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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1399
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 11 4332-4339
Copyright © 2007 by The Endocrine Society

Glucocorticoid Modulation of Insulin Signaling in Human Subcutaneous Adipose Tissue

Laura L. Gathercole, Iwona J. Bujalska, Paul M. Stewart and Jeremy W. Tomlinson

Institute of Biomedical Research, Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Jeremy W. Tomlinson, Ph.D., M.R.C.P., Institute of Biomedical Research, Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TT, United Kingdom. E-mail: J.W.Tomlinson{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored.

Objective: We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle.

Design and Setting: This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues.

Patients: Biopsy specimens were from healthy volunteers who gave their full and informed written consent.

Interventions: Combinations of treatments, including GC, RU38486, and wortmannin, were used.

Main Outcome Measures: Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined.

Results: In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 ± 0.1 (50 nM); 2.2 ± 0.2 (250 nM), P < 0.01 vs. control; 3.4 ± 0.2 (1000 nM), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 ± 2.0 (6 h); 9.1 ± 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 ± 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 ± 0.1 (insulin) vs. 2.8 ± 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 ± 0.2 (insulin) vs. GC 1.3 ± 0.1 (insulin + GC); P < 0.05].

Conclusions: We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE LINK BETWEEN increased central adiposity and insulin resistance is established (1). In addition, increased cardiovascular morbidity and mortality as a consequence of intra-abdominal adipose deposition (as opposed to sc) are well described (2). Consequently, there is a need to identify factors that regulate adipose tissue distribution. Patients with glucocorticoid (GC) excess (both endogenous and exogenous), Cushing’s syndrome develop a phenotype characterized by hypertension, central obesity, and profound insulin resistance, emphasizing the potent effects of GC upon adipose tissue. Furthermore, the metabolic consequence of GC excess leads to significantly increased morbidity and mortality, principally through cardiovascular disease. The prevalence of the use of oral GCs may be as high as 2.5% of the population (3), and, therefore, the magnitude of the health burden from their adverse effects cannot be underestimated.

The mechanisms by which GCs cause obesity and insulin resistance remain unclear. The development of GC-induced obesity is complex. GC administration leads to increased food intake (4), without changes in energy expenditure (5). In addition, GCs have multiple effects upon the adipocyte, inducing lipolysis in mature adipocytes through induction of hormone-sensitive lipase, resulting in increased glycerol release (6). However, in contrast, GCs are essential for preadipocyte differentiation into mature adipocytes that have the ability to store triglyceride, and here GC and insulin act synergistically (7). GC excess results in preferential accumulation of intra-abdominal adipose tissue; increased glucocorticoid receptor (GR) expression (8) and increased expression of the pre-receptor-modulating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) (9) are putative mechanisms.

After binding of insulin to the cell surface insulin receptor and the subsequent tyrosine autophosphorylation, a complex intracellular signaling cascade is initiated. Although activation of the Ras/Raf MAPK pathway is crucial in the proliferative actions of insulin (10), the majority of its "metabolic" actions occur via the insulin receptor substrate (IRS), phosphatidylinositol-3 (PI3)-kinase pathway, which ultimately leads to activation of protein kinase B (PKB)/akt through phosphorylation at the S473 and Th308 positions (11). The functional effects of PKB/akt activation are well characterized, and include inhibition of glycogen synthase kinase 3 (12), inhibition of apoptosis (13), and GLUT4 translocation to the cell membrane with consequent glucose uptake (14).

GCs induce whole-body insulin resistance, as measured by hyperinsulinemic euglycemic clamps in both rodents and humans (15), largely reflecting the effect of GCs upon muscle. The specific interactions between GCs and the insulin signaling cascade in adipose tissue have only been investigated in a relatively small number of studies, and almost all have used rodent models and rodent cell lines (16, 17, 18, 19). Extrapolating directly from rodents to man must be done with caution, and we have, therefore, performed a detailed characterization of the activation of the insulin signaling cascade and investigated the impact of GCs (both endogenous and synthetic) in human tissues. We have used a novel human preadipocyte cell line (Chub-S7) that retains the ability to differentiate into mature adipocytes and store lipid. We have endorsed our findings in primary cultures of human adipose tissue and compared them with the effects of GCs upon differentiated human skeletal myocytes as a well-characterized model of GC-induced insulin resistance.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Chub-S7 cell line

