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Endocrinology Vol. 139, No. 10 4147-4154
Copyright © 1998 by The Endocrine Society

ARTICLES

{alpha}2-Heremans Schmid Glycoprotein Inhibits Insulin-Stimulated Elk-1 Phosphorylation, But Not Glucose Transport, in Rat Adipose Cells1

Hui Chen, Pothur R. Srinivas, Li-Na Cong, Yunhua Li, George Grunberger and Michael J. Quon

Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute (H.C., L.-N.C., Y.L., M.J.Q.), National Institutes of Health, Bethesda, Maryland 20892; and the Department of Internal Medicine, Wayne State University School of Medicine, Center for Molecular Medicine and Genetics (P.R.S., G.G.), Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-103, 10 Center Drive MSC 1754, Bethesda, Maryland 20892-1754. E-mail: quonm{at}gwgate.nhlbi.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}2-Heremans Schmid glycoprotein ({alpha}2-HSG) is a member of the fetuin family of serum proteins whose biological functions are not completely understood. There is a consensus that {alpha}2-HSG plays a role in the regulation of tissue mineralization. However, one aspect of {alpha}2-HSG function that remains controversial is its ability to inhibit the insulin receptor tyrosine kinase and the biological actions of insulin. Interestingly, some studies suggest that {alpha}2-HSG differentially inhibits mitogenic, but not metabolic, actions of insulin. However, these previous studies were not carried out in bona fide insulin target cells. Therefore, in the present study we investigate the effects of {alpha}2-HSG in the physiologically relevant rat adipose cell. We studied insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in transfected rat adipose cells overexpressing human {alpha}2-HSG. In addition, we measured insulin-stimulated glucose transport in adipose cells cultured with conditioned medium from the transfected cells as well as in freshly isolated adipose cells treated with purified human {alpha}2-HSG. Compared with control cells, we were unable to demonstrate any significant effect of {alpha}2-HSG on insulin-stimulated translocation of GLUT4 or glucose transport. In contrast, we did demonstrate that overexpression of {alpha}2-HSG in adipose cells inhibits both basal and insulin-stimulated phosphorylation of Elk-1 (a transcription factor phosphorylated and activated by mitogen-activated protein kinase and other related upstream kinases). Interestingly, we did not observe any major effects of {alpha}2-HSG to inhibit insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate-1, -2, or -3, in either transfected or freshly isolated adipose cells. We conclude that {alpha}2-HSG inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in adipose cells by a mechanism that may involve effector molecules downstream of insulin receptor substrate proteins. .


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
{alpha}2-HEREMANS Schmid glycoprotein ({alpha}2-HSG) is a serum protein synthesized and secreted predominantly by the liver that belongs to the fetuin family and the cystatin superfamily of proteins (for review, see Ref. 1). Although the structure of {alpha}2-HSG is well characterized, and {alpha}2-HSG may influence a wide variety of biological processes, including insulin and growth factor signaling (2, 3, 4, 5), lipid transport (6, 7), opsonization (8, 9), and fetal development (10), there is a growing consensus that an important physiological role of {alpha}2-HSG is to regulate tissue mineralization (4, 11, 12, 13, 14, 15, 16). Among the earliest described biological effects of {alpha}2-HSG was inhibition of the insulin receptor tyrosine kinase (2, 3, 17, 18, 19). Activation of the insulin receptor tyrosine kinase, one of the most proximal events in insulin signaling, is thought to be required for most, if not all, of the biological actions of insulin (for review, see Ref. 20). Therefore, it is intriguing that some studies report an inhibitory effect of {alpha}2-HSG on mitogenic, but not metabolic, actions of insulin (2, 3, 17). However, it should be noted that the inhibitory effect of {alpha}2-HSG on insulin signaling is somewhat controversial and may depend on the phosphorylation state of {alpha}2-HSG or other posttranslational modifications (17, 18, 21, 22, 23, 24). In addition, these previous studies were performed in cell types such as CHO cells that are not bona fide targets for insulin’s metabolic actions. Indeed, transgenic mice homozygous for a null allele in the {alpha}2-HSG gene have no obvious physiological abnormalities related to insulin signaling (15).

In the present study, we have investigated the effects of {alpha}2-HSG on metabolic and mitogen-activated protein kinase (MAPK)-dependent actions of insulin in the physiologically relevant rat adipose cell. Overexpression of human {alpha}2-HSG in transiently transfected adipose cells or treatment of freshly isolated adipose cells with purified {alpha}2-HSG had no detectable effect on either insulin-stimulated translocation of GLUT4 or glucose transport. In contrast, overexpression of {alpha}2-HSG in transfected adipose cells significantly inhibited insulin-stimulated phosphorylation of Elk-1 (Elk-1 is a transcription factor that is phosphorylated and activated by MAPK). Interestingly, this inhibitory effect of {alpha}2-HSG was also observed with platelet-derived growth factor (PDGF)-stimulated phosphorylation of Elk-1. To investigate potential mechanisms for the inhibitory effects of {alpha}2-HSG, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1 (IRS-1), -2, and -3 in the presence or absence of {alpha}2-HSG. We did not observe any obvious effects of {alpha}2-HSG on phosphorylation of these signaling proteins in response to insulin. Taken together, our data suggest that {alpha}2-HSG does not inhibit metabolic effects of insulin such as increased glucose uptake, but does impair MAPK-dependent effects of insulin such as Elk-1 phosphorylation in the metabolically responsive adipose cell. Furthermore, it is possible that the inhibitory effects of {alpha}2-HSG on MAPK-dependent pathways occur downstream from the IRS family of proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression vectors
pCIS2. An expression vector that generates high levels of expression in transfected rat adipose cells was used as the parent vector for subsequent constructions (25).

