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Endocrinology Vol. 144, No. 6 2388-2395
Copyright © 2003 by The Endocrine Society

Glucosamine Induces Resistance to Insulin-Like Growth Factor I (IGF-I) and Insulin in Hep G2 Cell Cultures: Biological Significance of IGF-I/Insulin Hybrid Receptors

K. Sakai and D. R. Clemmons

Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., 6111 Thurston Bowles, CB 7170, Division of Endocrinology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599. E-mail: endo{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I stimulates insulin-like actions directly through its receptor, and it also enhances sensitivity to insulin-mediated effects in vivo. These studies were undertaken to analyze the role of IGF-I, insulin, and insulin/IGF-I hybrid receptors (HRs) in mediating IGF-I and insulin signaling in cells that had been made insulin-resistant by treatment with glucosamine. Human HepG2 cells, which express IGF-I receptors, insulin receptors (IRs), and IGF-I/insulin HRs, were exposed to 20 mM glucosamine; and the effects of IGF-I and insulin in stimulating glycogen synthesis were determined. An overnight exposure to glucosamine markedly attenuated the effects of insulin and IGF-I in stimulating glycogen synthesis. To determine which receptors were mediating this effect, the ability of insulin and IGF-I to stimulate phosphorylation of their respective receptors was analyzed. An 18-h exposure to glucosamine (20 mM) caused a 75% reduction in the ability of IGF-I to phosphorylate its receptor but no change in receptor abundance. Glucosamine also caused a major reduction in insulin-stimulated receptor phosphorylation, although, unlike IGF-I, there was also a 50% reduction in IR abundance. Exposure to glucosamine also resulted in a reduction in the ability of IGF-I or insulin to stimulate phosphorylation of insulin IGF-I/HRs. The combination of insulin plus IGF-I was a more potent stimulus of HR phosphorylation than either agent alone, and this combination was also more potent in partially reversing the inhibitory effect of glucosamine. Taken together, these findings indicate that glucosamine induces a loss of sensitivity to stimulation of insulin, IGF-I, or HR tyrosine kinase activity by insulin or IGF-I. Although insulin is able to partially reverse the effect of glucosamine on IR phosphorylation, it has a very minimal effect on glucosamine-induced inhibition of HR phosphorylation. However, the combination of IGF-I and insulin induces a major increase in HR phosphorylation, even in the presence of glucosamine, suggesting that it is improving the sensitivity of the HR to insulin activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BOTH IGF-I AND INSULIN stimulate metabolic effects that are mediated through their cell surface receptors. Both the IGF-I receptor (IGF-IR) and the insulin receptor (IR) are tyrosine kinase receptors and are composed of two extracellular {alpha}-subunits that contain the ligand binding sites, and two ß-subunits that have tyrosine kinase activity contained within their cytoplasmic domains. Both IGF-IR and IR activate common intracellular signal transduction pathways including the members of the IR substrate (IRS) family, and signaling components of the PI3 kinase and MAP kinase pathways. In addition to the IGF-I and the IR; an {alpha}ß-dimer of the IR can form a tetramer with an {alpha}ß-dimer of the IGF-IR to form the IGF-I/insulin hybrid receptor (HR) (1, 2). HR is widely expressed in many mammalian tissues and in several cell lines (1, 2, 3, 4). Recently, it has been demonstrated that the abundance of HR is increased in skeletal muscle of patients with type 2 diabetes (5), in placenta from insulin-resistant mothers with gestational hypertension (6), in skeletal muscle of 90% pancreatectomized diabetic rats (7), and in skeletal muscle of glucosamine-infused rats (7). HR binds IGF-I with an affinity similar to that of IGF-IR, whereas it binds insulin with a substantially lower affinity, compared with native IR (1, 8, 9). Therefore, the expression of HR might contribute to changes insulin and IGF-I sensitivities in target tissues, but the variables that regulate such changes in pathophysiologic states are not well defined.

