Suppressor of Cytokine Signaling-3 (SOCS-3), a Potential Mediator of Interleukin-6-dependent Insulin Resistance in Hepatocytes*

Joseph J. SennDagger, Peter J. Klover§, Irena A. Nowak, Teresa A. Zimmers||, Leonidas G. Koniaris||, Richard W. Furlanetto**, and Robert A. MooneyDaggerDagger

From the Departments of  Pathology and Laboratory Medicine, || Surgery, and ** Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, October 18, 2002, and in revised form, January 28, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin-6 (IL-6) is one of several pro-inflammatory cytokines implicated in insulin resistance during infection, cachexia, and obesity. We recently demonstrated that IL-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (2002) Diabetes 51, 3391-3399). Members of the suppressors of cytokine signaling (SOCS) family associate with the insulin receptor (IR), and their ectopic expression inhibits IR signaling. Since several SOCS proteins are induced by IL-6, a working hypothesis is that IL-6-dependent insulin resistance is mediated, at least in part, by induction of SOCS protein(s) in insulin target cells. To examine the involvement of SOCS protein(s) in IL-6-dependent inhibition of insulin receptor signaling, HepG2 cells were treated with IL-6 (20 ng/ml) for periods from 1 min to 8 h. IL-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal levels by 2 h. SOCS-3 induction by IL-6 paralleled IL-6-dependent inhibition of IR signal transduction. Ectopically expressed SOCS-3 associated with the IR and suppressed insulin-dependent receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, and activation of Akt. SOCS-3 was also a direct inhibitor of insulin receptor autophosphorylation in vitro. In mice exposed to IL-6 for 60-90 min, hepatic SOCS-3 expression was increased. This was associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of IL-6-mediated insulin resistance.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin resistance is a critical component in the pathogenesis of type 2 diabetes. Several cellular lesions have been associated with insulin resistance, including decreased insulin receptor tyrosine kinase activity and decreased phosphorylation of target proteins such as insulin receptor substrate-1 (IRS-1)1 (1, 2). Elevated levels of cytokines such as IL-6, IL-1, and TNFalpha and hormones such as growth hormone and insulin have been linked to insulin resistance (3-6). The mechanisms for these effects, however, have not been clearly defined. One proposed mechanism, which is currently being actively investigated, is the serine phosphorylation of IRS-1 and its subsequent direct inhibitory effect on insulin receptor kinase activity (7-9). An alternate hypothesis is that cytokines and hormones induce the expression of cellular proteins that inhibit IR signal transduction. Our laboratory and others have proposed that the suppressors of cytokine signaling (SOCS) family of regulatory proteins may indeed fulfill this role (10-12).

SOCS proteins are a recently discovered family of proteins, identified simultaneously by several groups that were screening for negative regulators of cytokine signaling, particularly, inhibitors of JAK/STAT signal transduction (13-15). There are currently eight members of this family (CIS and SOCS1-7) that share a similar three domain structure. These proteins are defined by an N-terminal variable length region followed by a central Src homology domain 2 (SH2) and a highly conserved C-terminal domain of 40-50 amino acids termed the SOCS box (16). Because SOCS proteins inhibit cytokine-induced signaling pathways and because the expression of some SOCS genes is induced by the corresponding cytokines, SOCS proteins are believed to play a role in the negative feedback control of cytokine signaling. SOCS proteins appear to employ several mechanisms to inhibit cytokine signaling (17). SOCS-1 inhibits several cytokine receptor signaling pathways by binding directly to and inhibiting the associated Janus kinases (JAKs) (14-20). In contrast, SOCS-3 binds more weakly to JAK kinases and inhibits several cytokine receptor signaling pathways by binding to phosphotyrosine residues on these receptors. This association allows it to interact with and inhibit the receptor-bound JAKs (21-23). The JAK inhibitory activity of SOCS-1 has been mapped to a motif it shares with SOCS-3 in the N-terminal regions of these proteins, which is distinct from the phosphotyrosine binding determinants (18-20). Additionally, it has been shown that the SOCS proteins can inhibit signaling by targeting their binding partners for proteosomal degradation (24-27). This activity appears to be mediated by the SOCS box, which contains a functional BC box similar to that in Elongin A and in the von-Hippel Lindau protein (24).

