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Endocrinology, doi:10.1210/en.2005-1602
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Endocrinology Vol. 147, No. 7 3555-3562
Copyright © 2006 by The Endocrine Society

Kinome Analysis Reveals Nongenomic Glucocorticoid Receptor-Dependent Inhibition of Insulin Signaling

Mark Löwenberg, Jurriaan Tuynman, Meike Scheffer, Auke Verhaar, Louis Vermeulen, Sander van Deventer, Daniel Hommes and Maikel Peppelenbosch

Laboratory of Experimental Internal Medicine (M.L., J.T., M.S., A.V., L.V.S, S.v.D.) and Department of Gastroenterology and Hepatology (M.L., D.H.), Academic Medical Center, NL-1105 AZ Amsterdam, The Netherlands; and Department of Cell Biology (M.P.), University of Groningen, NL-9713 AV Groningen, The Netherlands

Address all correspondence and requests for reprints to: Mark Löwenberg, Laboratory of Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail: m.lowenberg{at}amc.uva.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids (GCs) are powerful immunosuppressive agents that control genomic effects through GC receptor (GR)-dependent transcriptional changes. A common complication of GC therapy is insulin resistance, but the underlying molecular mechanism remains obscure. Evidence is increasing for rapid genomic-independent GC action on cellular physiology. Here, we generate a comprehensive description of nongenomic GC effects on insulin signaling using peptide arrays containing 1176 different kinase consensus substrates. Reduced kinase activities of the insulin receptor (INSR) and several downstream INSR signaling intermediates (i.e. p70S6k, AMP-activated protein kinase, glycogen synthase kinase-3, and Fyn) were detected in adipocytes and T lymphocytes due to short-term treatment with dexamethasone (DEX), a synthetic fluorinated GC. Western blot analysis confirmed suppressed phosphorylation of the INSR and a series of downstream INSR targets (i.e. INSR substrate-1, p70S6k, protein kinase B, phosphoinositide-dependent protein kinase, Fyn, and glycogen synthase kinase-3) after DEX treatment. DEX inhibited insulin signaling through a GR-dependent (RU486 sensitive) and transcription-independent (actinomycin D insensitive) mechanism. Overall, we postulate here a molecular mechanism for GC-induced insulin resistance based on nongenomic GR-dependent inhibition of insulin signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS (GCs) ARE widely used therapeutically for their immunosuppressive and antiinflammatory properties. Among the most common and serious clinical complications of GC therapy is the induction of insulin resistance (1, 2, 3). However, the molecular mechanism responsible for GC-induced insulin resistance remains to be defined and is of obvious clinical relevance. The insulin receptor (INSR) signaling pathway regulates growth and metabolic responses in many cell types (4). INSR-mediated signaling is initiated by insulin binding to the {alpha}-subunit of the cell surface INSR, which leads to autophosphorylation of the ß-subunit and activation of INSR tyrosine kinase activity. Downstream signaling targets are subsequently activated, including INSR substrate (IRS), p70S6 kinase (p70S6k), AMP-activated protein kinase (AMPK), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase (GSK)-3 (5, 6, 7, 8, 9, 10). Although it has been reported that prolonged treatment with the synthetic fluorinated GC dexamethasone (DEX) reduced IRS-1, PI3K, and PKB cellular contents in adipocytes (11, 12), the mechanism underlying GC-induced insulin resistance remains obscure at best.

Because the kinome regulates virtually all major cellular metabolic pathways, it is reasonable to assume that a full description of the kinome should enable the identification of rapid DEX-induced effects on cellular metabolism possibly involved in mediating insulin resistance. The aim of this study was to investigate rapid effects of DEX on the adipocyte and T lymphocyte kinome. Here, we have generated a comprehensive description of early DEX-induced effects on the kinome signal transduction, employing a peptide array containing 1176 spatially addressed mammalian kinase consensus substrates (13, 14, 15). These results were validated with conventional techniques revealing nongenomic GC receptor (GR)-dependent inhibition of insulin signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
3T3-L1 cells (American Type Culture Collection, Manassas, VA; CCL 92.1) were provided by F. Falix (Liver Centre, Academic Medical Center, Amsterdam, The Netherlands) and maintained in DMEM (Life Technologies, Rockville, MD), supplemented with 10% fetal calf serum (Gemini Bio-Products, Woodland, CA), 2 mM L-glutamine (Life Technologies, Inc., Breda, The Netherlands), and penicillin/streptomycin in a humidified 5% CO2 environment at 37 C. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers by Ficoll-Isopaque density gradient centrifugation (Amersham Biosciences, Roosendaal, The Netherlands). Monocytes present in the PBMC pellet were removed by an adherence procedure. Cells were plated out in six-well plates (CellStar, Greiner Bio-One, Alphen a/d Rijn, The Netherlands) at a final concentration of 5.106 cells/well for 2 h at 37 C, and soluble cells were harvested for subsequent magnetic cell sorting (MACS), described below. CD4+ T cells were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin.

