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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0595
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6588-6595
Copyright © 2005 by The Endocrine Society

Insulin-Like Growth Factor Binding Protein-3 Leads to Insulin Resistance in Adipocytes

Sophie S. Y. Chan, Stephen M. Twigg, Sue M. Firth and Robert C. Baxter

Kolling Institute of Medical Research (S.S.Y.C., S.M.F., R.C.B.), Royal North Shore Hospital and Discipline of Medicine (S.M.T.), University of Sydney, NSW 2065, Australia

Address all correspondence and requests for reprints to: Sophie S. Y. Chan, Kolling Institute of Medical Research, Royal North Shore Hospital, Pacific Highway, St. Leonards 2065, NSW, Australia. E-mail: ssychan{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Transgenic mice overexpressing IGF binding protein-3 (IGFBP-3) have insulin resistance with reduced uptake of 2-deoxyglucose in muscle and adipose tissue.

Objective: Our aim was to investigate the effects of IGFBP-3 on glucose uptake in adipocytes.

Results: In 3T3-L1 adipocytes, IGFBP-3 reduced insulin-stimulated but not basal glucose uptake. This was independent of IGF binding because IGFBP-2 and IGFBP-1 had no effect, whereas two non-IGF binding mutants of IGFBP-3 were inhibitory. The effect of IGFBP-3 was independent of the blockade of the IGF-I receptor. A mutant form of IGFBP-3 that does not translocate to the nucleus or bind retinoid X receptor-{alpha} was able to inhibit insulin-stimulated glucose uptake, indicating that nuclear translocation and retinoid X receptor-{alpha} binding are not essential for this IGFBP-3 action. IGFBP-3 reduced insulin-stimulated glucose transporter-4 translocation to the plasma membrane and reduced threonine phosphorylation of Akt. Collectively, our data indicate that IGFBP-3 impacts on the insulin signaling pathway to inhibit insulin-stimulated glucose uptake independent of IGFs and through nonnuclear mechanisms. Finally, we showed that IGFBP-3 inhibited insulin-stimulated glucose uptake in omental but not sc adipose tissue explants.

Conclusion: IGFBP-3 may contribute to insulin resistance in adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEIN-3 (IGFBP-3) is one of six high-affinity binding proteins for IGFs. It can mediate effects on cells through controlling the access of IGFs to the type I IGF receptor (IGFR1) (1). IGFBP-3 can exert a number of IGF-independent effects on cell growth and apoptosis (2, 3, 4). IGFBP-3 contains a nuclear localization sequence (NLS) and is translocated to the nucleus of various human cell types (5, 6, 7). Recently, IGFBP-3 has been shown to bind to the nuclear transcription factor retinoid X receptor-{alpha} (RXR-{alpha}) and to modulate RXR-{alpha}-mediated signaling (8).

IGFBP-3 is produced by a wide variety of tissues including both rodent and human adipocytes where it is up-regulated during adipocyte differentiation (9, 10). However, the role of IGFBP-3 in adipose tissue has not been well studied. It has recently been shown that transgenic mice overexpressing IGFBP-3 develop insulin resistance and impaired glucose tolerance (11). It was found that uptake of 2-deoxyglucose in vivo was reduced in muscle and adipose tissue in the IGFBP-3 transgenic mice.

Peroxisome proliferator activated receptor-{gamma} is a transcription factor that forms an obligate heterodimer with RXR-{alpha} to affect adipocyte differentiation and glucose regulation. It was reported in preliminary form that IGFBP-3 could inhibit basal and insulin-stimulated glucose transport in 3T3-L1 adipocytes. Shim et al. (12) hypothesized that this may be because of IGFBP-3 binding to RXR-{alpha} and thereby interfering with peroxisome proliferator activated receptor-{gamma} signaling.