Glucosamine has been extensively used to model the role of the hexosamine synthesis pathway in glucose-induced insulin resistance. Glucosamine exposure to cells and infusions into animals have been shown to induce insulin resistance (10, 11, 12, 13, 14). Recently, it has been shown that glucosamine infusion into rats up-regulates the expression of HR in skeletal muscle (7). However, another study showed that increased insulin resistance in normal rats, induced by glucosamine infusion, was not altered by infusion of IGF-I (15).

Importantly, the receptors mediating these responses and the relative contributions of hybrid IGF-I/IR have not been determined. Our laboratory has recently shown that human Hep G2 cells contain easily detectable IGF-I, insulin, and HRs and that HRs seem to be the predominant subtype. We demonstrated that release of free IGF-I from IGF-binding protein-1 resulted in enhancement of the cellular response to insulin and this was mediated primarily through the HR (16). The current studies were undertaken to determine whether glucosamine altered IGF-I, insulin, or IGF/insulin HR abundance in human hepatoma (Hep G2) cells and whether glucosamine exposure induced changes in insulin, IGF-I, or HR activation after IGF-I or insulin stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human hepatoma (Hep G2) cells were obtained from the American Type Culture Collection (Rockville, MD). Tissue culture media, penicillin, streptomycin, and insulin were purchased from Life Technologies, Inc. (Grand Island, NY). Polyvinylidene difluoride transfer membrane (Immobilon-P) was from Millipore Corp. (Bedford, MA). The antiphosphotyrosine antibody (PY99), antibody for ßIGF-IR (C-20), antibody for ß-IR (C-19), and anti-IRS-1 (A-19) antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal antibody for {alpha}-IR(83–14) that has no cross-reactivity with IGF-IR was from NeoMarker (Fremont, CA). The monoclonal antibody for the {alpha}-subunit of IGF-IR ({alpha}IR-3) was prepared as described previously (3). Recombinant human IGF-I was a gift from Genentech, Inc. (South San Francisco, CA).

Tissue culture
Hep G2 cells were maintained in DMEM with high glucose (4500 mg/liter) supplemented with 10% (vol/vol) fetal calf serum (Life Technologies, Inc.) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). The cells were grown in 5% CO2-95% air at 37 C and passaged using a split ratio of 1:4 after trypsinization.

Immunoprecipitation, immunodepletion, and immunoblotting
Hep G2 cells were grown to 80% confluency on 10-cm tissue culture dishes. The cells were rinsed three times with serum-free DMEM with low glucose (1000 mg/liter) (DMEM-L) and incubated overnight with the same medium containing concentrations of glucosamine that varied from 0–20 mM. The medium was replaced with 5 ml DMEM-L, and the incubation was continued for 2 h. The cultures were exposed to the indicated concentration of insulin or IGF-I for 5 min or 10 min, respectively. The cells were washed once with ice-cold PBS, and the cultures were solubilized in lysis buffer [1% Nonidet P-40, 2 mM EGTA, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 mg/ml pepstatin A, 1 mg/ml leupeptin, 1 mg/ml aprotinin]. The insoluble material was removed by centrifugation at 15,000 x g for 10 min, and the supernatant (1 mg protein) was incubated with anti-{alpha}-IR antibody (1 mg) for 18 h at 4 C. The immune complexes were incubated with antimouse IgG agarose for 3 h at 4 C. The immobilized antimouse IgG was sedimented by centrifugation at 7000 x g for 1 min and washed with lysis buffer without phosphatase inhibitors four times, and the remaining bound proteins were resuspended in 30 µl Laemmli sample buffer. The supernatants from each sample were incubated with either ß-IR antibody (1 µg) or ßIGF-IR (1 µg) antibody for 14 h at 4 C. The immune complexes were incubated with protein-A Sepharose for 3 h at 4 C. The immobilized protein-A Sepharose was sedimented by centrifugation at 7000 x g for 1 min and washed with lysis buffer without phosphatase inhibitors four times, and the proteins were resuspended in 30 µl Laemmli sample buffer. The proteins were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (0.45-mm pore size). The membrane was probed with the indicated antibody and visualized with enhanced chemiluminescence (Super-Signal CL-H substrate system; Pierce Chemical Co., Rockford, IL) and exposed to X-AR film (Eastman Kodak Co., Rochester, NY). For IRS-1 immunoprecipitation, the same lysis buffer that contained 0.25% sodium deoxycholate was used. The results of the immunoblotting experiments were quantified by scanning densitometry using a AGFA DuoScan Densitometer (Brussels, Belgium). The results were analyzed using NIH Image, version 1.61. Each experiment was repeated three times, and the results are expressed as the mean ± SD. Statistical analyses were performed using a paired Student’s t test.