CD4+ purification
CD4+ T cells were purified by negative selection using the MACS system. In short, non-CD4+ cells were indirectly magnetically labeled with a cocktail of biotin-conjugated monoclonal antibodies (Abs) bound to MicroBeads as secondary labeling agent (Miltenyi Biotec Inc., Auburn, CA). The magnetically labeled non-CD4+ T cells were depleted by retaining them on a MACS Column in the magnetic field of the autoMACS Separator (Miltenyi Biotec Inc.), and the unlabeled CD4+ T cells were collected. The sample purity was assessed by fluorescence-activated cell sorter (Becton Dickinson, San Jose, CA) with phosphatidylethanolamine-conjugated CD3 and fluorescein isothiocyanate-conjugated CD4 monoclonal Abs (purity > 95% CD3+CD4+; data not shown), according to routine procedures.

Reagents and Abs
Phospho-specific Abs recognizing INSRTyr1131/1146, IRS-1Ser636/639, GSK-3Ser21/9, PKBSer473, p70S6kThr389, phosphoinositide-dependent protein kinase (PDK)-1Ser241, GRSer211, ERK1/2Thr202/Tyr204, signal transducer and activator of transcription (STAT) 3Tyr705, c-Jun N-terminal kinase (JNK) 1/2/3Thr183//Tyr185, and I{kappa}B kinase (IKK) {alpha}/ßSer176/180, as well as Abs against total IRS-1, GSK-3{alpha}/ß, PDK, p70S6k, and PKB were purchased from Cell Signaling Technology (Beverly, CA). Abs specific for the INSR, GR, Fyn, ERK1, STAT3, JNK, IKK, actin, and phosphorylated-FynThr12 were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Abs against phosphorylated-Janus kinase (JAK) 2Tyr1007/1008 and total JAK2 were from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat-antirabbit, goat-antimouse, and rabbit-antigoat were purchased from DakoCytomation (Heverlee, Belgium). Antihuman CD3 (CD3-{epsilon} mouse) was kindly provided by the group of Prof. Dr. H. Spits (Academic Medical Center), and anti-CD28 Ab (mouse IgG1) was from Sanquin (Amsterdam, The Netherlands). Phosphatidylethanolamine-conjugated CD3 and fluorescein isothiocyanate-conjugated CD4 Abs were from BD Biosciences (Alphen a/d Rijn, The Netherlands). DEX, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and actinomycin D were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). RU486 (Mifepristone) was from LKT Laboratories Inc. (St. Paul, MN). {gamma}-33P-ATP was purchased from Amersham Biosciences. Insulin (Actrapid, 100 U/ml) was from Novo Nordisk A/S (Copenhagen, Denmark). Recombinant IL-6 and TNF{alpha} were obtained from R&D Systems (Minneapolis, MN), and recombinant IGF-I was from Invitrogen (Breda, The Netherlands). Lysis buffer and kinase buffer were purchased from Cell Signaling Technology. Lysis buffer was supplemented with protease and phosphatase inhibitors, including 1 µg/ml NaF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 mM Na3VO4, and 1 mM Pefabloc, obtained from Merck BV (Amsterdam, The Netherlands).