We now show that IGFBP-3 reduces insulin-stimulated but not basal glucose uptake via an IGF-independent mechanism in 3T3-L1 adipocytes. Insulin resistance in these cells is also induced by a NLS mutant form of IGFBP-3 that is unable to translocate to the nucleus (13) or bind to RXR-{alpha}. We show that IGFBP-3 reduces insulin-stimulated glucose transporter-4 (GLUT-4) translocation and phosphorylation of Akt. Lastly, we show that IGFBP-3 inhibits insulin-stimulated glucose uptake in human omental adipose tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Except where specified, reagents were purchased from Sigma-Aldrich (St. Louis, MO). Actrapid, recombinant human insulin, was purchased from Novo Nordisk Pharmaceutical (Bagsvaerd, Denmark).

3T3-L1 adipocytes

3T3-L1 cells (obtained from American Type Culture Collection, Manassas, VA) were maintained in DMEM with 10% newborn calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine and passaged before confluence. For differentiation, confluent cells were treated with 0.1 µg/ml dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.7 µM insulin in DMEM supplemented with 10% fetal calf serum (FCS) for 48 h. They were then grown in DMEM containing 10% FCS and 0.7 µM insulin for an additional 48 h. Cells were maintained in DMEM with 10% FCS. Cells were used for experiments at d 8–12 after differentiation when there were greater than 95% of cells displaying the fully differentiated adipocyte phenotype as assessed by lipid accumulation using oil red O (Proscitech, Thuringowa, Australia).

Human adipose tissue explants

The study was approved by the institutional ethics committee, and patients gave informed consent. Patients with diabetes mellitus or on medications known to affect glucose metabolism were excluded. Subjects were fasted overnight before their elective abdominal surgery. Adipose tissue was obtained during surgery from omental and abdominal sc fat deposits. Adipose tissue was placed in DMEM with 1000 mg/liter glucose and 2% BSA. Adipose tissue explants were isolated as described (14). Briefly, whole-tissue adipose explants (~15–20 mg), excluding visible connective tissue and blood vessels, were removed from the biopsy material and placed in DMEM with 1000 mg/liter glucose, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% BSA and incubated at 37 C with 5% CO2.

IGFBPs and other compounds

Recombinant human IGFBP-3 and IGFBP-3 NLS mutant (228MDGEA) were produced in our laboratory by a replication-deficient adenovirus-mediated expression system, as previously described (15). The non-IGF binding IGFBP-3 mutants (80G81G and 80G81G217S223A) were generated by site-directed mutagenesis as described (16). Recombinant human IGFBP-2 was provided by Sandoz (now Novartis; Basel, Switzerland). Human IGFBP-1 was purified from amniotic fluid as previously described (17). Recombinant human IGF-I and IGF-I receptor antibody {alpha}IR-3 were obtained from Genentech (San Francisco, CA) and Oncogene Research Products (Cambridge, MA), respectively. Cycloheximide was purchased from A.G. Scientific, Inc. (San Diego, CA).

Cellular localization of labeled IGFBP-3

Fluorescent-labeled IGFBP-3 was generated by conjugating protein to dichlorotriazinylaminofluorescein I HCl (DTAF) as described for Cy3 conjugation (5). The 3T3-L1 adipocytes were cultured on glass coverslips, washed twice, and placed into serum-free DMEM with or without labeled IGFBP-3 for 2–24 h. Cells were then processed, fixed, and examined under confocal microscopy as previously described (5).

2-Deoxyglucose uptake

Differentiated 3T3-L1 cells in 12-well plates were rendered serum free for up to 24 h with or without indicated compounds. They were washed twice and placed into Krebs buffer with or without indicated compounds for an additional 2 h. Insulin (10 ng/ml) was added for 20 min. Samples were incubated for 5 min with radiolabeled glucose (final concentration, 0.2 mM 2-[3H]deoxyglucose; 0.1 µCi/ml) (PerkinElmer Life Sciences, Inc., Boston, MA), and cells were washed three times with ice-cold PBS and lysed with 0.5% SDS. Three milliliters of Ultima Gold scintillant (Packard, Groningen, The Netherlands) were added, and radioactivity was counted in an A290001 ß-counter (Packard).