D-[3H]glucose incorporation into Hep G2 cells
Assays of [3H]glucose incorporation into glycogen were performed using a modification of the method described by Widmer et al. (17). Hep G2 cells were seeded at a density of 6 x 104 cells/cm2 in 24-well culture plates with growing medium. After 48 h, cells were washed twice with serum-free DMEM-L and incubated for 18 h with the same medium in the presence and absence of 20 mM glucosamine. The medium was replaced by 200 µl DMEM-L and either IGF-I (0–15 nM) or insulin (0–100 nM); 0.5 µCi D-[3H]glucose (specific activity, 34.0 Ci/mmol) was added, and the incubation was continued. After 3 h, the medium was aspirated, and 200 ml 1.0 M KOH with 1 mg glycogen was added and incubated for 40 min at 60 C. The cell lysates were transferred to 12 x 75-mm polypropylene tubes, and the incubation was continued for 1 h at 60 C or until clear. The tubes were placed in ice water, and 1 ml cold ethanol (-20 C) was added. Glycogen was separated by centrifugation at 5,000 x g for 5 min and washed with 1 ml cold ethanol containing 0.1% LiBr and 0.02 N KOH twice. The final precipitable radioactivity was solubilized in 0.2 N HCl and scintillation cocktail and counted in a liquid scintillation counter. In some experiments, the antibody {alpha}-IR3 (10 nM) and/or {alpha}-IR (50 nM) was preincubated with the cultures, for 30 min before the addition of insulin or IGF-I, and [3H]glucose.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the effect of glucosamine on IGF-I and insulin action in this cell type, glycogen synthesis was analyzed. IGF-I caused a major increase in glycogen synthesis at both concentrations that were tested (Fig. 1Go). Similarly, insulin caused substantial increases at the concentrations that were tested. The combination of insulin and IGF-I induced the greatest increase. Pretreatment with glucosamine substantially inhibited the response of these cells to either insulin or IGF-I. When the effect of glucosamine on the combination of insulin plus IGF-I was determined, it was found to be less inhibitory (46 ± 7% inhibition, compared with 60 ± 5% with 3.3 nM IGF-I and 97 ± 4% with 1 nM insulin). To determine the effect of glucosamine exposure on the ability of IGF-I to activate IGF-IR phosphophorylation, IGF-I was added with glucosamine (20 mM) for variable time periods. Exposure to glucosamine for 18 h resulted in a 75 ± 7% (n = 3) reduction in the ability of IGF-I to stimulate receptor kinase activation (Fig. 2AGo). To determine the optimum concentration of glucosamine, the experiment was repeated with increasing concentrations of glucosamine (1–50 mM) that were added to the cultures for 18 h. There was marked inhibition of IGF-IR phosphorylation when concentrations of either 20 (66 ± 7%) or 50 mM (52 ± 6%, n = 3) glucosamine were added (Fig. 2BGo). There was no significant change in the abundance of the IGF-IR with any concentration that was tested.



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  		Figure 1. Glucosamine inhibits IGF-I and insulin-stimulated glycogen synthesis in Hep G2 cells. Hep G2 cells were seeded in 24-well culture plates. After 48 h, the medium was replaced with DMEM-L (with or without 20 mM glucosamine), and the incubation continued for 18 h. The medium was replaced with 200 µl DMEM-L, containing 3H-glucose and either IGF-I or insulin, and the incubation was continued for 2 h; then the activity of [3H] glucose incorporation into glycogen was measured ({blacksquare}, no glucosamine; , glucosamine, 20 mM). Lane 1, No addition; lanes 2 and 3, IGF-I (3.3 nM); lanes 4 and 5, IGF-I (13 nM); lanes 6 and 7, insulin (1 nM); lanes 8 and 9, insulin (10 nM); lanes 10 and 11, insulin (1 nM) plus IGF-I (3.3 nM). The results represent the mean ± SD of three separate experiments.