The Chub-S7 cell line is a transformed human sc preadipocyte cell line derived from an obese female. In cell culture, it retains the ability to proliferate, and upon confluence, in chemically defined media, differentiates into mature adipocytes expressing well-characterized markers of adipocyte differentiation as well as lipid droplets (20). Proliferating cells were cultured in DMEM/Nutrient Mixture F-, DMEM-F12 (Sigma, Poole, UK) with 10% fetal calf serum (FCS), and seeded into 12-well plates and grown until confluent. In some experiments, cells were differentiated in DMEM-F12 media with biotin 33 µM, pantothenate 17 µM, T3 0.2 nM, insulin 167 nM, cortisol 1 µM, and rosiglitazone 1 µM for 14 d. During the experimental protocol, before treatment all cells (both undifferentiated and differentiated) were cultured in media (DMEM-F12) without additives for 24 h.

Human preadipocytes

Findings from the experiments using the Chub-S7 cell line were endorsed using primary cultures of human adipose tissue. Human sc preadipocytes were isolated as previously reported (9) from nonobese patients undergoing elective total abdominal hysterectomy (n = 5; mean body mass index 26.6 ± 1.3 kg/m2; mean age 40 ± 3 yr) without evidence of malignancy. Patients with diabetes and those who had taken GC therapy within the last 12 months were excluded from the study. All subjects were on no regular medications. Informed, written consent was obtained in all cases, and the study had the approval of the local research ethics committee. Briefly, sc adipose tissue was washed in PBS containing 50,000 U penicillin and 50,000 µg streptomycin (Invitrogen, Paisley, UK). The tissue was then prepared and digested with collagenase class 1 (2 mg/ml; Worthington Biochemical Corp., Reading, UK) in 1x Hanks’ balanced salt solution (Invitrogen) for 45 min at 37 C. Samples were then centrifuged at 90 g for 5 min, the pellet-containing preadipocytes were removed, and cells were washed with DMEM-F12 containing 15% FCS and seeded on 12-well plates (Corning, Schiphol-Rijk, The Netherlands). Cells were left overnight and washed the following day with 1x Hanks’ balanced salt solution. Cells were then either cultured to confluence in DMEM F-12 medium containing 15% FCS or differentiated to mature adipocytes in chemically defined media without thiazolidinedione supplementation as previously described (21). Preparations containing greater than 5% endothelial cell contamination were discarded. Before treatment, cells were incubated in serum free media without additives for 24 h (for specific treatment times and doses, see Results).

Human myoblasts

Primary human myoblasts were obtained from PromoCell (Heidelberg, Germany). Myoblasts were cultured to confluence as per the manufacturer’s guidelines using the supplied media. Once confluent, media were changed to a chemically defined media (PromoCell), and cells were differentiated into myotubes for 14 d. After differentiation, cells were incubated with serum free media for 24 h before treatment (for specific treatment times and doses, see Results).

In all cell culture experiments investigating insulin signaling cascade protein phosphorylation, media were spiked with human insulin (0.1 µg/ml; Sigma) for the final 15-min treatment period. In experiments using the GR antagonist, RU38486, cells were pretreated with RU38486 (10 µM) for 10 min before adding dexamethasone (Dex) (1 µM). When used, wortmannin (100 nM) was added 15 min before the addition of insulin to the cell culture media. All treatments and reagents were supplied by Sigma unless otherwise stated.

RNA extraction and RT

Total RNA was extracted using the Tri-reagent system. RNA integrity was assessed by electrophoresis on 1% agarose gel. Concentration was determined spectrophotometrically at OD260. One microgram of total RNA was initially denatured with 200 ng random primers in a volume of 10 µl. Twenty units of avian myeloblastosis virus, 20 U ribonuclease inhibitor, 1 µM deoxynucleotide triphosphates, and 5 x reaction buffer were added to the volume of 20 µl. The RT reaction was performed at 37 C for 1 h. The reaction was terminated by heating the cDNA to 95 C for 5 min.