Whole-tissue adipose explants were incubated in serum-free media with or without IGFBP-3 for 24 h. Medium was removed and explants were washed three times and placed in Krebs buffer (1% BSA) with or without IGFBP-3 for an additional 2 h before 100 ng/ml insulin was added to the appropriate samples for 20 min. Samples were incubated for 5 min with radiolabeled glucose. Explants were washed in ice-cold Krebs buffer, blotted, and weighed. Data were analyzed as pmol 2-deoxyglucose/mg wet weight and expressed as fold change over basal glucose uptake.

Plasma membrane lawn assays

Translocation of GLUT-4 was assessed by plasma membrane (PM) lawn assays according to the method of Robinson et al. (18) and Whitehead et al. (19). Briefly, 3T3-L1 cells were differentiated on glass coverslips and incubated in Krebs buffer with or without IGFBP-3. Insulin (10 ng/ml) was then added to the appropriate wells for 20 min. Cells were washed and sonicated with a probe sonicator (Kontes, Vineland, NJ) leaving a lawn of PMs attached to the coverslip. The coverslips were blocked in PBS with 5% skim milk, rinsed in PBS, and incubated with GLUT-4 antibody (1:200) (kind gift from Prof. D. James, Garvan Institute of Medical Research, Australia) followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody (1:100) (Molecular Probes, Eugene, OR). Coverslips were washed with PBS, mounted onto glass microscope slides, and viewed with a Leica DMLB fluorescent microscope using excitation 480 ± 30 nm and emission 535 ± 40 nm. Images were obtained using a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Duplicate coverslips were examined for each condition, and seven random images of PMs were taken. The images were then quantitated by an observer blinded to the treatment conditions using Image J version 1.33j (National Institutes of Health, Bethesda, MD).

Immunoprecipitations

Immunoprecipitation of the insulin receptor (IR) was performed as previously described (20). Cells were cultured and differentiated in 80-cm2 flasks. Briefly, cell lysate was mixed with rabbit polyclonal ß-subunit IR antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and protein A beads (Amersham Pharmacia, Uppsala, Sweden) overnight at 4 C. Tyrosine phosphorylation of the IR-ß was assessed with PY20 (Transduction Laboratories, Inc., Lexington, KY), followed by antimouse IgG horseradish-peroxidase conjugate (Amersham Pharmacia) and detection using chemiluminescence (ECL plus; Amersham Pharmacia). After stripping with buffer [2% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100 mM ß-mercaptoethanol] for 30 min at 65 C, total IR-ß was assessed with a ß-subunit-IR antibody (Oncogene, Boston, MA) and detected as described above. The level of IR phosphorylation was quantified by densitometry, and results were normalized to total IR protein.

Immunoblots

Cells were grown to confluence and differentiated in six-well plates and lysed with 1x SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, and 0.01% bromophenol blue) containing 50 mM dithiothreitol. Prepared lysates were subjected to SDS-PAGE (10% separating gel) and transferred to Hybond-C nitrocellulose (Amersham Pharmacia) over 1.5 h at 12 V using a Novablot transfer unit (Amrad, Richmond, Australia). Blots were blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween 20, pH 7.4, for 1 h at room temperature and then probed with phospho-Akt threonine-308 (Thr-308) and phospho-Akt Serine-473 (Ser-473) antibody (Cell Signaling, Beverly, MA) 1:1000 in 5% BSA/Tris-buffered saline with 0.1% Tween 20, pH 7.4, overnight at 4 C. Blots were incubated with antirabbit IgG horseradish-peroxidase conjugate (Amersham Pharmacia), washed, and detected using ECL before autoradiography. The same blot was stripped as described above and probed with Akt antibody (Cell Signaling) for total Akt analysis. The level of Akt phosphorylation was quantified by densitometry, and results were normalized to total Akt protein.

Statistical analysis

All experiments were conducted in triplicate at least three times independently (unless otherwise stated). Figures show representative experiments. Statistical analysis was performed with StatView, version 5 (SAS Institute, Inc., Cary, NC). Results were analyzed using ANOVA followed by Fisher’s protected least-significant difference test. All graphs show means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGFBP-3 inhibits insulin-stimulated glucose uptake

We showed that IGFBP-3 is transported from the extracellular medium into 3T3-L1 adipocytes. Compared with the low background signal in the absence of added fluorescent-labeled IGFBP-3 (Fig. 1AGo, left), DTAF-labeled IGFBP-3 (500 ng/ml) was seen in both the cytoplasm and the nucleus by confocal microscopy after 2 h (Fig. 1AGo, right). It was still present after 24 h (data not shown).