 


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  		Figure 2. Glucosamine inhibits phosphorylation of IGF-IR and HRs after IGF-I stimulation. Hep G2 cells were grown to 80% confluency, and medium was replaced by serum-free DMEM-L in the presence of 20 mM glucosamine (lanes 3–8) for 1–18 h (A) or with increasing concentrations of glucosamine (1–50 mM) (lanes 3–8) for 18 h (B). The medium was changed to serum-free DMEM-L, and the incubation was continued for 2 h. The cells were stimulated with 13 nM IGF-I for 10 min (lanes 2–8, both panels) and solubilized in lysis buffer. The cell lysates were immunoprecipitated (IP) with the ßIGF-IR antibody and analyzed by SDS-PAGE with immunoblotting. The membranes were immunoblotted (IB) with phosphotyrosine antibody (PY99, upper panel) and then reprobed with ßIGF-IR antibody (lower panel). The scanning densitometry values for the bands shown in A were, from left to right: 0, 4822, 4714, 4447, 4471, 4064, 3930, and 652; and for B, they were: 5424, 5445, 5001, 5487, 5029, 5118, 4675, and 4802. For C, they were: 0, 4107, 4121, 4100, 3228, 2889, 1469 and 2084, and for the lower panel (D) they were: 8600, 8535, 7912, 7798, 7275, 7698, 7615, and 7841. The experiment was repeated three times, with similar results.

 
To determine the effect of glucosamine on insulin stimulation of IR phosphorylation, the experiment was repeated, and the cell lysates were immunoprecipitated with an antibody that is specific for the IR ß-subunit, followed by immunoblotting with the antiphosphotyrosine antibody. As shown in Fig. 3Go, insulin (1 nM) stimulated an increase in IR phosphorylation, and this increase was progressive up to 100 nM. An 18-h exposure to 20 mM glucosamine resulted in complete attenuation of IR phosphorylation in the cultures that had been exposed to 1 nM insulin and in significant inhibition in the cultures that had been exposed to 25 and 100 nM insulin (e.g. 25 nM, 88 ± 7%; and 100 nM, 57 ± 5%) (n = 3). In contrast to the IGF-IR data shown in Fig. 2Go, glucosamine exposure was associated with a major reduction in the abundance of the IR. Receptor abundance was reduced 58 ± 8% when no insulin was added and 52 ± 6%, 56 ± 5%, and 57 ± 7% (n = 3) when 1, 25, or 100 nM insulin was added, respectively.



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  		Figure 3. Glucosamine inhibits phosphorylation of IR and HR after stimulation with insulin. Hep G2 cells were grown to 80% confluency, and medium was replaced with serum-free DMEM-L in the presence (lanes 2, 4, 6, and 8) and absence (lanes 1, 3, 5, and 7) of 20 mM glucosamine, and the incubation continued for 18 h. The medium was replaced with serum-free DMEM-L, and the incubation was continued for 2 h. The cells were stimulated with insulin [1 nM (lanes 3 and 4), 10 nM (lanes 5 and 6), and 100 nM (lanes 7 and 8)] for 10 min and solubilized in lysis buffer. The cell lysate was immunoprecipitated with ß-IR antibody. The proteins were analyzed by SDS-PAGE and immunoblotting. The membranes were probed with phosphotyrosine antibody (PY99) and reprobed with ß-IR antibody. The scanning densitometry values for the bands shown in the upper panel were: 0, 0, 1143, 0, 3653, 301, 6702, and 2899. For the lower panel, they were: 6776, 3458, 6456, 3124, 5859, 3524, 6382, and 3599. The experiment was repeated three times, with similar results.