GLUT4-HA. pCIS2 vector containing the DNA coding for human GLUT4 with the influenza hemagglutinin epitope (HA1) inserted in the first exofacial loop of GLUT4 (26).

pCIS-{alpha}2HSG. A 1360-bp BamHI/EcoRI fragment containing the complementary DNA for human {alpha}2-HSG (2) was blunt ended and ligated in the sense orientation into the HpaI site of pCIS2.

pFR-Luc. A reporter plasmid containing the luciferase gene under the control of a synthetic promoter containing the yeast GAL4 binding site was obtained from Stratagene (La Jolla, CA).

pFA-Elk. Fusion activator plasmid containing complementary DNA for a fusion protein consisting of the DNA-binding domain of yeast GAL4 and the activation domain of the Elk-1 transcription factor was obtained from Stratagene.

pCIS-Luc. Plasmid containing luciferase gene driven by cytomegalovirus promoter/enhancer was used (25).

Isolated rat adipose cell preparation
Isolated adipose cells were prepared from the epididymal fat pads of male rats (170–200 g; CD strain, Charles River Breeding Laboratories, Wilmington, MA) by collagenase digestion as previously described (25, 27). These procedures were approved by the animal care and use committee of our institution.

Electroporation and assay for cell surface epitope- tagged GLUT4
Isolated adipose cells were transfected by electroporation as described previously (25, 28, 29). Cells from multiple cuvettes were pooled to obtain the necessary volume of cells for each experiment. For experiments in which cell surface epitope-tagged GLUT4 was measured by a double antibody binding assay (26), we pooled cells from 20 cuvettes for groups cotransfected with GLUT4-HA (2 µg DNA/cuvette) and either pCIS2 or {alpha}2-HSG (4 µg DNA/cuvette). A group transfected with pCIS2 alone (10 cuvettes, 6 µg DNA/cuvette) was used to determine nonspecific antibody binding. Thus, all cells were exposed to a total DNA concentration of 6 µg/cuvette. Twenty hours after electroporation, adipose cells were treated with insulin (0–60 nM) for 25 min. The cells were then treated with KCN (2 mM) to prevent redistribution of GLUT4, and the cell surface GLUT4-HA was quantified using the mouse monoclonal antibody HA-11 (Berkeley Antibody Co., Richmond, CA) in conjunction with an 125I-labeled secondary antibody as described previously (26, 28, 29). Total cellular levels of expression of GLUT4-HA in each group of transfected cells were determined by immunoblotting as previously described (29).

Immunodetection of {alpha}2-HSG in adipose cells and conditioned media
Adipose cells were transfected with either the empty expression vector pCIS2 or pCIS-{alpha}2HSG (4 µg DNA/cuvette, 12 cuvettes/group). After transfection, cells were maintained in culture overnight in 6-cm tissue culture dishes at 37 C in 5% CO2. The following morning, conditioned medium was collected from each dish, and the cells were washed once and resuspended in 2.5 ml TES buffer (20 mM Tris, 1 mM EDTA, and 8.73% sucrose, pH 7.4, containing 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml soybean trypsin inhibitor) at 18 C. The cells were homogenized, and a cell lysate was prepared as previously described (29). The cell lysates (200 µg total protein) from each group were subjected to SDS-PAGE (10% gel), the contents of the gel were transferred to nitrocellulose, and immunoblotting was performed using a goat polyclonal antibody against {alpha}2-HSG (1:1000 dilution of 13.7 mg/ml; Incstar, Stillwater, MN). A second antibody against goat IgG conjugated with horseradish peroxidase (Incstar) was used in conjunction with an enhanced chemiluminescence detection system (ECL, Amersham, Arlington Heights, IL) to visualize the immunolabeled bands.

For detection of {alpha}2-HSG in the conditioned media, a cocktail of protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml soybean trypsin inhibitor) was added to 2.5 ml conditioned medium, and the samples were immunoprecipitated with the anti-{alpha}2-HSG antibody (10 µg) for 2 h at 4 C. The immunocomplexes were then incubated with prewashed protein A-agarose for 1 h at 4 C and washed three times with lysis buffer. The samples were pelleted by centrifugation, eluted by boiling in Laemmli sample buffer for 5 min, and then subjected to SDS-PAGE and immunoblotting with the antibody against {alpha}2-HSG as described above.