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  		FIG. 1. Cellular uptake and effects of IGFBP-3. A, Confocal images of 3T3-L1 adipocytes not treated (left) or treated with DTAF-labeled IGFBP-3 (500 ng/ml) (right) for 2 h. B–D, The effects of IGFBP-3 on basal and insulin-stimulated 2-deoxyglucose (DOG) uptake in 3T3-L1 adipocytes. B, IGFBP-3 at 1 µg/ml for 24 h had no effect on basal glucose uptake; C, dose-dependent inhibition of insulin-stimulated glucose uptake by IGFBP-3 (24 h) with doses as indicated in ng/ml; D, time course of inhibition with 50 ng/ml IGFBP-3. Treatment with insulin at 10 ng/ml was for 20 min. Statistical significance is denoted as follows: **, P < 0.01; ***, P < 0.001.

 
Insulin (10 ng/ml) significantly increased glucose uptake (Fig. 1BGo). IGFBP-3 (1 µg/ml) had no effect on basal glucose uptake (Fig. 1BGo) but significantly reduced insulin-stimulated glucose uptake in a dose-dependent manner (Fig. 1CGo). Exposure to 50 ng/ml of IGFBP-3 for 24 h maximally inhibited insulin-stimulated glucose uptake by approximately 40%. Figure 1DGo shows that IGFBP-3 (50 ng/ml) significantly decreased insulin-stimulated glucose uptake by 36% at 90 min and by 44% at 24 h.

The inhibitory effect of IGFBP-3 is IGF-I and IGFR1 independent

IGFBP-2 also binds to IGFs with high affinity. IGFBP-2 had no effect on insulin-stimulated glucose uptake when tested at 200 ng/ml (Fig. 2AGo) or at any dose from 30–1000 ng/ml (data not shown). As was found for IGFBP-2, IGFBP-1 at increasing doses also had no effect on insulin-stimulated glucose uptake. There was no effect of IGFBP-1 on basal glucose uptake (data not shown). This suggested that the inhibitory effect of IGFBP-3 on insulin-stimulated glucose uptake was not a result of IGFBP-3 sequestering any possible endogenous IGF-I or IGF-II.



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  		FIG. 2. Inhibition of insulin-stimulated 2-deoxyglucose (DOG) uptake by IGFBP-3 is IGF and IGFR1 independent. A, IGFBP-2 at 300 ng/ml (24 h) had no effect on insulin-stimulated glucose uptake. B, IGFBP-1 at indicated doses (24 h) had no effect on insulin-stimulated glucose uptake. C, Non-IGF-binding IGFBP-3 mutants, 80G81G and 80G81G217S223A (both at 200 ng/ml for 24 h) had a similar inhibitory effect as wild-type IGFBP-3 (200 ng/ml for 24 h). D, IGFBP-3 (300 ng/ml for 24 h) significantly inhibited insulin-stimulated glucose uptake in the presence of the IGFR1 receptor blocker {alpha}IR3 (15 µg/ml for 24 h). Treatment with insulin at 10 ng/ml was for 20 min. Statistical significance is denoted as follows: **, P < 0.01; ***, P < 0.001; NS, not significant.

 
High-affinity binding of IGFBP-3 to IGFs requires both the N- and C-terminal domains of IGFBP-3 (21). Our laboratory has recently generated mutants of IGFBP-3 that have reduced or negligible binding to IGF-I and -II (16). In particular, the combined N- and C-terminal mutant IGFBP-3 80G81G217S223A showed undetectable binding to the IGFs. Despite such greatly reduced IGF binding, IGFBP-3 mutants 80G81G and 80G81G217S223A were still able to significantly reduce insulin-stimulated glucose uptake (Fig. 2CGo). This provides additional compelling evidence that the inhibitory effect of IGFBP-3 is independent of the binding of IGFs.