DEX in vitro stimulations and preparation of cell lysates
3T3 adipocytes were cultured at 37 C in six-well plates (concentration, 5 x 106 cells per well) and serum starved overnight. Next, cells were pretreated for 1 h with insulin (100 IE/ml) or IGF-I (20 ng/ml), followed by DEX treatment for 10, 20, or 30 min (1 µM) dissolved in dimethylsulfoxide or dimethylsulfoxide-supplemented media (control). To monitor the effect of prolonged DEX treatment on insulin signaling, insulin-pretreated adipocytes (100 IE/ml, 1 h) were incubated in the presence of 1 µM DEX up to 4 h. IL-6-stimulated adipocytes served as a control; e.g., cells were pretreated for 30 min with IL-6 (100 ng/ml), followed by 10, 20, or 30 min DEX incubation (1 µM). Furthermore, adipocytes were pretreated for 15 min with 0.1 µM actinomycin D (16, 17, 18), and insulin (100 IE/ml) was added for 45 min before addition of 1 µM DEX for 30 min. PBMC-derived CD4+ T cells were incubated at 37 C in six-well plates (final concentration, 5 to 10 x 106 cells per well) and pretreated for 10 min with DEX (1 µM). Cells were then stimulated for 15 min with anti-CD3 Abs (immobilized on plastic) and soluble anti-CD28 Abs (3 µg/ml). In vitro stimulations were terminated by an ice-cold PBS wash. Cell extracts were prepared by scraping cells into 100 µl lysis buffer supplemented with the indicated protease and phosphatase inhibitors. Cell lysates were used for PepChip array analysis or Western blotting. Support that 1 µM DEX did not exert a toxic effect in adipocytes and T cells was provided by trypan blue exclusion tests verifying that cells were viable after culture (data not shown).

Cell transfection and reporter assay
3T3 adipocytes were transfected using Effectene reagent (QIAGEN, Hilden, Germany) and a plasmid containing a nuclear factor (NF) {kappa}B binding site enhancing expression of a firefly luciferase gene (0.5 µg/well) (CLONTECH Laboratories Inc., Mountain View, CA). Cells were incubated at 37 C for 24 h, fresh medium was added to the wells, and cells were pretreated with actinomycin D for 45 min (0.1 µM), followed by TNF{alpha} stimulation (100 ng/ml) for 45 min. Transfection efficiency was evaluated by cotransfecting cells with a plasmid encoding a Renilla luciferase gene under the cytomegalovirus promoter (Promega, Leiden, The Netherlands), after which cell lysates were recovered for luciferase assay. The firefly and Renilla luciferase activities were assayed on a Lumat Berthold LB 9501 Luminometer. Each firefly luciferase value was corrected for its cotransfected Renilla luciferase value to correct for transfection efficiency or dilution effects. Values are expressed as percent increase over non-TNF-stimulated cells.

MTT viability assay
Cells were incubated at 37 C for 3 h with or without 0.1 µM actinomycin D, and cell viability was assessed with MTT colorimetric assay. In short, 0.5 mg/ml MTT was added to the media for 1–2 h at 37 C; subsequently, isopropanol/0.04 N HCl was added. The OD560 was determined using an ELISA plate reader (Bio-Rad Benchmark, Hercules, CA). Actinomycin D treatment did not affect cell viability compared with control cells: 0.1 µM actinomycin D-treated cells, +1.7 ± 3.7% SEM; and control cells, 0 ± 3.8% SEM.

Peptide array analysis
The protocol of the kinome array is described in detail on the web site: http://www.pepscan.nl/pdf/Manual%20PepChip%20Kinase%200203.pdf. Adipocyte and T cell extracts were corrected for protein concentrations using Bradford analysis (Bio-Rad, Veenendaal, The Netherlands) and used for subsequent kinome array analysis. Activation mix (10 µl), containing 50% glycerol, 50 µM ATP, 60 mM MgCl2, 0.05% (vol/vol) Brij-35, 0.25 mg/ml BSA, and 2000 µCi/ml {gamma}-33P-ATP was added to 60 µl cell lysate. The (second generation) peptide arrays, containing 1176 different kinase pseudosubstrates in duplicate (Pepscan, Lelystad, The Netherlands) (15), were incubated with these mixtures (i.e. cell lysates together with activation mix) for 2 h in a humidified stove at 37 C. The PepChips were washed (2 M NaCl, 1% Triton X-100, and 0.1% Tween in H2O), exposed to a phosphor imager plate for 24–72 h (Fuji, Stamford, CO), and the density of the spots was measured and analyzed with array software.

Data acquisition and statistical analysis of peptide arrays
Acquisition of the peptide arrays was performed with a phosphor imager (Fuji) and ArrayVision 6.0 software (Molecular Dynamics, Sunnyvale, CA). After acquisition and quantification using median spot densities, the data were exported to a spreadsheet program (Microsoft Excel 2002; Microsoft, Redmond, WA). Normalization was achieved by correction of the spot density for the individual background to diminish interarray variances. In addition, the variation between arrays and individual experiments was reduced by normalization to the 90th percentile of the intensity of each array. Inconsistent data (i.e. SD between the data points > 1.96 of the mean value) were excluded from further analysis. Spots were averaged and included for dissimilarity measurement to extract kinases of which activity was either significantly induced or reduced. Differential kinase activities in lysates from cells incubated in the presence or absence of DEX were determined by significant fold change ratios of the combined values of phosphorylated peptides resembling a substrate for kinase activity. Significance analysis was performed using a minimal modification for the algorithm originally developed for microarray analysis (http://www-stat.stanford.edu/~tibs/SAM/). The full list of peptides spotted on the peptide array can be found online: http://www.pepscan.nl/index5.htm.