Real-time PCR

PKB/akt1, PKB/akt2, IRS-1, IRS-2, fatty acid binding protein 4 (FABP4), and glycerol-3-phosphate dehydrogenase (G3PDH) mRNA levels were determined using an ABI 7500 sequence detection system (Perkin-Elmer Applied Biosystems, Warrington, UK). Reactions were performed in 25 µl volumes on 96-well plates in reaction buffer containing 2 x TaqMan Universal PCR Master mix (Applied Biosystems, Foster City, CA). Probes and primers for IRS-1, IRS-2, PKB/akt1, PKB/akt2, FABP4, and G3PDH were supplied by Applied Biosystems’ "assay on demand." All reactions were normalized against the housekeeping gene 18S rRNA, provided as a preoptimized control probe.

Data were expressed as ct values (ct = cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine {Delta}ct values [{Delta}ct = (ct of the target gene) – (ct of the housekeeping gene)], with high {Delta}ct values reflecting low mRNA expression levels. Fold changes were calculated using transformation (fold increase = 2–difference in {Delta}CT).

Protein extraction and immunoblotting

Cells were scraped into 100 µl radioimmunoprecipitation assay buffer [50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail; Roche, Lewes, UK], and incubated at –80 C for 10 min, incubated on ice for 30 min, and centrifuged at 4 C for 10 min at 14,000 rpm. The supernatant containing the soluble proteins was transferred to a fresh tube and total protein concentration determined by a commercially available protein assay (Bio-Rad Laboratories, Hercules, CA).

Fifteen micrograms of protein sample were resolved on a 12.5% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membrane, Hybond ECL (GE Healthcare, Chalfont St. Giles, UK). Primary [anti-PKB/akt, IRS-1, and IRS-2 (Upstate, Dundee, UK), antiphosphoIRS (serine 612) (Biosource, Nivelles, Belgium), and antiphosphoPKB/akt (serine 473) (R&D Systems, Abingdon, UK] and secondary antibodies (Dako, Glostrop, UK) were used at a dilution of 1:1000. Membranes were reprobed for ß-actin and primary and secondary antibodies used at a dilution of 1:5000 (Abcam plc, Cambridge, UK). Bands were quantified with Genesnap by Syngene (Cambridge, UK).

Glucose transport assay

Glucose uptake activity was analyzed by measuring the uptake of 2-deoxy-D-[3H] glucose, as described previously (22). After treatment, cells were washed three times with Krebs-Ringer-HEPES (KRP) buffer and incubated with 0.9 ml KRP buffer at 37 C for 30 min. Insulin (0.5 ng/ml) was then added, and the cells were incubated at 37 C for 15 min. Glucose uptake was initiated by the addition of 0.1 ml KRP buffer and 37 MBq/liter 2-deoxy-D-[3H] glucose (GE Healthcare) and 0.1 mmol/liter glucose as final concentrations. After 15 min, glucose uptake was terminated by washing the cells three times with cold PBS. Cells were lysed, and radioactivity was retained by the cell lysates determined by scintillation counting.

11ß-HD1 assay

Briefly, intact cells were incubated with 100 nM cortisone and tritiated tracer for 16 h. Steroids were then extracted using dichloromethane, separated using a mobile phase consisting of ethanol and chloroform (8:92) by thin layer chromatography, and scanned using a Bioscan 3000 image analyzer (Lablogic, Sheffield, UK). Protein levels were assayed using a commercially available kit (Bio-Rad Laboratories), and activity was expressed as picomole cortisol generated per milligram of protein per hour.

Statistical analysis

Where data were normally distributed, the unpaired Student’s t test was used to compare single treatments with control. If normality tests failed, then nonparametric tests were used. One-way ANOVA on ranks was used to compare multiple treatments, doses, or times (SigmaStat 3.1; Systat Software, Inc., Point Richmond, CA). Statistical analysis on real-time PCR data was performed on mean {Delta}ct values, and not fold changes.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Chub-S7 cells characterization–comparison with primary sc adipocytes

Chub-S7 cells have been characterized in detail previously (20, 23). We have performed our own "in-house" characterization of their ability to differentiate and serve as a model for human adipocyte biology. After differentiation in chemically defined media, lipid droplets were clearly visible in both Chub-S7 cells and primary cultures of human sc preadipocytes (Fig. 1Go). Using real-time PCR, mRNA expression of G3PDH increased 153-fold in primary cultures of sc cells and 1554-fold in Chub-S7 cells. Similarly, FABP4 increased 19-fold in primary cultures of sc cells and 7960-fold in Chub-S7 cells. GR mRNA expression did not change significantly in either cell type across differentiation (data not shown).