Similar experiments were performed to determine intracellular and medium levels of {alpha}2-HSG in freshly isolated cells treated with exogenous {alpha}2-HSG. Cells were incubated with 3 µM {alpha}2-HSG for 30 min. Medium was collected, and the cells were washed several times and lysed as described above. Media and cell lysates were then immunoprecipitated and immunoblotted with anti-{alpha}2-HSG antibody as described above.

D-[U-14C]Glucose uptake
Freshly isolated adipose cells were prepared as previously described (25, 27) and diluted to a cytocrit of 5% for the determination of insulin-stimulated glucose uptake in the presence or absence of {alpha}2-HSG. Two hundred-microliter aliquots of the 5% cell suspension were added to 7-ml polyethylene vials containing 200 µl KRBH buffer [Krebs-Ringer medium containing 10 mM NaHCO3, 30 mM HEPES, 200 nM adenosine, and 1% (wt/vol) BSA, pH 7.4] with or without insulin (60 nM). Half of the samples also contained purified human {alpha}2-HSG protein (3 µM; Calbiochem-Novabiochem Co., La Jolla, CA). After incubation for 30 min at 37 C, 100 µl KRBH buffer containing D-[U-14C]glucose [12.5 µl D-[U-14C]glucose (DuPont-New England Nuclear, Boston, MA) with a specific activity of 294 mCi/mmol diluted in 5 ml KRBH] were added to each vial. After an additional 30 min at 37 C, 300-µl aliquots were placed in polypropylene microcentrifuge tubes (4 x 45 mm) containing 100 µl dinonylphthalate oil (ICN Biomedicals, Costa Mesa, CA). The cells were rapidly separated from the aqueous buffer by centrifugation at 10,000 x g for 30 sec. Cell-associated radioactivity (a measure of glucose uptake by the cell) was counted in a liquid scintillation counter. The lipid weight from a 200-µl aliquot of cells from the original 5% suspension was determined as previously described (30) and used to normalize the data for each sample. The glucose uptake for each group of cells was determined in triplicate for each experiment. Each experiment was repeated independently at least four times.

Elk-1 phosphorylation assay in transfected adipose cells
We used the PathDetect kit from Stratagene to investigate the effects of overexpression of {alpha}2-HSG on insulin-stimulated Elk-1 phosphorylation in adipose cells. In this assay, insulin-stimulated phosphorylation of the transfected Gal4-binding domain/Elk-1 activation domain fusion protein (presumably mediated by MAPK) will result in binding and activation of the cotransfected Gal4-binding sequence/luciferase plasmid and drive expression of the luciferase reporter. Thus, the effect of overexpression of {alpha}2-HSG on insulin-stimulated MAPK activity can be inferred. Adipose cells were cotransfected with pFA-Elk (0.5 µg DNA/cuvette), pFR-Luc (1 µg DNA/cuvette), and either pCIS-{alpha}2HSG or the empty vector pCIS2 (4 µg DNA/cuvette). After electroporation, the contents of the cuvettes were transferred to 1.5-ml polypropylene tubes and incubated overnight at 37 C in 5% CO2. The following morning, cells were treated without or with 100 nM insulin for 5 h at 37 C. The cells were then lysed, and the luciferase activity in each sample was determined as previously described (25). Each experiment was performed in triplicate, and the lipid weight from an aliquot of each sample was determined to normalize the data for cell number. We also performed similar experiments using PDGF-BB (100 ng/ml) as the agonist. Control experiments in which pFA-Elk was omitted or pCIS-Luc was substituted for pFR-Luc were also performed.

Insulin-stimulated phosphorylation of cellular substrates in freshly isolated and transfected adipose cells
Freshly isolated adipose cells (1.5 ml at 40% cytocrit) were preincubated without or with purified {alpha}2-HSG protein (see figure legends for concentrations) for 30 min at 37 C, followed by treatment without or with insulin (100 nM) for 2 min at 37 C. Total membrane fractions were prepared from each group as previously described (29) and subjected to immunoprecipitation using monoclonal antibodies against the insulin receptor (C-19, Santa Cruz Biotechnology, Santa Cruz, CA) or IRS-1 or -2 (both from Upstate Biotechnology, Lake Placid, NY), as described above. Immunoprecipitates were subjected to SDS-PAGE, and immunoblotting was performed using an antiphosphotyrosine antibody (4G10, Upstate Biotechnology). In some cases, the membrane fractions were subjected to immunoblotting with 4G10 without immunoprecipitation.

We performed similar immunoprecipitation and immunoblotting experiments in total membrane fractions prepared from cells transfected with either pCIS-{alpha}2HSG or the empty expression vector pCIS2 (4 µg DNA/cuvette, 12 cuvettes for each group). After electroporation and overnight incubation, the cells were treated without or with insulin (100 nM) for 2 min, and the total membrane fractions were isolated and processed as described above.