Using a monoclonal antibody ({alpha}IR3) that selectively blocks IGFR1, we showed that the effect of IGFBP-3 on reducing insulin-stimulated glucose uptake was independent of the blockade of the IGFR1. {alpha}IR3 (15 µg/ml) was able to inhibit glucose uptake stimulated by 10 ng/ml IGF-I but not by insulin (Fig. 2DGo). In contrast, in the presence of {alpha}IR3 (15 µg/ml), IGFBP-3 (300 ng/ml) was still able to significantly inhibit insulin-stimulated glucose uptake, indicating that signaling through the IGFR1 is not involved in the IGFBP-3 effect.

The effect of IGFBP-3 on insulin-stimulated glucose uptake does not require nuclear localization or new protein synthesis

The basic carboxyl-terminal domain of IGFBP-3 contains a nuclear localization sequence. In the IGFBP-3 NLS mutant (228MDGEA), five critical residues for nuclear localization have been replaced with corresponding residues from IGFBP-1, which is known not to translocate to the nucleus. The IGFBP-3 NLS mutant (228MDGEA) is unable to translocate to the nucleus (5) or bind to the transcription factor RXR-{alpha} (Schedlich, L., personal communication). As shown in Fig. 3AGo, IGFBP-3 NLS mutant (100 ng/ml) significantly inhibited insulin-stimulated glucose uptake by 41%, indicating that the inhibitory effect of IGFBP-3 does not require IGFBP-3 to be translocated to the nucleus or to bind to RXR-{alpha}.



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  		FIG. 3. IGFBP-3 inhibition of insulin-stimulated 2-deoxyglucose (DOG) uptake does not require nuclear localization or new protein synthesis. A, The IGFBP-3 NLS mutant 228MDGEA at 100 ng/ml for 24 h inhibited insulin-stimulated glucose uptake. B, Cells were preincubated with cycloheximide (10 µg/ml) for 4 h before adding IGFBP-3 (300 ng/ml) for an additional 4 h. IGFBP-3 inhibited insulin-stimulated glucose uptake in the presence of cycloheximide. Treatment with insulin at 10 ng/ml was for 20 min. Statistical significance is denoted as follows: **, P < 0.01; ***, P < 0.001.

 
To determine whether new protein synthesis was required for the inhibitory effect of IGFBP-3, cycloheximide was used. As shown in Fig. 3BGo, 3TGo3-L1 adipocytes were preincubated with cycloheximide (10 µg/ml) for 4 h before adding IGFBP-3 (300ng/ml) for an additional 4 h. In the presence of cycloheximide, IGFBP-3 was still able to inhibit insulin-stimulated glucose uptake. This suggests that the inhibitory effect of IGFBP-3 is independent of new protein synthesis.

IGFBP-3 reduces GLUT-4 translocation to the PM of 3T3-L1 adipocytes

Insulin acts through the insulin receptor in adipocytes, and through complex intracellular cascades it leads to the translocation of GLUT-4 transporters to the PM. We therefore examined the effects of IGFBP-3 on GLUT-4 translocation to the PM using PM lawn assays (Fig. 4Go). There is little GLUT-4 at the PM detectable by this technique under basal conditions (Fig. 4Go, A and F). Insulin (10 ng/ml) for 20 min caused a significant 3-fold increase in GLUT4 translocation to the PM (Fig. 4Go, B and F). Exposure to IGFBP-3 (100 ng/ml) or IGFBP-3 NLS mutant 228MDGEA (100 ng/ml) for 24 h caused significant 39 or 35% reductions, respectively, in GLUT-4 translocation to the PM in the presence of insulin (Fig. 4Go, C and D). As a negative control, exposure to IGFBP-2 (300 ng/ml) for 24 h had no significant effect on insulin-stimulated GLUT-4 translocation to the PM (Fig. 4Go, E and F). These data are consistent with the effects of IGFBP-3 on inhibiting insulin-stimulated glucose uptake in adipocytes.