Western blot analysis
Total cell extracts were supplemented with sodium dodecyl sulfate (SDS) sample buffer [62.5 mM Tris-HCl (pH 6.8 at 25 C), 2% (wt/vol) SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% (wt/vol) bromphenol blue], sonificated, and heated at 90 C for 5 min. Samples were loaded on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Amsterdam, The Netherlands). The membranes were blocked with 5% BSA in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST) and incubated overnight at 4 C with the indicated primary Abs diluted in 5% BSA TBST. The membranes were subsequently incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary Abs diluted in 5% BSA TBST. All Abs were used in accordance with the supplier’s protocol, and images were revealed with a Lumi-Imager (Roche Applied Science, Almere, The Netherlands) using the chemoluminescence substrate LumilightPLUS (Roche, Woerden, The Netherlands). Blots were stripped with strip buffer [62.5 mM Tris-HCl (pH 6.8), 100 mM ß-mercaptoethanol, 2% SDS] and reprobed with appropriate Abs for evaluation of equal protein loading.

Supplemental data (published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org)
The supplemental data disclose the results obtained with the PepChip experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Short-term DEX treatment reduces kinase activities of INSR signaling intermediates in adipocytes
Because adipocytes are important in the pathogenesis of GC-induced insulin resistance, we studied rapid effects of DEX on the adipocyte kinome. 3T3 adipocytes were stimulated with insulin for 1 h and subsequently treated for 30 min with DEX or solvent-supplemented media (control). Kinome profiles of cell lysates revealed significant differences between DEX- and non-DEX-treated cells (Fig. 1Go). In particular, it was found that short-term DEX treatment influenced insulin-induced kinase activities in adipocytes. We next analyzed the individual components of the kinome to examine the underlying determinants of the observed changes in kinase activities. Significant decreased phosphorylation of INSR kinase consensus substrates was detected in DEX-treated cells (Fig. 2AGo), indicating reduced INSR enzymatic activity. Furthermore, GSK-3 (Fig. 2BGo), Fyn (Fig. 2CGo), AMPK (Fig. 2DGo), and p70S6k (Fig. 2EGo) kinase consensus substrates showed suppressed phosphorylation in DEX-treated adipocytes, pointing out suppressed kinase activities of these downstream INSR targets. Thus, these results demonstrate rapid DEX-induced inhibition of kinase activities of the INSR and several downstream signaling intermediates, providing evidence for DEX-induced inhibition of insulin signaling.


Figure 1
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  		FIG. 1. Rapid DEX-induced changes of kinome profiles in adipocytes and T cells. 3T3 adipocytes were stimulated with insulin (100 IE/ml) for 1 h followed by a 30-min DEX treatment (1 µM). CD4+ T cells were pretreated for 10 min with or without DEX (1 µM) and subsequently activated for 15 min using anti-CD3 and anti-CD28 Abs. Cells were lysed, and cell extracts were incubated on peptide arrays in the presence of {gamma}-33P-ATP. Differential kinase activities in lysates prepared from activated adipocytes (upper panel) or T cells (lower panel) incubated with or without DEX using median densities of the spots are shown. Dot plots, Phosphorylation status of 1176 different kinase consensus substrates spotted in duplicate on the PepChip arrays. Each spot reflects the amount of phosphorylation of a specific peptide substrate of which phosphorylation is significantly decreased (A), increased (B), or unchanged (C) due to DEX treatment.

 

Figure 2
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  		FIG. 2. Reduced enzymatic activities of the INSR and downstream signaling intermediates due to short-term DEX treatment. Adipocytes were stimulated for 1 h with insulin (100 IE/ml) and incubated for 30 min in the absence or presence of DEX (1 µM). T cells were pretreated for 10 min with or without 1 µM DEX and activated with anti-CD3 and anti-CD28 Abs for 15 min. Cells were lysed, and the lysates were incubated on PepChip arrays in the presence of {gamma}-33P-ATP. The original scanned pictures were used for quantification and statistical analysis of the arrays. The graphs show the phosphorylation status of kinase consensus substrates that are specific for the INSR (A), GSK-3 (B), Fyn (C), AMPK (D), and p70S6k (E). For each peptide substrate, the annotation number is depicted, and average changes of phosphorylation are indicated by the dotted lines. The peptide arrays used for these experiments do not contain kinase consensus substrates specific for other relevant kinases involved in insulin signaling, such as IRS and PKB. Results for human kinase consensus substrates are shown, and peptide substrates obtained from other species are not indicated. The supplemental data disclose the information obtained with the peptide array experiments.