Statistical analysis
The insulin dose-response curves were fit to the equation y = a + b [x/(x + k)] using a Marquardt-Levenberg nonlinear least squares algorithm (a = basal response, a + b = maximal response, k = half-maximal dose, and x = concentration of insulin). Insulin dose-response curves were compared using multivariate ANOVA. Paired t tests were used to compare individual points where appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of {alpha}2-HSG in transfected adipose cells
To characterize possible interactions between {alpha}2-HSG and insulin signal transduction pathways in a metabolic target tissue of insulin, we overexpressed human {alpha}2-HSG in adipose cells using our previously established transient transfection system for rat adipose cells in primary culture. Immunoblot analysis of cell lysates prepared from adipose cells transfected with pCIS-{alpha}2HSG demonstrated high levels of overexpression of the recombinant {alpha}2-HSG (Fig. 1Go). We were unable to detect expression of endogenous {alpha}2-HSG in control cells transfected with the empty expression vector pCIS2. As {alpha}2-HSG is known to be a secreted protein, we also used immunoprecipitation and immunoblotting with an antibody against {alpha}2-HSG to demonstrate that recombinant {alpha}2-HSG was present in conditioned medium collected from primary cultures of adipose cells transfected with pCIS-{alpha}2HSG (Fig. 1Go). Thus, although only 5% of cells are actually transfected using our transient transfection protocol (26), all of the cells in culture (both transfected and nontransfected) from the group electroporated with pCIS-{alpha}2HSG were exposed to {alpha}2-HSG in the medium. As expected, we were unable to detect {alpha}2-HSG in the conditioned medium derived from control cells transfected with pCIS2. These results were consistently reproducible, with similar levels of {alpha}2-HSG detected by immunoblotting in multiple independent experiments.



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  		Figure 1. Overexpression of recombinant {alpha}2-HSG in transfected rat adipose cells and conditioned media. Adipose cells were transfected with the empty expression vector pCIS2 or pCIS-{alpha}2HSG. After overnight incubation, cell lysates were prepared, and the conditioned media were collected as described in Materials and Methods. From each group, cell lysates containing 200 µg total protein were subjected to SDS-PAGE and immunoblotted with an antibody against {alpha}2-HSG (lanes 1 and 2). In addition, 2.5 ml conditioned media from each group were immunoprecipitated using the anti-{alpha}2HSG antibody and then immunoblotted with the same antibody (lanes 4 and 5). Ten micrograms of purified {alpha}2-HSG protein were run on the gel as a positive control (lane 3).

 
As some of our experiments depend on exogenous treatment of adipose cells with {alpha}2-HSG rather than overexpression of transfected {alpha}2-HSG, we compared intracellular and medium levels of {alpha}2-HSG from cells transfected with {alpha}2-HSG with those from cells exogenously treated with 3 µM {alpha}2-HSG for 30 min (Fig. 2Go). The levels of {alpha}2-HSG in the media of exogenously treated cells was greater than that seen in the conditioned media of transfected cells. In addition, intracellular levels of {alpha}2-HSG in the treated cells were comparable to those observed in the group of cells that was transfected with {alpha}2-HSG. These experiments demonstrate that {alpha}2-HSG in the media can be internalized effectively by adipose cells.



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  		Figure 2. Intracellular and medium levels of {alpha}2-HSG from cells treated with {alpha}2-HSG or transfected with {alpha}2-HSG. Adipose cells were treated for 30 min with 3 µM {alpha}2-HSG (lanes 2 and 4) or transfected with pCIS-{alpha}2HSG (Tx; lanes 3 and 5). Medium (1.5 ml) and 300 µg total protein from whole cell lysates from each group were immunoprecipitated using an anti-{alpha}2HSG antibody and then immunoblotted with the same antibody. Purified {alpha}2-HSG protein was run on the gel as a positive control (lane 1).

 
Effect of {alpha}2-HSG on insulin-stimulated translocation of GLUT4 and glucose transport
After demonstrating the presence of recombinant {alpha}2-HSG in both transfected adipose cells and conditioned media, we next examined the effects of overexpression of {alpha}2-HSG on metabolic actions of insulin such as the translocation of GLUT4 to the cell surface and insulin-stimulated glucose transport. With respect to translocation of GLUT4, the insulin dose-response curve for control cells cotransfected with pCIS2 and GLUT4-HA showed a 2.5-fold increase in cell surface epitope-tagged GLUT4 upon maximal insulin stimulation (ED50 = 0.1 nM; Fig. 3Go). This result is similar to what we have previously observed (28, 29). Adipose cells cotransfected with pCIS-{alpha}2HSG and GLUT4-HA had an insulin-dose-response curve that was not significantly different from that of the control cells (P > 0.6). Cotransfection of an epitope-tagged GLUT4 allowed us to follow GLUT4 translocation exclusively in the small fraction of cells that were actually transfected. As a control experiment, to rule out the remote possibility that {alpha}2-HSG was affecting the level of expression of GLUT4-HA, we used immunoblotting with an anti-HA antibody to show that the total cellular levels of GLUT4-HA in both the control group and the group overexpressing {alpha}2-HSG were similar (data not shown). Therefore, overexpression of {alpha}2-HSG does not appear to significantly affect insulin-stimulated translocation of GLUT4 in adipose cells.