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  		FIG. 4. Reduction of GLUT-4 translocation to the PM by IGFBP-3 and IGFBP-3 NLS mutant. GLUT-4 translocation to the PM was measured using PM lawn assays. A–E, Representative fluorescent microscope images: A, control; B, insulin alone; C, IGFBP-3 (100 ng/ml for 24 h) and insulin; D, IGFBP-3 NLS mutant 228MDGEA (100 ng/ml for 24 h) and insulin; E, IGFBP-2 (300 ng/ml for 24 h) and insulin. F, Quantification of seven images from each duplicate coverslip was performed. Representative experiment from two independent experiments is shown. Treatment with insulin at 10 ng/ml was for 20 min. Statistical significance is denoted as follows: ***, P < 0.001.

 
IGFBP-3 does not affect insulin receptor phosphorylation but decreases phospho-Akt

Given that IGFBP-3 inhibits GLUT-4 translocation to the PM, the classical insulin signaling pathway was studied. In this pathway, insulin elicits an initial effect by binding to the IR and causing IR autophosphorylation. We initially studied whether IGFBP-3 could affect phosphorylation of the IR by insulin. Figure 5AGo shows that IGFBP-3 (300 ng/ml) for 24 h had no effect on tyrosine phosphorylation of the IR by insulin. Thus, it is unlikely that IGFBP-3 is down-regulating insulin-stimulated glucose uptake via an effect on IR phosphorylation status.



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  		FIG. 5. Effects of IGFBP-3 on IR phosphorylation and phospho-Akt. A, IR phosphorylation was examined by immunoprecipitating IRs with a ß-subunit antibody (Ab). Tyrosine phosphorylation of the IR-ß was assessed with PY20 antibody. After stripping, blots were examined for total IR-ß with a ß-subunit-IR antibody. Treatment with insulin at 10 ng/ml was for 5 min. IGFBP-3 (300 ng/ml for 24 h) had no effect on insulin-induced phosphorylation of the IR. B, IGFBP-3 reduced threonine phospho-Akt. The top panel shows blots probed with antibodies to phospho-Akt Thr-308 and Ser-473. After stripping, blots were probed with total Akt antibody. Treatment with insulin at 10 ng/ml was for 5 min and IGFBP-3 at 100 ng/ml was for 24 h. The bottom panel shows data obtained from three representative blots quantified by densitometry with the amount of phospho-Akt corrected for total Akt. The results are expressed relative to the amount of phospho-Akt induced by insulin alone. C, IGFBP-3 reduced threonine phospho-Akt in a dose-dependent manner. Treatment with insulin at 10 ng/ml was for 5 min and IGFBP-3 at indicated doses was for 24 h. Statistical significance is denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

 
The downstream serine/threonine protein kinase Akt is important in insulin action. We studied the effects of IGFBP-3 on threonine phosphorylation of Akt. IGFBP-3 (100 ng/ml) for 24 h led to a significant reduction in Thr308 phosphorylation of Akt by insulin (Fig. 5BGo). There was no significant decrease in Ser473 phosphorylation of Akt by insulin in the presence of IGFBP-3 (Fig. 5BGo). IGFBP-3 dose-dependently reduced Thr308 phosphorylation of Akt by insulin (Fig. 5CGo). These data suggest that a decrease in phospho-Akt may account for the effect of IGFBP-3 to inhibit GLUT-4 translocation to the PM and insulin-stimulated glucose uptake, independently of a change in IR phosphorylation.

IGFBP-3 inhibits insulin-stimulated glucose uptake in human omental but not sc fat explants

Similar to published data (14, 22), insulin stimulated an approximately 1.8-fold increase in glucose uptake in both human sc and omental fat explants. We demonstrated that IGFBP-3 was able to inhibit insulin-stimulated glucose uptake in a depot-specific manner in human fat explants. In omental adipose tissue (Fig. 6AGo), IGFBP-3 (300 ng/ml) had no effect on basal glucose uptake but significantly inhibited insulin-stimulated glucose uptake by approximately 31%. However, in sc adipose tissue (Fig. 6BGo), IGFBP-3 (300 ng/ml) had no significant effect on either basal or insulin-stimulated glucose uptake.