 
Rapid decreased catalytic activities of INSR signaling intermediates in DEX-treated T cells
To investigate whether the observed effects of DEX on adipocyte INSR signaling were cell type specific, we extended our studies to CD4+ T lymphocytes that are known to be important target cells of GC action. Cells were pretreated for 10 min with or without DEX and activated for 15 min with anti-CD3 and anti-CD28 Abs. Cell lysates were used for PepChip array analysis, and the phosphorylation patterns of DEX- and non-DEX-treated T cells were compared (Fig. 1Go). In line with the adipocyte data, reduced phosphorylation of INSR kinase consensus substrates was seen in activated T cells due to short-term DEX treatment (Fig. 2AGo). Moreover, impaired phosphorylation of GSK-3 (Fig. 2BGo), Fyn (Fig. 2CGo), and AMPK (Fig. 2DGo) consensus substrates was detected after DEX incubation. These findings indicate that DEX-induced inhibition of enzymatic activities of kinases involved in insulin signaling is not limited to adipocytes but is of a more general significance.

DEX-induced inhibition of INSR-mediated signal transduction
To confirm that the observed changes in kinase activities indeed affect phosphorylation of INSR signaling elements, the effect of short-term DEX treatment on insulin signaling was studied in 3T3 adipocytes and CD4+ T cells. Adipocytes were pretreated for 1 h with insulin and subsequently treated for 10, 20, or 30 min with DEX. T cells were pretreated for 10 min with DEX and stimulated for 15 min with anti-CD3 and anti-CD28 Abs. Total cell extracts were immunoblotted employing phospho-specific Abs to study the phosphorylation status of the INSR and several downstream INSR signaling intermediates. Short-term treatment with DEX resulted in reduced INSR phosphorylation in adipocytes (Fig. 3AGo) and T cells (Fig. 3BGo). The effect of DEX on various kinases involved in insulin signaling was investigated in more detail in adipocytes. Western blot analysis revealed suppressed phosphorylation of IRS-1, p70S6k, PKB, GSK-3, PDK, and Fyn due to short-term DEX treatment (Fig. 3AGo). These observations are in accordance with the PepChip array results and indicate that DEX rapidly interferes with INSR-dependent signal transduction.


Figure 3
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  		FIG. 3. DEX inhibits INSR-mediated signal transduction. Adipocytes were stimulated for 1 h with insulin (100 IE/ml) and incubated for 10, 20, or 30 min with DEX (1 µM). A, Phosphorylation status of the INSR and several downstream INSR signaling elements was analyzed on Western blot using phospho-specific Abs against the INSR, IRS-1, p70S6k, PKB, GSK-3, PDK, and Fyn. Blots were analyzed for equal protein loading using the indicated Abs. B, CD4+ T cells were pretreated for 10 min with or without 1 µM DEX and subsequently activated for 15 min with anti-CD3 and anti-CD28 Abs. Cell lysates were immunoblotted using Abs against the (non)phosphorylated INSR. C, Adipocyte lysates, prepared as described above, were immunoblotted using phospho-specific Abs against JNK and IKK. Total JNK and IKK were studied on Western blot to evaluate for equal loading. These experiments were performed three times, and similar results were obtained. p, Phosphorylated.

 
DEX induces JNK phosphorylation but does not interfere with IKK phosphorylation
Because previous reports have indicated that JNK and IKK, two serine kinases, are able to induce insulin resistance (19, 20, 21, 22, 23, 24), the effect of short-term DEX treatment on JNK and IKK phosphorylation was studied. Adipocytes were pretreated for 1 h with insulin followed by DEX treatment for 10, 20, or 30 min. Cell extracts were analyzed on Western blot using phospho-specific Abs against JNK and IKK (associated with activation). Increased phosphorylation of JNK was seen after short-term DEX treatment, whereas DEX did not interfere with IKK phosphorylation (Fig. 3CGo). Altogether, these experiments demonstrate that DEX has opposing effects on insulin signaling, that is to say impaired phosphorylation of several INSR signaling targets, but enhanced JNK phosphorylation.