Figure 1
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  		FIG. 1. Chub-S7 cells and primary cultures of human sc preadipocytes differentiate in chemical defined media (14 d) and acquire a mature adipocyte phenotype with clearly visible lipid droplets.

 
Insulin signaling in differentiated Chub-S7 cells

Insulin stimulated PKB/akt phosphorylation in differentiated Chub-S7 cells in a dose-dependent manner as measured by semiquantitative densitometry of Western blotting relative to internal control, ß-actin [1.0 (0.1 ng/ml), 2.5 ± 0.9 (0.5 ng/ml), 3.1 ± 1.0 (1 ng/ml)] (Fig. 2AGo).


Figure 2
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  		FIG. 2. Insulin stimulation (15 min) causes a dose-dependent increase in PKB/akt phosphorylation in differentiated Chub-S7 cells (A). Dex pretreatment (1 µM, 48 h) does not change IRS-1 (B) or PKB/akt protein expression (C) but enhances insulin-stimulated phosphorylation (B and C). Representative Western blots are shown with the quantification relative to ß-actin of three to five experiments shown below. *, P < 0.05 vs. control. C, Control; D, Dex.

 
Dex pretreatment (1 µM, 48 h) in differentiated Chub-S7 cells did not regulate total IRS-1 or PKB/akt protein expression (Fig. 2Go, B and C). Interestingly, IRS-2 protein expression increased [1.0 (control) vs. 2.22 ± 0.42 (Dex); P < 0.05]. However, Dex increased both insulin-stimulated IRS-1 phosphorylation [1.0 (control) vs. 2.5 ± 0.84 (Dex); P < 0.05] and PKB/akt phosphorylation [1.0 (control) vs. 6.3 ± 2.4 (Dex); P < 0.05]. Furthermore, Dex induction of insulin-stimulated PKB/akt phosphorylation was blocked by both the GR antagonist, RU38486 [1.6 ± 0.6 (Dex + RU38486); P < 0.05 vs. Dex], and by the PI3 kinase inhibitor, wortmannin [1.1 ± 0.7 (Dex + wortmannin); P < 0.05 vs. Dex] (Fig. 3Go, A and B). Wortmannin treatment alone abolished insulin-stimulated PKB/akt phosphorylation; treatment with RU38486 alone was without effect (data not shown).


Figure 3
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  		FIG. 3. Dex (D) pretreatment (1 µM, 48 h) increases insulin-stimulated (0.5 ng/ml, 15 min) PKB/akt phosphorylation in both undifferentiated (A) and differentiated Chub-S7 cells (B), and in primary cultures of differentiated sc human preadipocytes (C). In Chub-S7 cells, these effects are blocked by coincubation with the GR antagonist, RU38486 (R) (10 µM), or the PI3 kinase inhibitor, wortmannin (W) (100 nM). Data are presented as the mean ± SE of three to five experiments and quantified relative to ß-actin. Representative Western blots and light microscopic images are shown inserted. *, P < 0.05 vs. control (C). {dagger}, P < 0.05 vs. Dex.

 
Insulin signaling in undifferentiated Chub-S7 cells

To determine whether the differentiation status plays a role in determining the response to GC pretreatment, similar experiments were performed in undifferentiated Chub-S7 cells (Fig. 3AGo). Replicating our findings in differentiated cells, Dex pretreatment (1 µM, 48 h) increased insulin-stimulated PKB/akt phosphorylation [undifferentiated Chub-S7: 1.0 (control) vs. 5.5 ± 1.5 (Dex); P < 0.05] without altering PKB/akt protein expression (data not shown). This was blocked by both the GR antagonist, RU38486 [2.7 ± 0.9 (Dex + RU38486); P < 0.05 vs. Dex], and by the PI3 kinase inhibitor, wortmannin [0.6 ± 0.4 (Dex + wortmannin); P < 0.05 vs. Dex].