Recent reports describe the interaction of several of the SOCS family members with both the IR and the insulin-like growth factor receptors in vivo, in vitro, and in the yeast two-hybrid system (10, 11, 28, 29). Since SOCS proteins are induced by cytokines and hormones associated with insulin resistance, the SOCS proteins are excellent candidates for mediating cytokine-induced insulin resistance. Our laboratory has reported that SOCS-1 and SOCS-6 can associate with the insulin receptor in HepG2 cells and inhibit insulin receptor signal transduction (11). These SOCS proteins can also inhibit in vitro receptor kinase activity (11). Emanuelli et al. (10) recently demonstrated that SOCS-3 is induced by insulin in adipocytes and inhibits insulin-dependent activation of STAT5. They propose that an association of SOCS-3 with Tyr-960 of the insulin receptor antagonizes STAT5 activation based on yeast two-hybrid studies. While this group could not demonstrate association between SOCS-3 and the insulin receptor in vivo, they report an inhibition of insulin-mediated IRS-1 tyrosine phosphorylation and p85 association by SOCS-3 in COS cells overexpressing IRS-1 and SOCS-3 (30). Emanuelli et al. (30) also report increased SOCS-3 expression in adipose tissue in ob/ob mice without changes in liver or muscle. These results suggest that SOCS proteins may play a negative regulatory role in insulin signal transduction in response to obesity and other conditions promoting insulin resistance.

IL-6 is the primary pro-inflammatory cytokine affecting the liver. This cytokine has also been shown to be elevated in the circulation of type 2 diabetics and to correlate most closely with insulin resistance (3, 31-34). IL-6 has recently been shown by our laboratory to inhibit insulin signal transduction in hepatocytes and HepG2 cells (35). The IL-6-induced inhibition is apparent after 1 h of exposure to the cytokine. The current investigation posed the question of whether SOCS-3 could play a role in mediating the observed IL-6-dependent insulin resistance. The results indicate that IL-6 induces SOCS-3 in a temporal pattern that is consistent with its role in inhibiting insulin receptor signaling. Additional data demonstrate that SOCS-3 is a potent in vivo and in vitro inhibitor of insulin receptor signaling. Together these results provide evidence that SOCS-3 can be an important mediator of IL-6-dependent insulin resistance in hepatocytes.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Reagents-- The isolation of the cDNA for human SOCS-3 has been described previously (29). The pBIG-2i expression plasmid was a gift from Dr. Craig Strathdee (The John P. Robarts Research Institute, London, Ontario) (36). alpha -IR-1 (37) was a gift of Dr. S. Jacobs (Wellcome Research Laboratories, Research Triangle Park, NC). The antibodies to phospho-Akt (Ser-473) were purchased from Cell Signaling Technology (Beverly, MA). The anti-FLAG antibodies M2 and M5 were from Sigma. The anti-phosphotyrosine antibody (4G10), recombinant rat IRS-1, anti-IRS-1 antibody, and anti-p85 antibody were from Upstate Biotechnology Inc., Lake Placid, NY. Anti-SOCS-3 antibody (M20) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The transfection reagent FuGENE 6 was purchased from Roche Molecular Biochemicals. Other reagents were obtained from commercial sources as indicated in the text and figure legends.

Transient Expression of SOCS-3 in Cell Lines-- HepG2 human hepatocarcinoma cells were grown to 75% confluence in a humidified atmosphere of 5% CO2, 95% air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected with the pBIG-2i-FLAG-SOCS-3 constructs or pBIG-2i-FLAG-null vector using FuGENE-6 transfection reagent according to the manufacturer's instructions. FLAG-tagged SOCS-3 was also transiently expressed in COS cells using the same expression plasmids and protocols.