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  		FIG. 6. Inhibition of insulin-stimulated 2-deoxyglucose (DOG) uptake by IGFBP-3 in human omental but not sc adipose tissue explants. A, In human omental adipose tissue, IGFBP-3 at 300 ng/ml (24 h) significantly inhibited insulin-stimulated glucose uptake but had no effect on basal glucose uptake. B, In sc adipose tissue, IGFBP-3 had no effect on basal or insulin-stimulated glucose uptake. Treatment with insulin at 100 ng/ml was for 20 min. Statistical significance is denoted as follows: *, P < 0.05; **, P < 0.01; NS, not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is now clear that IGFBP-3 can have effects on cellular function that are independent of IGFs (23). The 3T3-L1 cells express insignificant amounts of endogenous IGF-I (9), providing a model to study IGF-independent effects of IGFBP-3. We demonstrated that IGFBP-3 inhibited insulin-stimulated but not basal glucose uptake. This was not simply a result of IGFBP-3 binding any endogenous IGF-I or -II that may be present, because high-affinity IGF binders, IGFBP-2 and IGFBP-1, had no effect on insulin-stimulated glucose uptake. In addition, we showed that novel non-IGF-binding IGFBP-3 mutants were able to inhibit insulin-stimulated glucose uptake as effectively as IGFBP-3. This again indicated that the effects of IGFBP-3 were not through binding to any endogenous IGFs. We also demonstrated that IGFBP-3 was able to induce insulin resistance in adipocytes despite blockade of the IGFR1, indicating that the effect was independent of IGF-I acting through the IGFR1. Together these data provide strong evidence that the inhibitory action of IGFBP-3 on glucose uptake occurs independently of effects on IGF binding or IGFR1 activation. We then confirmed that IGFBP-3 was able to induce insulin resistance in human omental but not sc adipose tissue.

These data are consistent with the report that transgenic mice overexpressing IGFBP-3 develop insulin resistance and impaired glucose tolerance even in the presence of elevated total and free IGF-I (11). The IGFBP-3 transgenic mice develop elevated fasting insulin and glucose levels with reduced in vivo uptake of 2-deoxyglucose in muscle and adipose tissue.

IGFBP-3 can be taken into cells via mechanisms involving caveolin and transferrin (24, 25). It can translocate to the nucleus via an importin-mediated pathway (5). We have shown in this study that exogenous labeled IGFBP-3 is taken into both the cytoplasm and nucleus of adipocytes. Liu et al. (8) showed that IGFBP-3 directly bound the transcription factor RXR-{alpha} and that this interaction could regulate transcriptional signaling and apoptosis in F9 embryonal carcinoma cells. They also demonstrated that the IGFBP-3 residues required for binding to RXR are located within the 18-residue basic domain sequence, residues 215–232. We have also shown that our NLS mutant (228KGRKR->MDGEA) is unable to translocate to the nucleus (13) or bind RXR-{alpha} (Schedlich, L., personal communication). The short time course (90 min to 2 h) of the IGFBP-3 effect on insulin-stimulated glucose uptake in our study and the fact that cycloheximide did not abolish the IGFBP-3 effect make a transcriptional/translational effect less likely. More importantly, the observation that the NLS mutant form of IGFBP-3 inhibited insulin-stimulated glucose uptake in this study demonstrates that neither nuclear translocation nor binding to RXR-{alpha} is required for IGFBP-3-induced insulin resistance in 3T3-L1 adipocytes. Instead, rapid insulin signaling pathways are implicated for the observed effect of IGFBP-3. IGFBP-3 causes apoptosis in breast cancer cells (26). However, we found by cell counting and trypan blue exclusion that IGFBP-3 (up to 1 µg/ml for 24 h) had no effect on the number of viable 3T3-L1 adipocyte cells (data not shown).