Insulin signaling in primary cultures of human adipocytes

Endorsing our findings in Chub-S7 cells, Dex pretreatment increased insulin-stimulated PKB/akt phosphorylation in primary cultures of differentiated sc adipocytes [1.0 (control) vs. 1.6 ± 0.2 (Dex); P < 0.05] (Fig. 3CGo). However, although RU38486 decreased Dex induction of insulin-stimulated PKB/akt phosphorylation, this failed to reach statistical significance [1.3 ± 0.2 (Dex + RU38486); P = 0.2 vs. Dex], but the effect of Dex was completely abolished by wortmannin [0.7 ± 0.2 (Dex + wortmannin); P < 0.05 vs. Dex].

Time and dose dependency

To determine whether the observations with Dex could be extrapolated to endogenous GCs, dose and time course experiments were performed upon differentiated Chub-S7 cells using cortisol (Fig. 4Go, A and B). Cortisol pretreatment increased insulin-stimulated PKB/akt phosphorylation in both a dose-dependent [48-h treatment: 1.0 (control); 1.2 ± 0.1 (50 nM); 2.2 ± 0.2 (250 nM), P < 0.01 vs. control; 3.4 ± 0.2 (1000 nM), P < 0.001 vs. control] and time-dependent manner [1 µM cortisol: 1.0 (1 h); 3.2 ± 2.0 (6 h); 9.1 ± 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 ± 2.2 (48 h)]. In both experiments there were no changes in PKB/akt protein expression.


Figure 4
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  		FIG. 4. Cortisol induces a dose- (A) and time- (B) dependent increase in insulin-stimulated PKB/akt phosphorylation (white bars) without alteration in total PKB/akt expression (black bars) in human differentiated Chub-S7 cells. Data are presented as the mean ± SE of five experiments performed in triplicate. Representative Western blots are shown inserted. *, P < 0.05; **, P < 0.01; {dagger}, P < 0.001 vs. control.

 
Chub-S7 cells express 11ß-HSD1 mRNA and have demonstrable enzyme activity (24). Consistent with the role of 11ß-HSD1 in human adipose tissue to locally activate GC (cortisone -> cortisol), cortisone pretreatment (1 µM, 24 h) enhanced insulin-stimulated PKB/akt phosphorylation (Fig. 5Go) [1.0 (control) vs. 1.69 ± 0.09 (cortisone); P < 0.05]. Coincubation with glycyrrhetinic acid (GE) (5 µM) (an inhibitor of 11ß-HSD1, thus preventing the activation of cortisone to cortisol) completely abolished this observation [1.0 (control) vs. 1.12 ± 0.14 (cortisone + GE); P = not significant (ns)]. Parallel, 11ß-HSD1 activity studies demonstrated complete inhibition of 11ß-HSD1 activity after incubation with GE [21.4 ± 4.4 (control) vs. 0.0 pmol cortisol/mg protein per hour (GE)].


Figure 5
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  		FIG. 5. Both cortisol and cortisone (1 µM, 24 h) pretreatment enhance insulin-stimulated PKB/akt phosphorylation. The effect of cortisone is completely blocked by the 11ß-HSD1 inhibitor, GE, which prevents the activation of cortisone to cortisol. Data are presented as the mean ± SE of five experiments performed in triplicate. Representative Western blots are shown inserted. *, P < 0.05; **, P < 0.01 vs. control.

 
mRNA expression

Using real-time PCR, IRS-1, IRS-2, and PKB/akt1 and 2 were all expressed in differentiated Chub-S7 cells. Dex treatment (1 µM, 48 h) induced IRS-2 expression in differentiated Chub-S7 cells [IRS-2: 2.8-fold 14.7 ± 0.6 (control); 13.2 ± 0.3 (Dex); P < 0.05). Similarly, the endogenous GC, cortisol induced IRS-2 expression in differentiated cells in a dose-dependent manner [14.1 ± 0.2 (control), 13.5 ± 0.3, 1.2-fold (50 nM); 13.1 ± 0.3, 2.2-fold (250 nM); 12.2 ± 0.3, 3.4-fold (1000 nM); P < 0.05 vs. control]. However, IRS-1 expression did not change significantly. GC pretreatment did not change PKB/akt1 or PKB/akt2 mRNA expression levels. Complete mRNA expression data in response to GC treatment and also coincubation with RU38486 are shown in Table 1Go.