Coimmunoprecipitation of SOCS-3 with the Insulin Receptor-- HepG2 cells were transiently transfected with the SOCS-3 construct as described above. After 6 h, the cells were placed in serum-free medium for 18 h. Cells were then treated with insulin at 10-7 M for 1 min. After two washes with cold phosphate-buffered saline, cells were lysed in Lysis Buffer A containing 50 mM Tris, pH 7.4,140 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM tetrasodium pyrophosphate, 25 mM benzamidine, protease inhibitor mixture (Calbiochem), 2.5 mM pervanadate, 2 mM phenylmethylsulfonyl fluoride, and 10% glycerol. Lysates were passed 10 times through an 18-gauge needle, centrifuged at 10,000 × g for 10 min, and then adjusted to contain equal amounts of protein as determined by the Bradford method (38). Insulin receptors were immunoprecipitated using an anti-human insulin receptor antibody (alpha -IR-1) bound to protein G-Sepharose. Immune complexes were washed four times with wash buffer (1% Triton X-100, 100 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and separated by SDS-PAGE, and associated FLAG-tagged SOCS-3 was detected by Western blot analysis using anti-FLAG antibody.

In Vivo Insulin Receptor Autophosphorylation, Insulin-induced IRS-1 Tyrosine Phosphorylation and p85 Association, and Activation of Akt-- HepG2 cells transiently expressing SOCS-3 were harvested after treatment with insulin for 5 min as described above. To analyze the phosphorylation of the insulin receptor, tyrosine-phosphorylated proteins were immunoprecipitated with alpha -IR-1, and autophosphorylation was detected by Western blot analysis using anti-phosphotyrosine antibody 4G10. To examine the effects of SOCS proteins on the insulin-dependent tyrosine phosphorylation of IRS-1 and p85 association, cell lysates obtained from insulin-treated (5 min) HepG2 cells transiently expressing SOCS-3 were immunoprecipitated using anti-IRS-1 (alpha -IRS-1) antibody bound to protein A-Sepharose. Immunoprecipitates were then subjected to SDS-PAGE and Western blot analysis using anti-phosphotyrosine antibody 4G10 or anti-p85 antibody. Lysates from the same HepG2 cells were also examined by Western blot analysis for the presence of activated Akt kinase using anti-phosphoserine 473 Akt antibody.

In Vitro Interactions between the Insulin Receptor and SOCS-3-- FLAG-tagged SOCS-3 was harvested from transiently transfected COS cells by immunoprecipitation with anti-FLAG antibody. COS cells were used for the isolation rather than HepG2 cells because the former express few insulin receptors, thus avoiding the possible in vivo complexing of SOCS proteins with endogenous insulin receptors. Insulin receptors were isolated from HepG2 cells using wheat germ agglutinin-agarose affinity chromatography. Briefly, 5 × 106 cells were lysed in a buffer containing 50 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mg/ml bacitracin, 25 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. After homogenization in a Dounce homogenizer and centrifugation, the supernatant was applied to a column containing 1 ml of wheat germ agglutinin-conjugated agarose beads. After 30 min at 4 °C, the column was drained dry and washed three times with wash buffer (50 mM Tris, pH 7.4, and 0.1% Triton X-100), and the IR was eluted with buffer containing 300 mM N-acetylglucosamine, 50 mM Tris, pH 7.4, and 0.1% Triton X-100.

Insulin (10-7 M) and ATP (10 µM) were added to aliquots of the wheat germ agglutinin-purified receptors and incubated at room temperature for 30 min. The receptor preparations were then added to the SOCS protein immunoprecipitates and incubated for an additional 30 min. Finally, 10 µM [gamma -32P]ATP was added, and the reaction mixtures were incubated for an additional 30 min at room temperature. The reactions were stopped with Laemmli (39) sample buffer, and the constituents were separated by SDS-PAGE. The SDS-polyacrylamide gel was treated with 1 N NaOH for 60 min at 55 °C, fixed, and dried. Analysis was by autoradiography.

