity by approximately 45% in the absence of dexamethasone. In contrast, no effect of insulin on basal promoter activity is seen in cells transfected with reporter gene constructs in which the IRSs have been mutated, consistent with previous reports (13). As shown by the solid bar on the right in Fig. 2, the ability of insulin to suppress IGFBP-1 promoter activity is enhanced in cells that have been treated with dexamethasone (65% inhibition by insulin, P < 0.01 vs. no dexamethasone). Mutation of the GRE disrupts the ability of dexamethasone to enhance the effect of insulin on promoter activity (data not shown), indicating that this effect is mediated through the GRE. To our knowledge, this result provides the first evidence that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that this effect is mediated through the GRE.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Effect of insulin (INS) on basal and dexamethasone-stimulated IGFBP-1 promoter activity. HepG2 cells were cotransfected with reporter gene constructs containing the 320-bp IGFBP-1 promoter with (BP1mut; open bar) or without (BP1.luc; solid bar) mutation of the IRSs or mutation of IRS/GRE (hatched bar), as in Fig. 1, and treated with serum-free medium (SFM) with or without 100 nM insulin and/or 10 µM dexamethasone (DEX) for 18 h before lysis and analysis of luciferase activity. The effect of insulin on basal promoter activity is shown on the left: for each reporter gene construct, luciferase activity in cells treated with SFM plus insulin is expressed relative to luciferase activity in cells treated with SFM alone. The effect of insulin on promoter activity in DEX-treated cells is shown on the right: for each construct, luciferase activity in cells treated with SFM plus DEX plus insulin is expressed relative to luciferase activity in cells treated with SFM plus DEX. Transient transfection studies were performed in triplicate, and the combined results of three separate experiments are shown.

IRS-independent effect of insulin is context-dependent
We next asked whether the ability of insulin to suppress dexamethasone-stimulated promoter activity through an IRS-independent mechanism is context independent or whether other elements within the IGFBP-1 promoter also might be required. Here we performed transient transfection studies with a luciferase reporter gene construct containing three copies of the GRE from the tyrosine aminotransferase gene immediately upstream of a minimal promoter (pTAT3.luc) or the reporter gene construct containing the IGFBP-1 promoter in which the IRSs have been mutated (BP1.IRSmut). As shown in Fig. 3, insulin inhibits the ability of dexamethasone to stimulate the activity of the BP1.IRSmut promoter by approximately 40% in HepG2 cells (Fig. 3A) but does not inhibit the ability of dexamethasone to stimulate the activity of the pTAT3 promoter (Fig. 3B). This result indicates that the ability of insulin to inhibit the effect of dexamethasone on promoter activity is context dependent and is not due solely to the ability of insulin to suppress transactivation by the glucocorticoid receptor.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effect of insulin (INS) on dexamethasone (DEX)-stimulated IGFBP-1 promoter activity is context dependent. HepG2 cells were transfected with 10 µg of plasmid DNA, including 200 ng of the human GR (hGR) expression vector, and 1 µg of the IRSmut (A) or the pTAT3.luc (B) reporter gene construct, which contains three copies of the GRE from the tyrosine aminotransferase gene. Cells were treated with or without 10 µM Dex and/or 100 nM insulin for 18 h before lysis and analysis of luciferase activity. The effect of insulin and Dex on IGFBP-1 promoter activity is shown relative to control.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Effects of kinase inhibitors on insulin-regulated IGFBP-1 promoter activity. HepG2 cells were transfected with reporter gene constructs with or without the p85 expression vector and treated with or without insulin (INS), glucocorticoids, LY294002 (LY), and/or PD98509 (PD) for 18 h before lysis and analysis of luciferase activity, as in Fig. 2. Solid bars show the effect of insulin on basal activity of the IGFBP-1 promoter in cells transfected with the BP1.luc reporter gene construct and treated with or without kinase inhibitors. Hatched bars show the ability of insulin to inhibit stimulation of promoter activity by dexamethasone (Dex) in cells transfected with the IRSmut reporter gene construct and treated with or without kinase inhibitors.