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  		TABLE 1. mRNA expression of markers of adipocyte differentiation and key components of the insulin signaling cascade in differentiated Chub-S7 cells after treatment with Dex (1 µM, 48 h) and the GR antagonist, RU38486

 
The observed changes in response to GC pretreatment could not be attributed to changes in differentiation status of the cells. Using real-time PCR, markers of differentiation increased in comparison with undifferentiated cells [FABP4 6 x 106-fold induction, mean {Delta}ct ± SE 21.8 ± 0.4 (undifferentiated) vs. 4.5 ± 0.5 (differentiated), P < 0.001; G3PDH 446-fold induction, 23.1 ± 0.1 (undifferentiated) vs. 14.3 ± 1.0 (differentiated), P < 0.01]. However, in differentiated cells after the 24-h washout period and subsequent treatment with Dex (1 µM, 48 h), FABP4 and G3PDH mRNA expression did not change significantly.

Insulin signaling in primary cultures of human myocytes

To determine a tissue specificity of response, we have performed a small number of studies in primary cultures of skeletal myocytes. In contrast to our findings in adipocytes (primary cultures and Chub-S7 cells), Dex (1 µM, 48 h) had no effect upon PKB/akt phosphorylation or total PKB/akt protein expression in primary cultures of differentiated human skeletal myocytes [1.0 (control), 1.1 ± 0.3 (Dex), 1.0 ± 0.3 (Dex + RU38486)]. However, coincubation with wortmannin significantly decreased insulin-stimulated PKB/akt phosphorylation [0.6 ± 0.1 (Dex + wortmannin); P < 0.01 vs. Dex].

Dex induced both IRS-1 [2.6-fold, 14.4 ± 0.6 (control); 13.0 ± 0.6 (Dex); P < 0.05] and IRS-2 mRNA expression [4.3-fold, 16.7 ± 0.3 (control); 14.6 ± 0.2 (Dex); P < 0.05] in differentiated skeletal myocytes. There were no significant changes in PKB/akt mRNA expression (data not shown).

Glucose transport in differentiated Chub-S7 cells and human myocytes

To investigate the functional impact of the changes in the insulin signaling cascade, 2-deoxy-D-[3H] glucose uptake was mea-sured (Fig. 6Go). In differentiated Chub-S7 cells, insulin stimulated 2-deoxy-D-[3H] glucose uptake in a dose [data expressed relative to noninsulin stimulated control ± SE (insulin dose, ng/ml): 1.0 (control), 1.7 ± 0.2 (0.1), 1.9 ± 0.2 (0.5), and 1.9 ± 0.2 (1.0)] and time [2.5 ± 0.2 (5 min), 7.9 ± 0.2 (10 min), 13.4 ± 0.1 (20 min), 17.3 ± 0.1 (40 min), and 2.7 ± 0.2 (60 min)]-dependent manner. Wortmannin abolished insulin-stimulated glucose transport [1.1 ± 0.1 (insulin 1.0 ng/ml + wortmannin). In both undifferentiated and differentiated cells, Dex increased insulin-stimulated glucose uptake into Chub-S7 cells [undifferentiated: 1.0 (control), 1.6 ± 0.1 (insulin, 0.5 ng/ml) vs. 2.1 ± 0.03 (Dex, 1 µM, 48 h, insulin 0.5 ng/ml), P < 0.05; differentiated: 1.0 (control), 1.8 ± 0.1 (insulin) vs. 2.8 ± 0.2 (Dex + insulin), P < 0.05]. In contrast, in primary cultures of human skeletal muscle, Dex decreased insulin-stimulated glucose uptake [1.0 (control), 1.9 ± 0.2 (insulin) vs. 1.3 ± 0.1 (Dex + insulin); P < 0.05].


Figure 6
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  		FIG. 6. Insulin stimulated 2-deoxy-D-[3H] glucose uptake in undifferentiated and differentiated Chub-S7 cells and skeletal myocytes. Data are expressed as the fold increase in glucose uptake with basal unstimulated uptake set as one (white bars). In all cell systems, insulin treatment significantly increased glucose uptake (gray bars). Dex (1 µM, 48 h) pretreatment increased 2-deoxy-D-[3H] glucose uptake in undifferentiated and differentiated Chub-S7 cells, while decreasing uptake in human skeletal muscle cells (black bars). Data are presented as the mean ± SE of three to five experiments performed in triplicate. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
It is a widely held belief that GCs induce insulin resistance in all tissues, including adipose. However, although there is little doubt that globally they induce insulin resistance, tissue specificity of response has not been fully addressed. Although most studies would postulate inhibition of insulin signaling by GC in adipocytes, the data and mechanism are not clear. Importantly, studies have invariably focused upon rodent models and cell lines, and we would argue that simple extrapolation to human studies cannot be made. We have performed studies in a novel human cell line that resembles primary culture systems in its ability to acquire a mature adipocyte phenotype.