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  		Figure 3. Recruitment of GLUT4-HA to the cell surface in response to insulin stimulation in transfected rat adipose cells. Cells cotransfected with pCIS-{alpha}2HSG and GLUT4-HA (•) were compared with control cells cotransfected with the empty expression vector pCIS2 and GLUT4-HA ({circ}); (see Materials and Methods for the amounts of plasmid DNA used). Data are expressed as a percentage of the cell surface GLUT4-HA in the presence of a maximally effective insulin concentration for the control group. Results are the mean ± SEM of five independent experiments. The insulin dose-response curve for cells overexpressing {alpha}2-HSG is not statistically different from that of the control cells by multivariate ANOVA (P > 0.64).

 
Although translocation of GLUT4 was not affected by {alpha}2-HSG, it is possible that {alpha}2-HSG might alter glucose transport by influencing the intrinsic activity or accessibility of GLUT4 at the cell surface. Therefore, we also assessed insulin-stimulated glucose transport in freshly isolated adipose cells that were treated with or without exogenous purified {alpha}2-HSG. In control cells incubated without {alpha}2-HSG, we observed a 13-fold increase in glucose transport upon insulin stimulation (as assessed by D-[U-14C]glucose incorporation; Fig. 4Go). Similar to our experiments assessing translocation of GLUT4, we did not observe any significant differences between cells treated with {alpha}2-HSG (3 µM) and untreated control cells with respect to insulin-stimulated glucose transport. In addition, we examined glucose transport in cells transfected with either pCIS2 or pCIS-{alpha}2HSG. After transfection and overnight incubation, no significant differences in insulin-stimulated glucose transport were detected between the two groups of cells (data not shown).



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  		Figure 4. Effect of {alpha}2-HSG treatment on insulin-stimulated glucose uptake in freshly isolated rat adipose cells. A group of cells treated with purified {alpha}2-HSG protein (3 µM) was compared with an untreated control group. Each of the original two groups of cells was split further into two groups for incubation with or without insulin (60 nM, 30 min). The cells were then incubated with D-[U-14C]glucose for an additional 30 min, and the specific cell-associated radioactivity was determined. There was no significant difference between groups of cells treated with or without {alpha}2-HSG under basal conditions (P > 0.2) or after insulin stimulation (P > 0.8). Results are the mean ± SEM of five independent experiments.

 
Effect of {alpha}2-HSG on insulin-stimulated phosphorylation of Elk-1
In addition to examining insulin-stimulated glucose transport, we examined effects of {alpha}2-HSG on phosphorylation of Elk-1 in adipose cells. For this purpose, we used a transient transfection assay in which we cotransfected cells with pFA-Elk (the binding domain of Gal4 fused with the activation domain of Elk-1), pFR-Luc (Gal4-binding sequence upstream of luciferase reporter), and either pCIS2 or pCIS-{alpha}2HSG. Elk-1 is a transcription factor that can be phosphorylated and activated by MAPK and other related upstream signaling pathways. Therefore, insulin-stimulated MAPK activity will result in phosphorylation of the transfected Elk-1 fusion protein. The Gal4-binding domain of the phosphorylated Elk-1 fusion protein will then interact with the Gal4-binding sequence in the cotransfected pFR-Luc plasmid and drive transcription of the luciferase reporter. In control cells transfected with pCIS2/pFA-Elk/pFR-Luc, we observed a 3-fold increase in luciferase activity in response to insulin stimulation, consistent with the known effect of insulin to activate MAPK (Fig. 5AGo). Interestingly, in cells transfected with pCIS-{alpha}2HSG/pFA-Elk/pFR-Luc, overexpression of {alpha}2-HSG resulted in a significant, approximately 50% decrease in the basal level of luciferase activity (in the absence of insulin) compared with that in control cells (P < 0.003). In addition, in sharp contrast to the control cells, there was no significant increase in luciferase activity in response to insulin stimulation of cells overexpressing {alpha}2-HSG (P > 0.8). Interestingly, we found similar results when we used PDGF-BB as the agonist. In control cells transfected with pCIS2/pFA-Elk/pFR-Luc, we observed a 2-fold increase in luciferase activity in response to PDGF stimulation (Fig. 5BGo). Overexpression of {alpha}2-HSG resulted in an approximately 50% decrease in the basal level of luciferase activity (P < 0.03) and complete inhibition of the PDGF response (P > 0.8).