Suppression of PGC-1 is not required for IRS-independent effect of insulin
PGC-1 is a coactivating protein that interacts with and contributes to the ability of the glucocorticoid receptor to stimulate gene expression (21, 27), and recent studies have suggested that insulin can suppress hepatic expression of PGC-1 (25). To determine whether insulin may suppress glucocorticoid-stimulated promoter activity by limiting the availability of PGC-1 in HepG2 cells, we performed cotransfection studies with a PGC-1 expression vector. As shown in Fig. 5A, the expression of PGC-1 enhances the ability of dexamethasone to stimulate the IRSmut promoter in a dose-dependent fashion (Fig. 5A, solid bars), consistent with the concept that PGC-1 can interact with and enhance the function of the GR. However, overexpression of PGC-1 does not limit the ability of insulin to suppress promoter activity (Fig. 5A, hatched bars). This result indicates that the ability of insulin to inhibit dexamethasone-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism in HepG2 cells is not due to limited availability of PGC-1 .

View larger version (27K): [in this window] [in a new window]   FIG. 5. Effects of PGC-1 on IGFBP-1 promoter activity and regulation. A, Effect of PGC-1 on IRS-independent regulation of promoter activity by dexamethasone and insulin. HepG2 cells were cotransfected with the BP1.IRSmut reporter gene construct and the vector expressing the human GR (hGR), treated with or without 10 µM dexamethasone (Dex) and/or 100 nM human insulin (INS) for 18 h before lysis and analysis of luciferase activity. The effect of treatment with dexamethasone on promoter activity is shown in solid bars, and the effect of dexamethasone plus insulin on promoter activity is shown in hatched bars. B, Effect of insulin on transactivation by PGC-1. HepG2 cells were transfected with 10 µg plasmid DNA, including 1 µg of the pG5e1b.luc reporter gene construct, which contains five Gal4 binding sites immediately upstream of the e1b minimal promoter and luciferase cDNA, together with 1 µg of a vector expressing the Gal4 DNA binding domain in-frame with either full-length PGC-1 (G4.PGC-11–797) or the truncated PGC-1 (G4.PGC-11–482), in which the C-terminal region of PGC-1 has been deleted. Cells were treated with or without 100 nM insulin 18 h before lysis and analysis of luciferase activity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we sought to better understand the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression. Because glucocorticoids can exert effects on IGFBP-1 expression through effects on both IGFBP-1 gene transcription and IGFBP-1 mRNA stability, we used reporter gene constructs to directly assess the effects of glucocorticoids and insulin on IGFBP-1 promoter activity. Previous studies have shown that glucocorticoids and insulin regulate IGFBP-1 promoter activity through IRSs and a GRE located in the proximal IGFBP-1 promoter (12, 39). Mutating the IRSs has been shown to reduce the ability of glucocorticoids to stimulate IGFBP-1 promoter activity (14), and this has suggested that insulin may suppress IGFBP-1 glucocorticoid-stimulated promoter activity by disrupting the function of trans-acting factors that interact with these IRSs. Here we used IGFBP-1 promoter constructs with/without mutations of the IRSs or GRE to better define the mechanisms mediating the effects of insulin and glucocorticoids on IGFBP-1 gene expression.

Initial studies revealed that glucocorticoid treatment increases the ability of insulin to suppress the function of the intact IGFBP-1 promoter and that mutation of the GRE disrupts this effect, indicating that interaction between the GR and the GRE is required for this effect. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal IGFBP-1 promoter activity in HepG2 cells, consistent with previous studies (13). In contrast, mutation of the IRSs impairs but does not completely disrupt the ability of insulin to suppress glucocorticoid-stimulated promoter activity. Mutation of the GRE also disrupts this IRS-independent effect of insulin, indicating that this effect of glucocorticoids also is mediated through the GRE. Together these results indicate that insulin suppresses glucocorticoid-stimulated IGFBP-1 promoter activity through both IRS-dependent and -independent mechanisms. To our knowledge, these studies provide the first report that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that it does so by conferring the ability of insulin to suppress promoter activity through an IRS-independent mechanism that is mediated through the GRE.