In differentiated 3T3L1 cells, GCs have induced insulin resistance through decreased IRS-1 protein expression (16, 17). However, IRS-2 expression and phosphorylation are also increased after GC treatment, whereas PKB/akt phosphorylation remains unchanged. In addition, recent kinome analysis has suggested that nongenomic actions of GC in 3T3L1 cells may be important in inducing insulin resistance (25). The net effect of these changes is a decrease in insulin-stimulated glucose uptake (17). In primary cultures of rat adipocytes, GCs decrease expression of IRS-1, PI3 kinase, and PKB/akt, although interestingly, IRS-2 expression is increased (19). In addition, IRS-2 phosphorylation is increased after GC treatment in isolated rat hepatocytes (26). There is only a single published study in human adipose tissue that has used isolated and not cultured differentiated adipocytes (18). GC decreased IRS-1 expression, decreased insulin-stimulated glucose uptake, but had no effect upon PKB/akt phosphorylation in omental adipocytes. GCs were without effect in sc cells (18). In our study, acute exposure to GC (up to 48 h) increased insulin-stimulated IRS-1 and PKB/akt phosphorylation, and subsequent glucose uptake study in both undifferentiated and differentiated human sc adipocytes (both primary cultures and cell line), effects that are mediated by the GR, with insulin acting through PI3 kinase activation. The discrepancies between our observations and the previously published report may be a reflection of the different stage of adipocyte differentiation: proliferating preadipocytes, isolated and differentiated in culture vs. isolated intact mature adipocytes. The active, highly regulated process of adipocyte differentiation is dependent upon insulin (and GCs), and, therefore, once adipocytes are fully differentiated, insulin signaling (and its regulation) may differ substantially. The net effect upon insulin-stimulated glucose uptake in adipose tissue as a whole remains unclear. This may offer an explanation as to the lack of response in the previously published study. It is possible that this could represent a novel mechanism of GC induced obesity, sensitizing adipose tissue to the effects of insulin, and thus enhancing both insulin and GC stimulated adipocyte differentiation. In addition, GC mediated insulin sensitization can promote insulin-induced lipid storage through induction of sterol regulatory binding protein 1c (SREBP1c) (27). However, the fact that these cells are cultured ex vivo always needs to be considered when interpreting the data.

The data in skeletal muscle are clearer; insulin receptor autophosphorylation and IRS-1 expression and/or phosphorylation are decreased (28, 29, 30). In addition, PI3 kinase activity is decreased (31, 32), and this has been attributed to increased expression of the p85{alpha} regulatory subunit that impairs activation of the p110 catalytic subunit, leading to decreased IRS-1 phosphorylation (31). In agreement with the published literature (33), in our study GC induced insulin resistance as evidenced by decreased insulin-stimulated glucose uptake in primary cultures of human muscle.

Species specificity of response within adipose tissue may be important and could underpin some of the differences between published data and our observations. In contrast to humans, rodents treated with GC lose rather than gain weight (34, 35). Whereas this is initially through loss of muscle mass, there is subsequently a progressive loss of adipose tissue (36), although the mechanisms underpinning this process are not clear, which is a marked contrast to clinical observations in humans. It is possible that this may reflect true insulin resistance within adipose tissue, as well as in other organs, decreasing adipocyte differentiation and lipid accumulation in contrast to our postulated hypothesis of GC mediated insulin sensitization in human adipose tissue.