 
Hep G2 cells contain not only IRs and IGF-IRs, but they also contain IGF/insulin HRs. The experimental conditions that were used in the experiments shown in Figs. 2Go and 3Go would immunoprecipitate a combination of either homodimeric IGF-IRs and IGF/insulin heterodimers (Fig. 2Go) or homodimeric IRs and IGF/IR hybrids (Fig. 3Go). To determine the effect of glucosamine on HR abundance and activation, the experimental paradigm was repeated using a low concentration of IGF-I as a ligand that was not sufficient to stimulate IRs. To distinguish between HRs and IGF-IR homodimers, the initial immunoprecipitation was performed with {alpha}IR (an IR {alpha}-subunit-specific antibody), and immunoblotting was performed for both phosphotyrosine and for the IGF-IR ß-subunit. Because both antibodies are specific, this method should detect only HRs. To determine the effect of glucosamine on IGF-IR homodimer activation, the supernatant remaining after the first immunoprecipitation step was exposed to the ßIGF-IR antibody and was reimmunoprecipitated. This precipitate was then immunoblotted for phosphotyrosine or IGF-IR. As shown in Fig. 4Go, HRs that contained the IGF-IR ß-subunit were expressed in abundance, and their levels were comparable with those of IGF-IR homodimers. When stimulation of receptor tyrosine phosphorylation was analyzed, the HR was more sensitive to IGF-I, because 1.3 nM caused an increase in phosphorylation and the degree of stimulation increased progressively up to 13 nM. In contrast, when IGF-IR homodimer phosphorylation was analyzed, no effect was detectable at 1.3 nM. Although responses were noted with 3.3 and 13 nM IGF-I, there was less stimulation, compared with the HR. Glucosamine exposure completely blocked the effect of 1.3 nM IGF-I on HR phosphorylation, and it induced significant inhibition when 3.3 and 13 nM were added (decreases of 48 ± 7 and 45 ± 6%, respectively). When the IGF-IR homodimer was examined, glucosamine inhibited the responses to 3.3 or 13 nM IGF-I addition (e.g. 26 ± 2 and 30 ± 4%, respectively). Glucosamine induced variable changes in HR abundance (e.g. ranging between 5 and 26% reductions), and these changes were not significant. Similarly, it had no significant effect on IGF-IR homodimer abundance (e.g. changes ranged from a 2% decrease to a 5% increase).



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  		Figure 4. Glucosamine inhibits IGF-I stimulated phosphorylation of IGF-I/insulin HR. Hep G2 cells were grown to 80% confluency, and medium was replaced with serum-free DMEM-L with (+) or without (-) 20 mM glucosamine for 18 h. The medium was replaced with serum-free DMEM-L, and the incubation was continued for 2 h. The cells were stimulated with 1.5, 3.3, or 13 nM IGF-I for 10 min. The cells were lysed using lysis buffer, and the cell lysate was immunoprecipitated with {alpha}-IR antibody (1 ). The remaining supernatant was immunoprecipitated with ßIGF-IR antibody (2 ). The proteins in both sets of immunoprecipitates were analyzed by SDS-PAGE with immunoblotting. The membranes were probed with phosphotyrosine antibody (PY99) and then reprobed with ßIGF-IR antibody. The scanning densitometry values for the bands shown in A were: 0, 0, 0, 0, 3150, 0, 0, and 0; in B, they were: 5621, 4149, 5376, 4096, 7263, 5581, 5936, and 5507. For C, they were: 7501, 1334, 4369, 987, 9656, 4232, 6510, and 3157; and for D, they were: 4812, 1102, 3578, 2560, 5183, 2706, 4898, and 3072. The experiment was repeated three times, with similar results.