The final common outcome of insulin signaling pathways in the adipocyte is the translocation of GLUT-4 transporters to the PM. In our study, IGFBP-3 caused a significant reduction of GLUT-4 translocation to the PM. There are currently thought to be two distinct signaling pathways that lead to insulin-stimulated GLUT-4 translocation. We demonstrated that IGFBP-3 impacted on the classical insulin signaling pathway with reduced phospho-Akt, in particular threonine-308 phospho-Akt. Interestingly, insulin-stimulated phosphorylation of Akt on Thr308 and Ser473 showed different sensitivities to IGFBP-3 inhibition. Worrall and Olefsky (27) have also shown that a decrease in insulin-induced phosphorylation of Akt on Thr308 was more marked in response to Ca2+ depletion than the decrease seen in phosphorylation of Akt on serine-473. The 3'-phosphatidylinositol-dependent kinase 1 specifically phosphorylates Akt at Thr308 and not Ser473 (28). It is possible that IGFBP-3 affects preferentially the Akt Thr308 kinase, 3'-phosphatidylinositol-dependent kinase 1, over the Ser473 candidate kinases. Alternatively, IGFBP-3 may stimulate the dephosphorylation of phospho-Akt itself or of the IR targets insulin receptor substrate-1 or -2, as demonstrated in mink lung cells (29). Additional studies are needed to address the precise mechanisms of the IGFBP-3 effect on phospho-Akt.

Siddals et al. (30) found that IGFBP-1 inhibited IGF-I-stimulated glucose uptake at a 1:1 ratio presumably by binding IGF-I in 3T3-L1 adipocytes. However, this effect was in the presence of added IGF-I. We have shown that IGFBP-1 alone had no effect on basal or insulin-stimulated glucose uptake.

Finally, we examined the effects of IGFBP-3 on human adipose tissue. IGFBP-3 had no effect on basal glucose uptake but significantly decreased insulin-stimulated glucose uptake in omental but not sc adipose tissue. Clinically, there is a strong association between visceral adiposity (rather than general obesity) and insulin resistance (31). It is also well recognized that a variety of hormones act differently in omental compared with visceral fat. For example, insulin causes a higher rate of lipolysis in omental than sc fat (32). Interestingly, we have found that IGFBP-3 induced insulin resistance in human adipose tissue in a depot-specific manner. IGFBP-3 may contribute to human adipocyte insulin resistance in the omental fat, which plays a more important role in contributing to clinical insulin resistance. Lundgren et al. (22) recently found that glucocorticoid, another hormone known to induce insulin resistance, inhibited glucose uptake only in omental but not sc adipocytes.

In summary, we have found that IGFBP-3 inhibits insulin-stimulated glucose uptake in adipocytes in vitro. This process is independent of IGF and the IGFR1 and does not require nuclear localization of IGFBP-3. Consistent with these results, we have shown that IGFBP-3 down-regulated GLUT-4 transporters to the PM. We have also demonstrated that a possible mechanism of IGFBP-3-induced insulin resistance in 3T3-L1 adipocytes is through reduced Thr308 phospho-Akt, independent of a change in IR phosphorylation. IGFBP-3 caused insulin resistance in human omental but not sc adipose tissue. Whether IGFBP-3 may contribute to adipocyte insulin resistance in humans in vivo and thus may be a target for intervention requires additional assessment.


   Acknowledgments
 
The authors thank Professor David James and Dr. Juan Carlos Molero (Garvan Institute of Medical Research, Sydney, Australia) for assistance with the plasma membrane lawn technique and the kind gifts of the GLUT-4 and secondary antibodies. We also thank Dr. Jon Whitehead and Professor Johannes Prins (Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Woolloongabba, Australia) for advice on the culture of 3T3-L1 cells and technique of human adipose tissue explants.


   Footnotes
 
S.S.Y.C. is supported by a National Health and Medical Research Council of Australia Medical and Dental Postgraduate Scholarship.

First Published Online September 27, 2005

Abbreviations: DTAF, Dichlorotriazinylaminofluorescein I HCl; FCS, fetal calf serum; GLUT-4, glucose transporter-4; IGFBP-3, IGF binding protein-3; IGFR1, type I IGF-I receptor; IR, insulin receptor; NLS, nuclear localization sequence; PM, plasma membrane; RXR-{alpha}, retinoid X receptor-{alpha}.

Received March 17, 2005.

Accepted September 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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