Tissue specificity of response to GC is not a new concept. In rodents, GCs decrease IRS-1 and PKB/akt phosphorylation in liver, but not in muscle (29, 30). Furthermore, key metabolic enzymes may also be differentially regulated. In liver, GCs increase the expression of phosphoenolpyruvate carboxykinase, the rate-limiting step in gluconeogenesis, while decreasing expression in adipose tissue (37). In our cell systems, IRS-2 expression increased in response to GC treatment, and it is possible that this may represent the mechanism by which GCs exert their effect upon insulin signaling in human adipose tissue. Evidence to support the role of IRS proteins as determinants of tissue-specific insulin sensitivity is provided by IR, and IRS-1 and 2 knockout mice. Lack of IRS-1 appears to be more crucial in determining insulin resistance in skeletal muscle, whereas IRS-2 is more important in liver and adipose tissue (38, 39), although the detailed mechanisms underpinning this observation are not known. Interestingly, we also observed an increase in IRS-1 and IRS-2 expression after GC treatment in skeletal muscle cells. However, this did not translate to downstream activation of PKB/akt, and, indeed, insulin-stimulated glucose uptake was decreased in contrast to our observations in adipocytes, and, therefore, the significance of this observation in muscle is not clear. Phosphorylation status of the IRS proteins as markers of their activation was not examined in this study, and this may be important in explaining the discrepancy between IRS mRNA expression and insulin signaling cascade activation.

Insulin sensitization of adipose by GC would explain many clinical scenarios. In states of GC excess, there is a predilection for increased intra-abdominal adipose tissue, however, although the data are less clear, it is probable that there is an associated (albeit less dramatic) increase in abdominal sc adipose tissue (40). The primary preadipocyte cultures in this study were all taken from female patients. Sexually dimorphic expression of GR has been described in adipose tissue with increased expression in female sc vs. omental preadipocytes, but no such changes in men (41). Although there are no published data, it is interesting to speculate that in light of the differential GR expression, GC mediated changes in fat distribution may also be different between sexes. Certainly, after successful treatment of Cushing’s disease in women, sc fat mass decreases (42). Although we are unable to comment specifically about the intra-abdominal depot because our studies were exclusively performed on a sc derived cell line and endorsed in human sc primary cultures, GC-mediated increases in insulin sensitivity in sc adipose tissue may predispose to increases in sc fat mass, and contribute to the Cushing’s phenotype and health risks (43). Whereas it is plausible that this may have an impact upon insulin sensitivity in distant organs, including liver and muscle (44), sc adipose tissue deposition per se may not always be detrimental and, indeed, may be protective from some of the adverse consequences of obesity (45).

Tissue-specific differential regulation of insulin signaling by GC may have therapeutic implications. Local GC availability to bind to the GR is controlled by the isoenzymes of 11ß-HSD1 and 2. In adipose, liver, and to a lesser extent in muscle, the type 1 isoform predominates, converting inactive cortisone to active cortisol, thus amplifying local GC effects (46). Selectively inhibiting 11ß-HSD1 decreases local GC availability, and it is exciting to speculate that this may improve insulin sensitivity in muscle, while making adipose relatively more insulin resistant. Certainly, our observations would endorse this hypothesis. Initial studies, albeit in rodents, confirm global profound insulin sensitization (47), and recently, depot-specific changes in adipocyte biology have been shown with decreased fat cell size, decreased fatty acid synthesis, and evidence of enhanced lipid oxidation (48). However, results from human studies are still awaited.

In conclusion, we have characterized the first description of human tissue-specific regulation of insulin signaling by acute exposure to GC. As well as offering a putative mechanism for GC-induced obesity, therapeutic manipulation of local GC availability may offer a novel therapeutic strategy by enhancing global insulin sensitivity (through its actions in liver and muscle) and inducing adipose tissue-specific insulin resistance.


   Acknowledgments
 
We thank Dr. C. Darimont (Nestle, Switzerland) for providing the Chub-S7 cells.


   Footnotes
 
Funding for this study has been provided by the Wellcome Trust, United Kingdom (program grant to P.M.S. and Clinician Scientist Fellowship to J.W.T.).

Disclosure Statement: L.L.G., I.J.B., and J.W.T. have nothing to declare. P.M.S. has had a consultancy with Pfizer Global R&D.

First Published Online August 21, 2007

Abbreviations: Dex, Dexamethasone; FABP4, fatty acid binding protein 4; FCS, fetal calf serum; GC, glucocorticoid; GE, glycyrrhetinic acid; G3PDH, glycerol-3-phosphate dehydrogenase; GR, glucocorticoid receptor; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; IRS, insulin receptor substrate; KRP, Krebs-Ringer-HEPES; ns, not significant; PI3, phosphatidylinositol-3; PKB, protein kinase B.

Received June 22, 2007.

Accepted August 9, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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