The IGFBP-1 promoter has proved to be an important model for defining mechanisms by which insulin regulates basal promoter activity through IRS-mediated mechanisms. Studies in other promoters in which insulin regulates gene expression through both IRS-dependent and -independent mechanisms, including the catalytic subunit of glucose-6 phosphatase (23) and phosphoenolpyruvate carboxykinase (40, 41, 42), have largely been conducted in the presence of cAMP agonists. Because cAMP stimulates the expression of PGC-1, an important coactivator for the glucocorticoid receptor (34) and FoxO proteins (25), some of these effects may be mediated through changes in the expression of PGC-1. Here we performed our experiments in the absence of cAMP agonists. Also, we found that overexpression of PGC-1 does not disrupt IRS-independent effects of insulin on glucocorticoid-stimulated promoter activity and that insulin does not suppress transactivation of PGC-1 in reporter gene studies. These results indicate that the IGFBP-1 promoter may prove to be a useful model for defining IRS-independent effects of insulin on glucocorticoid-stimulated gene expression under conditions in which PGC-1 expression and/or function is not a limiting factor.

We performed additional studies to characterize this effect of insulin. Whereas insulin suppressed the effect of glucocorticoids on the BP1.IRSmut construct, it failed to suppress the effects of glucocorticoids on an artificial promoter driven by an array of three GREs from the tyrosine aminotransferase gene (pTAT3.luc), indicating that this effect of insulin on IGFBP-1 promoter activity is context dependent in HepG2 cells and that other elements in the IGFBP-1 promoter may be required for insulin to suppress the effects of glucocorticoids on promoter activity through an IRS-independent mechanism in this cell culture model. However, it is important to note that Lemaigre and coworkers (43) have reported that insulin can exert direct effects on transactivation by the glucocorticoid receptor in other cell lines. This suggests that insulin may modulate the effects of glucocorticoids on gene expression through multiple mechanisms and that different mechanisms may be important in different settings. In the context of the IGFBP-1 promoter, Suh et al. (44) found that two elements outside the IRS/GRE region also contribute to the ability of glucocorticoids to stimulate promoter activity. This includes a site for hepatocyte nuclear factor-1 located downstream from the GRE and a site located approximately 150 bp upstream from the GRE. The protein factor(s) that binds to this upstream site remains to be identified. Additional studies are needed to identify cis-acting elements and trans-acting factors required for the ability of insulin to suppress glucocorticoid-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism.

Previous studies have shown that signaling through the PI3K pathway is critical for the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (13). In the present study, we found that PI3K also is important for the ability of insulin to suppress glucocorticoid-stimulated promoter activity through an IRS-independent mechanism. Whereas the effects of insulin on basal IGFBP-1 promoter activity in HepG2 cells appear to be mediated primarily through protein kinase B (13), other PI3K-dependent kinases may contribute to the regulation of IGFBP-1 expression in liver-derived cells that have been treated with glucocorticoids and cAMP (45, 46). Additional studies will be required to identify the role of specific effectors downstream from PI3K that are involved in mediating IRS-independent effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. We also found that inhibition of MAPKK activity reduces basal promoter activity and limits the ability of insulin to exert its effects on the IGFBP-1 promoter in HepG2 cells. These results indicate that multiple signaling pathways contribute to the regulation of the IGFBP-1 promoter and are required for the full effect of insulin on promoter activity.

Together, these findings suggest that insulin regulates IGFBP-1 promoter activity through multiple mechanisms. Whereas the effects of insulin on basal promoter activity is mediated through an IRS-dependent mechanism, stimulation of the promoter with glucocorticoids creates a more complex environment in which insulin exerts effects on promoter activity through both IRS-dependent and -independent mechanisms. Defining specific mechanisms mediating IRS-independent effects of insulin will be important for a more complete understanding of how insulin acts to regulate gene expression in the liver. The IGFBP-1 promoter promises to continue to provide a valuable model in this regard.

	Footnotes

 
First Published Online July 7, 2005

Abbreviations: GR, Glucocorticoid receptor; GRE, glucocorticoid response element; IGFBP-1, IGF binding protein-1; IRS, insulin response sequence; IRS/GRE, mutation of both the IRSs and the GRE; IRSmut, mutation of the IRSs; MAPKK, MAPK kinase; PGC, peroxisomal proliferator-activated receptor- coactivator; PI3K, phosphatidylinositol-3' kinase; PKB, protein kinase B.

Received February 23, 2005.

Accepted for publication June 29, 2005.

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