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  		Figure 5. Effect of overexpression of {alpha}2-HSG on insulin- or PDGF-stimulated Elk-1 phosphorylation in transfected adipose cells. Cells were cotransfected with pFR-Luc, pFA-Elk, and either pCIS2 (control) or pCIS-{alpha}2HSG (see Materials and Methods). A, Overexpression of {alpha}2-HSG blocked the effect of insulin to stimulate Elk-1 phosphorylation (P > 0.8). In the absence of insulin, overexpression of {alpha}2-HSG significantly inhibited Elk-1 phosphorylation compared with that in the untreated control cells (P < 0.003). Results shown are the mean ± SEM of three independent experiments performed in triplicate. B, Experiments similar to those shown in A, except that PDGF-BB (100 ng/ml) was used as the agonist. Results shown are the mean ± SEM of four independent experiments performed in triplicate.

 
To demonstrate that we could achieve a similar inhibitory effect from treatment with exogenous {alpha}2-HSG, we transfected cells with pFA-Elk/pFR-Luc and treated the cells for 30 min with or without 3 µM {alpha}2-HSG followed by incubation in the presence or absence of insulin for 4 h. Similar to the results described above, we observed a 4-fold increase in Elk-1 phosphorylation in response to insulin in the control cells (no {alpha}2-HSG treatment) that was nearly completely blocked by exogenous {alpha}2-HSG treatment (data not shown). Finally, to help rule out the possibility that insulin or {alpha}2-HSG was having a nonspecific effect on promoter activity, we transfected cells with either pFR-Luc or pCIS-Luc along with pCIS2 or pCIS-{alpha}2-HSG (but without pFA-Elk) and then treated cells without or with insulin. Cells transfected with pFR-luc had low levels of basal activity that were unaffected by transfection with {alpha}2-HSG or treatment with insulin. Similarly, cells transfected with pCIS-Luc had high levels of basal activity that were unaffected by transfection with {alpha}2-HSG or treatment with insulin (data not shown).

Effects of {alpha}2-HSG on tyrosine phosphorylation of the insulin receptor and its cellular substrates
To investigate potential mechanisms for the inhibitory effect of {alpha}2-HSG on insulin-stimulated MAPK pathways, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor and proximal substrates such as IRS-1, and -2 in adipose cells treated with or without {alpha}2-HSG. Membrane fractions prepared from freshly isolated adipose cells treated with or without {alpha}2-HSG were subjected to immunoprecipitation with antibodies against the insulin receptor, IRS-1, or IRS-2 followed by immunoblotting with an antiphosphotyrosine antibody. As expected, in control cells (not exposed to {alpha}2-HSG), insulin stimulation caused a marked increase in tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 (Fig. 6AGo). Similarly, in cells treated with {alpha}2-HSG, insulin also stimulated phosphorylation of the insulin receptor, IRS-1, and IRS-2. When results from cells treated with {alpha}2-HSG were compared with results from control cells, there were no obvious differences in the levels of phosphorylation of the insulin receptor, IRS-1, or IRS-2 in either the basal or insulin-stimulated state. We performed similar experiments comparing membrane fractions isolated from cells transfected with either pCIS2 (control) or pCIS-{alpha}2HSG. As with the freshly isolated cells, insulin-stimulated phosphorylation of the insulin receptor, IRS-1, and IRS-2 was not significantly affected by overexpression of {alpha}2-HSG (compared with that in control cells; Fig. 6BGo).



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  		Figure 6. Antiphosphotyrosine immunoblots of immunoprecipitates of total membrane fractions derived from adipose cells. Samples from each group were immunoprecipitated (IP) with antibodies against the insulin receptor, IRS-1, or IRS-2. Representative blots are shown from experiments that were repeated independently at least twice. A, Freshly isolated adipose cells were treated without or with purified {alpha}2-HSG (10 µM, 30 min; 3 µM in the case of IP with IRS-1) followed by treatment without or with insulin (100 nM, 2 min). No significant differences in the pattern of phosphorylation of the insulin receptor, IRS-1, or IRS-2 were detected in samples derived from control cells compared with those in cells treated with {alpha}2-HSG. B, Cells transfected with either the empty expression vector (pCIS2) or pCIS-{alpha}2HSG were incubated overnight and then treated without or with insulin (100 nM, 2 min). In adipose cells pretreated or transfected with {alpha}2-HSG, no significant differences in the pattern of phosphorylation of the insulin receptor, IRS-1, or IRS-2 were detected in samples derived from control cells compared with that in cells transfected with {alpha}2-HSG.

 
To examine possible effects of {alpha}2-HSG on phosphorylation of other insulin receptor substrates (where we do not have an antibody for immunoprecipitation), we repeated the above antiphosphotyrosine immunoblotting experiments without prior immunoprecipitation. In freshly isolated cells treated with or without {alpha}2-HSG, insulin stimulation caused the phosphorylation of bands that correspond to the expected sizes of the insulin receptor, IRS-1/-2, and IRS-3 (Fig. 7AGo). As before, we could not detect any obvious differences in insulin-stimulated phosphorylation of the insulin receptor or receptor substrates as a result of treatment with {alpha}2-HSG. Similar results were obtained in transfected cells (Fig. 7BGo). Thus, in both freshly isolated adipose cells and transfected cells, {alpha}2-HSG does not appear to cause a major inhibition of insulin-stimulated phosphorylation of the insulin receptor, proximal substrates such as IRS-1 or -2, or proteins that are in the 60-kDa range under conditions where insulin-stimulated Elk-1 phosphorylation is inhibited.