In Vivo IL-6 Injection and Insulin Treatment of Mouse Liver-- Male C57BL/6 mice (6-8-month-old) were injected with 1 µg/kg IL-6 or vehicle by subcutaneous administration. After 30, 60, and 90 min, livers were rapidly harvested. To examine the hepatic response to insulin, additional mice were anesthetized in a halothane chamber 90 min after IL-6 injections. The portal vein was exposed, 30 ng of insulin or vehicle were injected, and after 60 s the livers were rapidly frozen in liquid nitrogen. Tissue extracts were prepared in ice-cold 2× homogenization buffer containing no detergent. Three 10-s cycles of a Polytron homogenizer were used, chilling homogenates between each cycle. The final 1× homogenization buffer contained 100 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 100 mM NaF, and 1× protease inhibitor mixture set I (Calbiochem). Extracts were kept on ice for 30 min prior to a 10-min centrifugation at 10,000 × g at 4 °C. The supernatant was then collected, protein content determined by the Bradford method (38), and subjected to immunoprecipitation and Western blot analysis. All procedures involving laboratory animals were approved by the University of Rochester Committee on Animal Resources.

Northern Blot Analysis of SOCS-3 Expression in HepG2 Cells and Mouse Liver-- Total RNA was extracted from HepG2 cells and mouse livers described above using the guanidium thiocyanate phenol/chloroform method. mRNA was purified from the total RNA preparation using poly(A) Quik mRNA isolation kit (Stratagene). RNA electrophoresis was then performed using 10 µg of RNA or 1 µg of mRNA per lane on a 1% agarose gel containing 2.2 M formaldehyde and then transferred to a Zeta-Probe GT membrane (Bio-Rad). RNA integrity was confirmed by ethidium bromide staining of the gel. An [alpha -P32]dCTP-labeled probe was created with the Random Primers DNA labeling system (Invitrogen) using the 650-bp human SOCS-3 cDNA as a template. The probe was then purified by ethanol precipitation. Hybridization was performed using the Express-Hyb solution (Clontech) according to the manufacturer's protocol. The relative intensity of SOCS-3 message was determined using a PhosphorImager (Storm 840, Molecular Dynamics) and autoradiography.

Statistical Analysis-- Densitometry of autorads determined by using the ChemImager (Alpha Innotech, San Leandro, CA). Statistical analysis was performed using the Student's t test in the Stataquest statistical software package.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IL-6 Induces SOCS-3 mRNA and Protein Expression in the HepG2 Cell Line-- The ability of IL-6 to induce expression of SOCS mRNA in HepG2 cells was examined at several time points during an 8-h incubation with the cytokine. Total cellular RNA was isolated, and the presence of SOCS-1, SOCS-2, SOCS-3, and SOCS-6 mRNA were examined by Northern blot analysis. Only SOCS-3 expression was elevated in response to IL-6. SOCS-3 mRNA levels increased after 15 min of IL-6 treatment and were maximal by 1 h followed by a nadir at 2 h and then a small 2-3-fold increase at 4 h (Fig. 1A). Following an identical time course of IL-6 treatment, SOCS-3 protein expression was detectable following 1 h of IL-6 treatment and then declined rapidly. No induction of SOCS-1, SOCS-2, or SOCS-6 was observed under these conditions (data not shown). These results indicate that IL-6 is capable of specifically inducing SOCS-3 mRNA and protein in HepG2 cells. Importantly, this induction closely correlates with our observations that IL-6 can inhibit insulin receptor signal transduction in hepatocytes and HepG2 cells after 1 h of pretreatment (35).