 
To determine whether glucosamine could also alter the IGF-I/insulin HR response to insulin, the experiment shown in Fig. 4Go was repeated, except that insulin was used to stimulate receptor phosphorylation, and the effect of glucosamine on insulin activation of IGF-I/insulin hybrid or insulin homodimers was analyzed. No stimulation of the HR was detected until 100 nM insulin was added, whereas a minimal (but significant) effect on IR homodimer phosphorylation was detected with 1.0 nM insulin (e.g. 365 ± 56 scanning units, compared with 20 ± 18; n = 3) (Fig. 5Go). Glucosamine-inhibited HR stimulation, by 100 nM insulin, by 64 ± 6%; and it inhibited IR homodimer activation at each of the three insulin concentrations that were tested (e.g. 65 ± 7, 45 ± 6, and 40 ± 5%, respectively). The changes in the abundance of the ßIR component of the HR were not consistent (ranging from 4–34%), whereas the IR homodimer was reduced by values ranging from 29 ± 4 to 39 ± 6%.



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  		Figure 5. Effect of glucosamine on insulin-stimulated IGF-I/insulin hybrid and IR phosphorylation. Hep G2 cells were grown to 80% confluency, and the medium was replaced with serum-free DMEM-L in the presence (+) or absence (-) of 20 mM glucosamine for 18 h. The medium was changed to serum-free DMEM-L, and the inhibition continued for 2 h. The cells were then exposed to medium alone (0), or insulin was added at the concentrations listed for 10 min. The cell lysates were immunoprecipitated with {alpha}IR3 (left panel), and the precipitates were analyzed by immunoblotting using either PY-99 or ßIR antiserum. The remaining supernatants were exposed to ßIR antiserum and immunoprecipitated (right panel). The pellets were analyzed by immunoblotting using PY-99 or ßIR antisera (right panel). The scanning densitometry values are: left upper panel, 29, 47, 0, 0, 0, 78, 5004, and 1635; left lower panel, 7638, 5079, 5048, 4854, 6937, 5906, 5710, and 5241; right upper panel, 19, 63, 339, 116, 4087, 2339, 10489, and6450; and right lower panel, 4705, 3113, 6332, 3058, 6076, 4805, 6198, and4549. The experiment was repeated three times, with similar results.

 
To determine whether IGF-I was altering hybrid or IR sensitivity to insulin plus IGF-I, the experiment was repeated using IGF-I or insulin alone and the combination of both peptides. As shown in Fig. 6Go, upper panel, IGF-I (3.3 nM) stimulated HR phosphorylation and glucosamine inhibited this response by 78 ± 6%. Insulin (3 nM) had a minimal effect. The combination of insulin plus IGF-I stimulated a much greater increase (e.g. 5.6-fold greater than IGF-I alone). Glucosamine caused much less inhibition (e.g. 21 ± 4%), compared with its effect on the response to IGF-I alone (P < 0.02). When their effects on ßIR phosphorylation were examined, IGF-I alone induced an increase, although the effect of insulin alone was greater and the combination stimulated an equivalent increase. Glucosamine exposure completely inhibited the IR response to both IGF-I and insulin alone and to the combination by 94 ± 6%.



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  		Figure 6. Hep G2 cells were grown to 80% confluency, then washed three times with serum-free DMEM-L and incubated overnight in medium containing 0 or 20 mM glucosamine. The medium was replaced with 5 ml containing 0 or 3.3 nM IGF-I for 2 h, then either 0 or 3 nM insulin was added directly for 5 min. The cells were lysed, and the insoluble material was pelleted as described previously. The soluble lysates were immunoprecipitated with {alpha}IR3 (1.0 µg), then immunoblotted for phosphotyrosine or ßIR. The remaining supernatants were reimmunoprecipitated after incubation with ßIR antibody (1.0 µg), then immunoblotted for phosphotyrosine or ßIR. The scanning densitometry values were: top left panel, 437, 15, 1513, 305, 356, 49, 8429, and 6542; top right, 0, 0, 723, 0, 4622, 253, 4735, and 227; bottom left, 6011, 4741, 3992, 4144, 3272, 3504, 3596, and 3424; and bottom right, 4250, 3023, 4131, 3299, 6761, 3804, 6306, and 3712. The experiment was repeated three times, with similar results.