DEX-induced inhibition of insulin signaling is independent of transcription
To determine whether DEX-induced inhibition of insulin signaling is indeed dependent on a nongenomic (i.e. transcription independent) mechanism, adipocytes were pretreated for 15 min with 0.1 µM actinomycin D (16, 17, 18), a transcription inhibitor. Next, insulin was added to the media for 45 min, followed by a 10, 20, or 30 min DEX stimulation. Cell lysates were immunoblotted using phospho-specific Abs against the INSR and IRS-1 (Fig. 4AGo). Actinomycin D was found to be ineffective at preventing rapid DEX-induced inhibition of INSR and IRS-1 phosphorylation. These results indicate that the inhibitory effects of DEX on INSR kinase activity are not influenced by actinomycin D treatment, providing evidence for a transcription-independent or nongenomic mechanism. Control experiments were performed to ensure that the actinomycin D treatment inhibited gene transcription under these conditions. Comparable time frames were used as to the DEX stimulation experiments, that is to say 45 min actinomycin D preincubation followed by 45 min TNF stimulation. Under these conditions, a TNF-dependent increase in the activity of a 3-{kappa}B sites-containing reporter construct was seen, which was abolished in the presence of actinomycin D (Fig. 4BGo). Hence, TNF-induced reporter activity was completely blocked in the presence of actinomycin D, demonstrating that actinomycin D inhibits transcription.


Figure 4
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  		FIG. 4. Nongenomic DEX-induced inhibition of insulin signaling. A, Adipocytes were preincubated for 15 min with actinomycin D (0.1 µM); thereafter, insulin (100 IE/ml) was added for 45 min, followed by 10, 20, or 30 min DEX (1 µM) treatment. Cells were lysed and analyzed on Western blot to determine the phosphorylation levels of the INSR and IRS-1. Total INSR and IRS-1 expression levels were measured to evaluate for equal protein loading. A representative experiment (of3) is shown. B, Inhibition of TNF-induced NF{kappa}B-reporter activity by actinomycin D treatment. Twenty-four hours after transfection with the NF{kappa}B-luciferase plasmid, actinomycin D (0.1 µM) was added to the cell culture media for 45 min, followed by 45 min stimulation with TNF{alpha} (100 ng/ml). Actinomycin D treatment abolished TNF-induced reporter gene activity, and this was not a consequence of reduced cell viability as assessed by MTT viability assay. Activity was normalized to that of Renilla and expressed relative to the untreated control. Results are expressed as the mean ± SEM of triplicate determinations. p, Phosphorylated; RLU, relative luciferase activity.

 
GR-dependent inhibition of INSR activity
To find out whether the observed DEX-induced inhibition of INSR tyrosine kinase activity is GR-dependent, experiments were employed using a pharmacological GR ligand (RU486, Mifepristone). Adipocytes were preincubated for 1 h with insulin, then treated for 5 min with increasing RU486 concentrations and subsequently stimulated for 30 min with or without DEX. Cell lysates were immunoblotted to monitor GR activity employing a phospho-specific Ab against the activated GR. Because IRS-1 is a direct downstream INSR target, phosphorylated IRS reflects INSR kinase activity. Increased IRS-1 phosphorylation was detected upon insulin stimulation, indicating increased INSR enzymatic activity (Fig. 5Go). Cells incubated with combinations of high concentrations of RU486 and DEX displayed decreased expression of phosphorylated GR (indicating reduced GR activation) and increased IRS-1 phosphorylation (reflecting increased INSR activity). Thus, these results demonstrate that high RU486 concentrations antagonize the inhibitory effects of DEX on INSR catalytic activity. In contrast, cells incubated with combinations of low RU486 concentrations together with DEX showed increased GR phosphorylation and reduced phosphorylated IRS-1. Accordingly, RU486 at low concentration is not able to antagonize the inhibitory action of DEX on INSR enzymatic activity. These experiments reveal a correlation between the activated GR and reduced INSR activity and therefore provide evidence for a GR-dependent mechanism of DEX-induced inhibition of INSR kinase activity.


Figure 5
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  		FIG. 5. GR-dependent inhibition of INSR kinase activity. A, Adipocytes were preincubated for 1 h with insulin (100 IE/ml), followed by a 5-min treatment with increasing RU486 concentrations (50 mM to 50 pM); thereafter, 1 µM DEX was added for 30 min. Catalytic activities of the GR and INSR were determined on Western blot employing phospho-specific Abs against the activated GR and IRS-1, respectively. Immunoblots were analyzed for total GR, IRS-1, and actin to evaluate for equal loading. Three independent experiments were performed, and comparable outcomes were obtained. B, Western blots were quantitated, and the phosphorylation status of the GR and IRS-1 is depicted on the y-axis. p, Phosphorylated.