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Fig. 1.   IL-6 induces SOCS-3 expression in HepG2 cells. HepG2 cells were exposed to IL-6 (20 ng/ml) for 15 min to 8 h before harvesting. A, mRNA (2 µg) from each sample was separated, transferred, and blotted with a 32P-labeled probe. Quantitation was performed on a phosphorimager. Plotted data represent the mean ± S.D. of four experiments. B, Western blot of SOCS-3 protein expression at time points after IL-6 treatment.

Transiently Expressed SOCS-3 Associates with the Endogenous Insulin Receptor in HepG2 Cells-- We have recently reported that SOCS-1 and 6 can be co-immunoprecipitated with the insulin receptor, and this association is markedly enhanced by insulin (11). It has been suggested by yeast two-hybrid and co-localization experiments that SOCS-3 may also interact with the insulin receptor (10, 29). To determine if SOCS-3 was capable of directly interacting with the insulin receptor, a FLAG epitope-tagged SOCS-3 was transiently expressed in HepG2 cells. Insulin receptor immunoprecipitates were examined by Western blot analysis for the presence of FLAG-tagged SOCS-3. As indicated in Fig. 2, SOCS-3 associated with the insulin receptor in the presence or absence of insulin. This differs from experimental results with SOCS-1 and SOCS-6, which required insulin for maximum association. Additionally, this association was weak when compared with total SOCS-3 expression (Fig. 2, lower panel) and when compared with identical experiments performed with SOCS-1 and SOCS-6 (data not shown). While SOCS-3 can directly associate with the insulin receptor, suggesting that it may be involved in insulin signal transduction, the mechanism of interaction appears to differ from that of SOCS-1 and SOCS-6.


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Fig. 2.   Ectopically expressed SOCS-3 associates with the endogenous insulin receptor. HepG2 cells at 50-75% confluence were transfected with either vector containing FLAG-tagged SOCS-3 cDNA or control vector using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Cells were then serum-starved overnight in Dulbecco's modified Eagle's medium with the addition of 1% bovine serum albumin. Following treatment with 100 nM insulin for 5 min, cells were harvested and immunoprecipitated using the anti-IR antibody, (alpha -IR-1). Immunoprecipitates (IP) were separated using SDS-PAGE, and the presence of FLAG-tagged SOCS-3 was detected by Western blot analysis (IB). Cell lysates were similarly probed for total SOCS-3 expression.

Ectopic Expression of SOCS-3 Inhibits Insulin Signal Transduction in HepG2 Cells-- Since SOCS-3 can directly interact with the IR when ectopically expressed, the following experiments examined whether SOCS-3 was capable of altering insulin signal transduction. We have recently shown that ectopic expression of SOCS-1 and SOCS-6 proteins can inhibit insulin signal transduction without effecting IR autophosphorylation (11). Interestingly, ectopic expression of SOCS-3 was found to inhibit insulin receptor autophosphorylation in response to 100 nM insulin by ~40-50% (Fig. 3). Ectopic expression of SOCS-3 appeared to elevate basal autophosphorylation; however, this increase was not statistically significant. SOCS-3 expression did not affect the total cellular mass of the insulin receptors in HepG2 cells. These data provide further evidence that the interaction of SOCS-3 with the insulin receptor differs from that of SOCS-1 and SOCS-6.


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Fig. 3.   Expression of SOCS-3 inhibits insulin receptor autophosphorylation. HepG2 cells at 50-75% confluence were transfected with SOCS-3 construct or controls and treated with insulin as in Fig. 2 except for a 2-min exposure to insulin. Insulin receptors were immunoprecipitated (IP) using the alpha -IR-1 antibody, and precipitates were separated using SDS-PAGE. Autophosphorylation was determined by Western blot analysis (IB) using anti-phosphotyrosine antibody (4G10). The plotted data represent the average ± S.D. of three independent experiments. Brackets and asterisks indicate bars that differ at p < 0.05. SOCS-3 alone had no statistically significant effect on basal IR autophosphorylation (comparison of gray bars, p > 0.1). Results of a representative experiment are also displayed.