 
To determine whether the effect of glucosamine on ligand-stimulated receptor phosphorylation was reflected by a change in downstream signaling elements, IRS-1 phosphorylation and PI-3 kinase association with IRS-1 were examined. Stimulation with IGF-I resulted in a major increase in IRS-1 phosphorylation and in the association of the p85 subunit of PI-3 kinase with IRS-1 (Fig. 7Go). Both responses were markedly inhibited by glucosamine exposure when 3.3 nM IGF-I was used as a stimulant, but glucosamine was less effective when 13 nM IGF-I was used. Glucosamine had no effect on IRS-1 abundance. This result is consistent with major inhibition of hybrid or IGF-IR activation by pretreatment with glucosamine and indicates that activation of these downstream signaling components is attenuated.



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  		Figure 7. Glucosamine inhibits IGF-I-stimulated phosphorylation of IRS-1 and p85 association with IRS-1. Hep G2 cells were grown to 80% confluency, and medium was replaced with serum-free DMEM-L in the presence or absence of 20 mM glucosamine for 18 h. The medium was changed to serum-free DMEM-L, and the incubation was continued for 2 h. The cells were stimulated with 0, 3.3, or 13 nM IGF-I for 10 min. The cell lysates were immunoprecipitated with anti-IRS-1 antibody. The proteins were analyzed by SDS-PAGE and immunoblotting using antiphosphotyrosine antibody. The membrane was reprobed with anti-p85 antibody and anti-IRS-1 antibody. The scanning densitometry values for the bands shown in A were: 148, 0, 4623, 593, 8082, and 6112; for B, they were 232, 0, 7500, 676, 7980, and 4180; and for C, they were: 7103, 6769, 6981, 8675, 8895, and 6881. The experiment was repeated three times, with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To determine whether glucosamine induced impairment in activation of the insulin, IGF-I, or hybrid IGF-I/IRs, this study used Hep G2 cells, a cell type that contains easily detectable amounts of the three receptor subtypes. This cell line is also useful because glucosamine exposure results in inhibition of insulin and IGF-I stimulated glycogen synthesis (Fig. 1Go). Our results show that this effect is mediated principally by a reduction in the sensitivity of both the IGF/insulin HR and IGF-IR homodimer phosphorylation responses to increasing concentrations of IGF-I and of the IGF-I/insulin hybrid and IR homodimer responses to increasing concentrations of insulin. In contrast to IGF-IR homodimers, glucosamine exposure was associated with a reduction in IR homodimer abundance. However, the inhibitory effect of glucosamine on IR phosphorylation was greater than the decrease in receptor number when 1 or 10 nM insulin was used, and this suggests that the effect of glucosamine was attributable to both inhibition of the sensitivity of the receptor to insulin stimulation and to receptor down-regulation. HR abundance was not consistently decreased in response to glucosamine, but there was a significant reduction in the ability of IGF-I or insulin to stimulate HR activation. In contrast to IR, the IGF-IR showed no change in receptor abundance in response to glucosamine; however, there was some impairment of the ability of IGF-I to stimulate IGF-IR phosphorylation. The changes in sensitivity to IGF-I stimulation of the IGF-I and HRs were also reflected by a reduction in the ability of IGF-I to stimulate IRS-1 phosphorylation and the association of the p85 subunit of PI-3 kinase with IRS-1. The ability of glucosamine to inhibit insulin or IGF-I stimulation of HR phosphorylation was greatest when lower concentrations of each ligand were used. Thus, there also seems to be a shift in the dose-response curve to the right. These findings are consistent with the findings of Virkamaki et al. (13), who showed that hexosamine infusion into rats reduced insulin responsiveness but, at the highest insulin concentrations, there was less impairment (on a percentage basis) than at lower concentrations. These findings are also consistent with published literature showing that in vivo infusion of glucosamine into animals results in impairment in IGF-I action (7), because blunting of the sensitivity of the hybrid and the IGF-IR to the activating effects of ligand occupancy by IGF-I would be predicted to result in impairment in IGF-I actions.