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  		Figure 7. Antiphosphotyrosine immunoblots of total membrane fractions derived from adipose cells treated without or with insulin. Representative blots are shown from experiments that were repeated independently at least twice. A, Freshly isolated adipose cells were treated without or with purified {alpha}2-HSG (3 µM, 30 min) and then treated without or with insulin (100 nM, 2 min). Insulin-stimulated phosphorylation of proteins was detected in regions of the gel corresponding to the expected sizes of the insulin receptor, IRS-1, IRS-2, and IRS-3. No significant differences were observed between the pattern of phosphorylation in samples derived from control cells and the cells treated with {alpha}2-HSG. B, Cells transfected with either the empty expression vector (pCIS2) or pCIS-{alpha}2HSG were incubated overnight and then treated without or with insulin (100 nM, 2 min). No significant differences were observed between the pattern of phosphorylation in samples derived from control cells and that in cells transfected with {alpha}2-HSG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{alpha}2-HSG is an abundant plasma protein (circulating concentration, ~8 µM) whose structural properties have been well characterized (1). However, a definitive understanding of the physiological role of {alpha}2-HSG is still lacking. Although the ability of {alpha}2-HSG to inhibit the insulin receptor tyrosine kinase was among the earliest described biological effects (17), this function remains controversial, and its physiological significance is uncertain (2, 3, 17, 18, 19, 21, 22, 23, 24). Indeed, the recently described {alpha}2-HSG knockout mouse does not have any obvious abnormalities related to insulin signaling (15). Because previous studies examining this issue were performed in cell types that are not classical insulin targets, we chose to study the effects of {alpha}2-HSG on insulin action in the physiologically relevant rat adipose cell.

Effect of {alpha}2-HSG on insulin-stimulated translocation of GLUT4 and glucose transport
Because our transient transfection system for adipose cells in primary culture has only a 5% transfection efficiency, we cotransfect an epitope-tagged GLUT4 to allow us to study GLUT4 translocation exclusively in the subpopulation of cells that are transfected. Previous studies suggest that the cotransfection efficiency of our system is quite high, and it is likely that more than 95% of cells expressing GLUT4-HA also express the cotransfected transgene (28, 29, 31). Despite high levels of {alpha}2-HSG in both the transfected cells and media, we were unable to observe any significant effects of {alpha}2-HSG on insulin-stimulated translocation of cotransfected GLUT4-HA. In addition, insulin-stimulated glucose transport was unaffected by overexpression of {alpha}2-HSG. Similarly, treatment of freshly isolated adipose cells with purified {alpha}2-HSG was without effect on insulin-stimulated glucose transport. Taken together, our data suggest that {alpha}2-HSG does not have a significant inhibitory effect on metabolic actions of insulin such as glucose transport. Previous studies using tissue culture cell lines have demonstrated that other metabolic actions of insulin, such as aminoisobutyric acid transport (3, 17) and induction of tyrosine aminotransferase (2, 17), are unaffected by {alpha}2-HSG. However, the effects of insulin per se on these other metabolic functions are minimal in the cell types studied. In the present study, we used primary cultures of rat adipose cells that are extremely responsive to insulin with respect to glucose transport and were still unable to detect any effects of {alpha}2-HSG on this major metabolic function of insulin.

Effect of {alpha}2-HSG on insulin-stimulated phosphorylation of Elk-1
Under normal conditions, the phosphorylation of Elk-1 is dependent on MAPK and other related upstream kinases that participate in the mitogenic actions of insulin. Overexpression of {alpha}2-HSG resulted in significant inhibition of Elk-1 phosphorylation even in the absence of insulin. Furthermore, the effect of insulin to stimulate Elk-1 phosphorylation was completely blocked by overexpression of {alpha}2-HSG. Importantly, acute exogenous treatment of adipose cells with {alpha}2-HSG had a similar effect to block insulin-stimulated Elk-1 phosphorylation. This helps to rule out the possibility that {alpha}2-HSG is having a nonspecific effect to inhibit expression of the pFR-Luc or pFA-Elk constructs. Our results are consistent with previous reports showing that {alpha}2-HSG inhibits mitogenic actions of insulin such as thymidine incorporation (2, 17, 19, 32), and Raf/MEK activation in tissue culture cells (3). Interestingly, we observed a similar inhibitory effect of {alpha}2-HSG on PDGF-stimulated Elk-1 phosphorylation, suggesting that this effect of {alpha}2-HSG may not be specific to insulin signaling pathways, but may involve signaling molecules that are common to many growth factor/cytokine signaling pathways. Indeed, {alpha}2-HSG has also been reported to inhibit the mitogenic functions of HGF in hepatocytes (5).