SOCS-3 expression also inhibited insulin-induced tyrosine phosphorylation of IRS-1 by a similar 40-50% (Fig. 4A). This SOCS-dependent inhibition was produced without affecting IRS-1 protein levels. As expected, insulin-induced p85 association with IRS-1 was also decreased by ~50% (Fig. 4B), although this change did not reach statistical significance. As with IR autophosphorylation, SOCS-3 alone appeared to modestly elevate IRS-1 tyrosine phosphorylation and its association with p85, but this observation was quite variable and was not statistically significant. More distal components in the insulin signal transduction pathway did not show this pattern. A 100 nM insulin concentration also produced a 3.5-fold increase in phosphorylation of Akt on Ser-473. In the presence of SOCS-3, however, this increase was reduced to ~2-fold (Fig. 5). These experiments illustrate that SOCS-3 can directly inhibit insulin receptor signal transduction. Additionally, it is not anticipated that the SOCS-3 construct was being expressed in all cells following transient expression. Therefore, the 40-50% inhibition of insulin signaling is probably an underestimation of the effectiveness of SOCS-3 in inhibiting signaling.


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Fig. 4.   Expression of SOCS-3 inhibits insulin-induced tyrosine phosphorylation of IRS-1 and formation of p85/IRS-1 complexes. HepG2 cells were treated as in Fig. 2 except for exposure to insulin for 3 min. IRS-1 was immunoprecipitated (IP) using alpha -IRS-1 antibody (UBI), and precipitates were separated using SDS-PAGE. A, tyrosine phosphorylation was determined by Western blot analysis (IB) using anti-phosphotyrosine antibody (4G10). IRS-1 mass was determined by stripping and reprobing the blot with an anti-IRS-1 antibody. B, association of the p85 subunit of phosphatidylinositol 3-kinase with IRS-1 immunoprecipitates was determined by Western blot analysis of the IRS-1 immunoprecipitates with an anti-p85 antibody. Data plots represent the average ± S.D. of three independent experiments. Brackets and asterisks indicate bars that differ at **, p < 0.01 and *, p = 0.06. SOCS-3 alone had no statistically significant effect on basal levels (comparison of gray bars, p > 0.1). Results of a representative experiment are also displayed.


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Fig. 5.   SOCS-3 inhibits insulin-induced Akt activation. HepG2 cells were treated as in Fig. 2 including exposure to insulin for 5 min. Lysates were separated by SDS-PAGE. Akt activation was determined by Western blot (IB) analysis using anti-phospho-Ser-473 antibody (CST). The data represent the average ± S.D. of three independent experiments. Brackets and asterisks indicate bars that differ at p < 0.05. SOCS-3 alone had no statistically significant effect on basal Akt activity (comparison of gray bars, p > 0.1). Results of a representative experiment are also displayed.

In Vitro Inhibition of Insulin Receptor Autophosphorylation by SOCS-3-- Ectopic expression of SOCS-3 inhibited IR autophosphorylation and signal transduction in transfected HepG2 cells. This suggests but does not prove that SOCS-3 is a direct inhibitor of the IR. In the following experiments, an in vitro kinase assay was used to examine whether SOCS-3 is a direct inhibitor of the IR kinase activity. IR was isolated from HepG2 cells using wheat germ agglutinin affinity chromatography. Receptors were pre-activated with unlabeled ATP and insulin (10-7 M) before being combined with radiolabeled ATP and SOCS-3 immunoprecipitated from COS cells that had been transfected with the SOCS-3 construct. As shown in Fig. 6, SOCS-3 inhibited autophosphorylation of the IR in this assay by ~40%. In contrast to the report by Peraldi et al. (40) no phosphorylation of SOCS-3 was detected in this assay.