The effect of the combination of insulin plus IGF-I on HR phosphorylation and stimulation of glycogen synthesis suggests that IGF-I is enhancing the response to insulin. Insulin (3 nM) had a minimal effect on HR phosphorylation, and the response to the combination of insulin plus IGF-I was substantially greater than the response to IGF-I or insulin alone. Glucosamine exposure had a much greater inhibitory effect on the response of the HR to IGF-I alone, compared with its effect on the response to IGF-I plus insulin. In contrast to the HR, glucosamine completely inhibited the response of the IR to the combination of insulin and IGF-I. These findings suggest that IGF-I enhances the effect of insulin on HR phosphorylation and that the inhibitory effect of glucosamine on insulin stimulation can be partially reversed if IGF-I is also present. We conclude that concomitant exposure to IGF-I and insulin can partially overcome the inhibitory effect of glucosamine on insulin signaling in these cells, and this effect of IGF-I is likely to be mediated through the HR.

The exact mechanism by which HR activation partially reverses the inhibitory effect of glucosamine needs to be determined. IGF-I could sensitize HRs to insulin by increasing the affinity of the HR for insulin, leading to an enhancement of the ability of insulin to overcome the negative effects of glucosamine on insulin signaling. Alternatively IGF-I could act at a more distal point in the signaling pathway to alter the effects of glucosamine on insulin signaling. Glucosamine has been shown to impair the IRS-1 phosphorylation response to insulin and the ability of the p85 subunit of PI-3 kinase to bind to IRS-1 (18). Similarly, the stimulation of PI-3 kinase activity by insulin is impaired (19). Because IGF-I also acts to stimulate each of these signaling events and IGF-I can activate IRS-1 through the HR (16), it is possible that it is directly counteracting these inhibitory effects of glucosamine. It is also possible that HR activation stimulates an intracellular event that directly counteracts an antiinsulin action of glucosamine that is unrelated to IRS-1 or PI-3 kinase activation (20).

The extent to which these in vitro findings can be extrapolated to in vivo studies cannot be definitively determined, because hepatocytes contain either very minimal (or no) IGF-IRs. However, skeletal muscle (an important insulin and IGF-I target tissue) contains IGF-I/IR hybrids (21). Infusion of glucosamine into whole animals results in impairment in insulin sensitivity in skeletal muscle (11, 13, 22). Several physiological responses to insulin are altered, such as stimulation of glucose transport, glut 4 translocation (22), and glycogen synthesis (23). Furthermore, glucosamine infusion to rats has been shown to induce resistance to insulin stimulation of tyrosine phosphorylation of IRS-1 and to induction of PI-3 kinase phosphorylation in skeletal muscle (18, 24). These changes correlated with a reduced glycogen synthesis response (25).

Of pertinence to our findings, studies in rats (7) and humans (5) have demonstrated that the presence of insulin resistance (26) and/or diabetes (27) is associated with an increase in HR formation in skeletal muscle (5) and that this change can be induced by a glucosamine infusion (7). The importance of modulating both IGF-I and HR sensitivity is further emphasized by the findings of Fernandez et al. (28), who showed that specific deletion of IGF-I and HRs in skeletal muscle resulted in severe insulin resistance. Their findings suggest that activation of IGF-I and/or HRs, presumably by IGF-I, may be necessary to attain a normal response to insulin. Our results suggest that glucosamine is acting to impair this response.

In summary, our data show that glucosamine induced impairment of IGF-IR, as well as IR-mediated signaling in hepatocytes, and that part of this attenuation that is induced by glucosamine is mediated by changes in HR activation. Although IGF-I or insulin alone can partially overcome glucosamine inhibition, when the combination is added, it can overcome much of the inhibitory effect of glucosamine on HR activation. Determining the mechanism that links HR activation or its ability to counteract glucosamine-induced resistance to insulin action is an important goal of future studies.


   Acknowledgments
 
The authors wish to thank Ms. Laura Lindsey for her help in preparing the manuscript.


   Footnotes
 
This work was supported by NIH Grant AG-023331.

Abbreviations: DMEM-L, DMEM with low glucose (1000 mg/liter); HR, hybrid receptor; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS, IR substrate.

Received October 30, 2002.

Accepted for publication February 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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