 
DEX inhibits IGF-I-induced signaling but does not interfere with IL-6-mediated signaling
The kinases evaluated in this study act as intermediates for the signal transduction of insulin and IGF-I (two IRSs), indicating that insulin and IGF-I share common signal transduction. The effect of DEX on IGF-I-induced signaling was monitored in adipocytes that were preincubated for 1 h with IGF-I followed by DEX treatment for 10, 20, or 30 min. Cells were lysed and analyzed on Western blot employing the same panel of phospho-specific Abs that were used to study insulin signaling. As can be seen in Fig. 6AGo, DEX treatment suppressed IGF-I-induced phosphorylation of the INSR, IRS-1, p70S6k, PKB, GSK-3, PDK, and Fyn. Altogether, these studies show that short-term DEX treatment interferes with insulin and IGF-I-induced signaling.


Figure 6
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  		FIG. 6. DEX inhibits IGF-I-induced signaling but does not interfere with IL-6 signaling. A, Adipocytes were preincubated for 1 h with IGF-I (20 ng/ml) and treated with 1 µM DEX for 10, 20, or 30 min. Whole lysates were immunoblotted using activation state-specific Abs against several kinases involved in insulin signaling, including the INSR, IRS-1, p70S6k, PKB, GSK-3, PDK, and Fyn. Total protein levels were determined by Western blot employing the indicated Abs. B, To ensure specificity in these experiments, adipocytes were pretreated with IL-6 (100 ng/ml) for 30 min and then incubated for 10, 20, or 30 min with 1 µM DEX (control). Cell lysates were analyzed on Western blot using phospho-specific Abs against STAT3, JAK2, and ERK1/2. Equal protein loading was verified employing Abs against proteins of interest. The immunoblots represent three independent experiments, and representative experiments are shown. p, Phosphorylated.

 
To examine the specificity of the observed effects of DEX on INSR-mediated signaling, we next investigated whether DEX interfered with a different kinase cascade. To this end, IL-6 (a gp130 receptor ligand) was added to adipocytes for 30 min followed by 30 min DEX incubation. Cell lysates were analyzed on Western blot for phosphorylated STAT3, JAK2, and ERK1/2 (Fig. 6BGo), key players in IL-6-induced signaling (25, 26). These results indicate that DEX does not interfere with IL-6 signaling in adipocytes, further underscoring the specificity of the observed effects.

Sustained DEX-induced inhibition of insulin signaling
The present study shows that DEX inhibits INSR-dependent signaling in adipocytes and T cells. These inhibitory effects of DEX are evident within the 30-min time frame. Finally, the effect of DEX at later time points was monitored to determine the temporal characteristics of DEX treatment on INSR tyrosine kinase activation. Adipocytes were pretreated for 1 h with insulin and subsequently stimulated up to 4 h with DEX. Cells were lysed at different time points, and total cell extracts were used for Western blot analysis employing phospho-specific Abs against the INSR and IRS-1 (Fig. 7Go). These findings indicate that the inhibitory effects of DEX on INSR and IRS phosphorylation are sustained for at least 4 h. Thus, these studies suggest that nongenomic GR-dependent inhibition of insulin signaling is not a transient effect, although involvement of genomic effects at later time points cannot be excluded.