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Fig. 6.   SOCS-3 inhibits in vitro insulin receptor autophosphorylation. Insulin receptors were isolated from HepG2 cells by wheat germ agglutinin affinity chromatography. Following addition of insulin and unlabeled ATP to activate the receptors, the reactions were combined with SOCS-3 immunoprecipitates. Radiolabeled ATP was subsequently added to initiate the reaction. Tyrosine-phosphorylated insulin receptors were visualized by autoradiography following SDS-PAGE and treatment of the gel with NaOH to hydrolyze serine/threonine phosphorylation. The data represent the average ± S.D. of three independent experiments. Brackets and asterisks indicate bars that differ at p < 0.05. SOCS-3 alone had no statistically significant effect on basal IR autophosphorylation (comparison of gray bars, p > 0.1). Results of a representative experiment are also displayed.

IL-6 Suppresses Hepatic Insulin Receptor Signaling in the Mouse-- To examine the relationship between IL-6 and SOCS-3 expression and inhibition of insulin signaling in the liver of mice, IL-6 (1 µg/kg) was injected subcutaneously into mice that had been fasted overnight. SOCS-3 expression increased severalfold at 90 min in the liver of animals injected with IL-6 (Fig. 7A). This is consistent with the IL-6-dependent induction of SOCS-3 expression that was observed in HepG2 cells after 60 min of IL-6 treatment (Fig. 1). After 90 min, insulin at a relatively low dose of 30 ng/mouse was injected into the portal vein, and livers were harvested 1 min later. Importantly, IL-6 caused a marked suppression of insulin receptor autophosphorylation under these conditions while receptor mass was unchanged (Fig. 7B). Insulin-dependent tyrosine phosphorylation of IRS-1 was also suppressed in IL-6-treated mice. However, tyrosine phosphorylation of IRS-1 in the mice that did not receive insulin was substantial, rendering the effect of exogenous insulin less pronounced (Fig. 7B). Nonetheless, these results demonstrate that IL-6 exerts an in vivo inhibitory effect on hepatic insulin receptor signaling, which corresponds with induction of SOCS-3 expression.


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Fig. 7.   IL-6 induces hepatic SOCS-3 expression and inhibits hepatic insulin receptor signal transduction in vivo. A, male C127/BL6 mice were injected subcutaneously with IL-6 at 1 µg/kg body weight or a saline control. Livers were rapidly harvested at 90 min and placed in liquid nitrogen. Total protein was isolated, and SOCS-3 expression was determined by immunoprecipitation (IP) with SOCS-3 (M-20) antibody and Western blot analysis using alpha -SOCS-3. (IgG is an immunoglobulin control). B, male C127/BL6 mice were injected subcutaneously with IL-6 at 1 µg/kg body weight. At 90 min after IL-6 injection, the portal vein was exposed, and 30 ng of insulin in 0.1 ml of saline or saline alone was infused. Livers were rapidly harvested and placed in liquid nitrogen. Insulin receptor was immunoprecipitated from homogenates, separated by SDS-PAGE, and tyrosine phosphorylation determined by Western blot analysis (IB). Blots were reprobed for receptor mass. Tyrosine phosphorylation of IRS-1 was similarly analyzed by Western blot analysis using an anti-phosphotyrosine antibody and reprobed for IRS-1 mass with an anti-IRS-1 antibody. Results are representative of three independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The recent observation that circulating IL-6 levels and adipose tissue secretion of IL-6 correlate with insulin resistance suggests that this cytokine could be a link between obesity and insulin resistance (3, 31-34, 41). IL-6 is the major cytokine mediator in the liver. As such, it may play an important role in hepatic insulin resistance. Its effect on skeletal muscle is less clear since IL-6 receptor content in skeletal muscle appears to be very low (42). The results of the current study demonstrate that acute exposure to IL-6 rapidly induces a transient expression of SOCS-3 in HepG2 cells and in the liver of mice. We also show that SOCS-3 is a potent inhibitor of insulin receptor signal transduction in HepG2 cells. In vitro kinase assays confirm that SOCS-3 directly inhibits insulin receptor autophosphorylation. These results complement other investigations of IL-6 by our laboratory that have shown that this cytokine inhibits insulin receptor signal transduction in primary mouse hepatocytes and HepG2 cells (35). The temporal effects of acute IL-6 on SOCS-3 expression and inhibition of insulin receptor signaling are tightly correlated including the marked effects at 30-90 min followed by a rapid decrease at 2 h. Similarly, we now demonstrate that IL-6 not only induces SOCS-3 expression in the livers of mice but also inhibits hepatic insulin receptor signaling in these animals. Together, these observations strongly suggest a role for SOCS-3 in mediating IL-6-dependent insulin resistance in the liver.