Effects of {alpha}2-HSG on tyrosine phosphorylation of the insulin receptor and its cellular substrates
We examined the effects of both overexpression of recombinant {alpha}2-HSG and treatment with purified {alpha}2-HSG on insulin-stimulated tyrosine phosphorylation of the insulin receptor and proximal substrates such as IRS-1, -2, and -3 because previous reports have demonstrated that {alpha}2-HSG causes significant inhibition of insulin receptor autophosphorylation (2, 17, 32) and IRS-1 phosphorylation (2). However, it seems unlikely that abnormalities in very proximal signaling events such as receptor autophosphorylation or IRS-1 phosphorylation would cause inhibition of distal effects such as Elk-1 phosphorylation without also inhibiting the effects of insulin on other downstream actions such as translocation of GLUT4 or glucose transport. Indeed, we have previously shown that the insulin receptor tyrosine kinase, IRS-1, and IRS-2 all play important roles in mediating the effects of insulin to stimulate translocation of GLUT4 in rat adipose cells (26, 33, 34). Furthermore, the fact that {alpha}2-HSG inhibits mitogenic signaling by other growth factors such as PDGF (this study) or hepatocyte growth factor (5) also suggests that {alpha}2-HSG affects convergent downstream signaling pathways that are not unique to insulin signaling.

In contrast to some previous reports (2, 17, 32), we were unable to demonstrate significant effects of either overexpression of {alpha}2-HSG or treatment with purified {alpha}2-HSG on insulin-stimulated phosphorylation of the insulin receptor, IRS-1, IRS-2, or a 60-kDa protein in rat adipose cells. However, our results are in agreement with those of another study that reports that human {alpha}2-HSG does not inhibit the tyrosine kinase activity of purified human insulin receptors in vitro (18). The concentration of {alpha}2-HSG we used for exogenous treatment (3 µM in most experiments, 10 µM in some experiments) is in the range that has previously been reported to maximally inhibit insulin receptor phosphorylation in other cell types (2–10 µM) (2, 19). By immunoblotting we have demonstrated that 3 µM {alpha}2-HSG is greater than the medium concentration of {alpha}2-HSG achieved by secretion from transfected cells. Furthermore, exogenous treatment with 3 µM {alpha}2-HSG is sufficient to inhibit insulin-stimulated Elk-1 phosphorylation without affecting insulin-stimulated glucose transport. It is possible that species differences in {alpha}2-HSG or differences in cell types may account for some of the discrepancies that exist between our data and those from previous reports regarding the effects of {alpha}2-HSG on insulin receptor autophosphorylation. For example, it is possible that in adipose cells there is preferential sequestration of {alpha}2-HSG in compartments that prevent interactions with the insulin receptor but still allow effects on MAP kinase-dependent pathways. In addition, it has been suggested that the phosphorylation state of {alpha}2-HSG is important for its inhibitory effects (5, 17, 24). However, it is unlikely that the phosphorylation state of {alpha}2-HSG could explain our inability to detect a significant decrease in phosphorylation of the insulin receptor, IRS-1, IRS-2, or the 60-kDa protein, because we observed an inhibitory effect of {alpha}2-HSG in our system on both insulin- and PDGF-stimulated Elk-1 phosphorylation. Nevertheless, we cannot rule out the possibility that a small change in phosphorylation of the insulin receptor, IRS-1, IRS-2, or the 60-kDa protein (that we could not appreciate by immunoblotting) could have functionally significant effects. For example, there are at least 21 phosphotyrosine motifs on IRS-1 that are predicted to interact with downstream SH2-domain containing signaling molecules. If phosphorylation of only one of these motifs was specifically inhibited by {alpha}2-HSG, we might not be able to detect this by immunoblotting even though it could conceivably have a functionally significant consequence. In addition, it is possible that {alpha}2-HSG is inhibiting other insulin substrates that we did not examine, such as Shc, which are known to activate MAPK pathways in response to insulin.

Insulin signaling pathways related to glucose transport and MAPK activity in adipose cells appear to be divergent after the level of the insulin receptor and substrates such as IRS-1, -2, and -3. Phosphatidylinositol 3-kinase-dependent pathways are crucial for insulin-stimulated glucose transport (28), while Ras/MAPK pathways are important for mediating mitogenic effects (20). As we were unable to demonstrate any effect of {alpha}2-HSG on metabolic insulin signaling pathways related to glucose transport, but did observe inhibitory effects on both insulin- and PDGF-stimulated Elk-1 phosphorylation, the simplest interpretation of our data would suggest that {alpha}2-HSG is inhibiting MAPK-dependent pathways downstream from IRS-1, -2, or -3. In summary, we have studied the effects of {alpha}2-HSG on insulin action in the physiologically relevant rat adipose cell and demonstrate inhibitory effects on MAPK-dependent pathways but not on important metabolic functions such as insulin-stimulated glucose transport.


   Acknowledgments
 
We thank Simeon I. Taylor for thoughtful reading of this manuscript and helpful suggestions.


   Footnotes
 
1 This work was supported in part by a Research Award grant from the American Diabetes Association (to M.J.Q.). Back

Received March 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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