Figure 7
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  		FIG. 7. Sustained DEX-induced inhibition of INSR kinase activity. Adipocytes were stimulated for 1 h with insulin (100 IE/ml); thereafter, cells were treated with 1 µM DEX up to 4 h. At the indicated time points (0, 30, 60, 120, and 240 min DEX treatment), cells were lysed and analyzed on Western blot using phospho-specific Abs against the INSR and IRS-1. Total lysates were analyzed for INSR and IRS-1 expression to evaluate for equal loading. Three independent experiments were performed, and reproducible results were obtained. p, Phosphorylated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GCs form the basis of current antiinflammatory and immunosuppressive therapy. GC treatment is however significantly hampered by side effects, such as GC-induced insulin resistance, but the underlying molecular mechanisms remain unclear. Well-defined GC-induced effects are mediated via GR-dependent transcriptional changes. Previous studies have explored long-term (i.e. ≥24 h) effects of DEX on glucose transport mechanisms and insulin signaling. It has been shown that DEX incubation results in decreased glucose uptake and impaired insulin signaling in 3T3 adipocytes and primary rat adipocytes (11, 12, 27, 28, 29, 30). Although of significant importance for our understanding of the molecular mechanism underlying GC-induced insulin resistance, we are not aware of previous studies on rapid nongenomic (i.e. ≤30 min) effects of DEX on insulin signaling. More generally, evidence for early GC effects on cellular physiology is accumulating, but the responsible molecular mechanisms remain to be defined (31, 32, 33). We have recently reported nongenomic GR-induced inhibition of T cell receptor signaling, providing further proof for rapid GC-effects on cellular function (15). In the current study, marked rapid differences in phosphorylation patterns between DEX- and non-DEX-treated cells were demonstrated using a peptide array approach. We demonstrate here DEX-induced effects on the signal transduction of adipocytes and CD4+ T cells. Phosphorylation of INSR consensus substrates was strikingly inhibited due to short-term DEX treatment. Furthermore, rapid reduced phosphorylation of AMPK, p70S6k, GSK-3 and Fyn kinase consensus substrates was detected in DEX-treated cells, and Western blotting confirmed that DEX impairs insulin signaling. Experiments employing a pharmacological transcription inhibitor (actinomycin D) and a GR ligand (RU486) provided evidence for a nongenomic GR-dependent mechanism of DEX-induced inhibition of INSR activity. We conclude that short-term DEX treatment interferes with INSR-mediated signaling, and the fact that these effects were seen in two different cell types suggests a general significance of DEX-induced inhibition of insulin signaling.

We anticipate that such decreased INSR signaling might play an important role in GC-induced insulin resistance. The notion that DEX directly influences INSR signaling rather than affecting cellular expression levels of the INSR or downstream signaling targets is supported by previous work (11). These authors showed that a 2- or 8-h DEX treatment did not affect IRS-1, PI3K, and PKB expression in primary rat adipocytes. This is consistent with our observation that the total cellular content of INSR and downstream signaling targets did not change due to 30 min DEX treatment, and it provides further evidence that DEX directly inhibits INSR-mediated signaling, rather than affecting absolute INSR expression levels.

An important question concerns the mechanism by which GR engagement through DEX interferes with activity of the INSR kinase. Previous studies have shown that activation of JNK, a MAPK family member, inhibits INSR-dependent signaling resulting in insulin resistance (21, 22, 23). We have investigated the effects of short-term DEX treatment on JNK phosphorylation. Interestingly, these findings showed increased JNK phosphorylation in DEX-treated adipocytes compared with nontreated cells. Furthermore, the effect of DEX on IKK phosphorylation was analyzed because this serine kinase has also been suggested to be involved in DEX-induced insulin resistance (19, 20, 24). We did not, however, detect a change in IKK phosphorylation status due to short-term DEX treatment (Fig. 3CGo). Further studies are needed to address the broader implications of JNK and IKK in generating DEX-induced insulin resistance.

Based on the present study, we postulate a novel molecular mechanism for nongenomic GR-mediated inhibition of insulin signaling that is independent of the classical GC action of regulation of gene expression. These findings could have clinical implications because this further underscores the importance of the development of GC analogs, which retain their immunosuppressive and antiinflammatory activities without having an accompanying effect on INSR tyrosine kinase activity. Characterization of novel GC analogs could mark an important step toward the development of a new class of safer GC preparations.


   Acknowledgments
 
We thank F.A. Falix (Academic Medical Center, Amsterdam, The Netherlands) for the kind gift of 3T3 cells and Hans Verdeurmen and Maarten Bijlsma (Academic Medical Center) for technical support.


   Footnotes
 
This work was supported by a grant from The Netherlands Organization for Health Research and Development (to D.H.) and by the Dutch Digestive Disease Foundation (to M.P.).

M.L., J.T., M.S., A.V., L.V., S.v.D., D.H., and M.P. have nothing to declare and do not have any conflicting financial interests.

First Published Online March 30, 2006

Abbreviations: Ab, Antibody; AMPK, AMP-activated protein kinase; DEX, dexamethasone; GC, glucocorticoid; GR, GC receptor; GSK, glycogen synthase kinase; IKK, I{kappa}B kinase; INSR, insulin receptor; IRS, INSR substrate; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; MACS, magnetic cell sorting; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NF, nuclear factor; PBMC, peripheral blood mononuclear cell; PDK, phosphoinositide-dependent protein kinase; PI3K, phosphatidylinositol 3 kinase; PKB, protein kinase B; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; TBST, Tris-buffered saline supplemented with 0.1% Tween 20.

Received December 16, 2005.

Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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