SOCS-3 is induced in liver by growth hormone, insulin, and TNFalpha as well as IL-6 (30, 43-46). Each has been implicated in hepatic insulin resistance. Thus, SOCS-3 may also contribute to hepatic insulin resistance associated with these agents. TNFalpha has been investigated most extensively as a potentially critical mediator of obesity-dependent insulin resistance (3, 5, 47-49). Kern et al. (3) have reported that human adipose tissue secretion of TNFalpha correlates better than IL-6 secretion with obesity-related insulin resistance while plasma levels of IL-6 have a more significant correlation to insulin resistance than plasma TNFalpha . Emanuelli et al. (30) have demonstrated that TNFalpha induces SOCS-3 expression in insulin responsive tissues when administered in vivo. Interestingly, TNFalpha induces IL-6 expression in the liver, although it is controversial whether TNFalpha induction of SOCS-3 requires expression of IL-6 (46, 50). It is clear, however, that TNFalpha and other cytokines and hormones that have been linked to obesity-dependent insulin resistance are capable of inducing SOCS proteins in the liver.

The hypothesis that members of the SOCS family play a role in mediating insulin resistance is accumulating experimental support. Our current data and that of Emanuelli et al. (30) indicate that SOCS-3 can inhibit insulin receptor signal transduction. The precise mechanism is not yet defined. We demonstrate here that SOCS-3 can associate with the insulin receptor and can be co-immunoprecipitated when ectopically expressed in cultured cells. This has not been previously demonstrated. We have also observed association between the insulin receptor and endogenously expressed SOCS-3 in HepG2 cells after IL-6 treatment, but this association has been weak and inconsistent (data not shown). Emanuelli et al. (10) propose that SOCS-3 associates with the insulin receptor at Tyr-960 based on cellular co-localization studies and yeast two-hybrid analyses. We too have identified SOCS-3 along with SOCS-1, SOCS-2, and SOCS-6 as binding partners for the insulin receptor by the yeast two-hybrid technique. The interaction of SOCS-3 with the insulin receptor displays some differences from that of SOCS-1 and SOCS-6 (11). The association of SOCS-3 with the insulin receptor does not require insulin. SOCS-3 inhibits insulin-dependent receptor autophosphorylation when ectopically expressed in HepG2 cells. SOCS-3 also inhibits insulin receptor autophosphorylation in vitro. This has not been previously reported. In contrast, SOCS-1 and SOCS-6 require insulin for maximum association with the insulin receptor and do not inhibit insulin receptor autophosphorylation when ectopically expressed in HepG2 cells. They do exert an inhibitory effect on insulin receptor autophosphorylation in vitro. Interestingly, SOCS-3 as well as the other SOCS proteins only associate with kinase active insulin receptor in the yeast two-hybrid system (data not shown). The nature of the SOCS-3-insulin receptor association in HepG2 cells in the absence of insulin is unclear. One explanation is that SOCS-3 binds to otherwise short-lived, insulin-independent phosphotyrosine residues on the insulin receptor. This would be expected to increase phosphorylation of the insulin receptor through trapping of the bound tyrosine residues in a phosphorylated state. In fact, tyrosine phosphorylation of insulin receptors is increased in HepG2 cells in the absence of insulin when the cells are transfected with SOCS-3 (Fig. 3). Insulin receptors from livers of mice exposed to IL-6 for 90 min also display a modest increase in receptor tyrosine phosphorylation in the absence of insulin when compared with the IL-6-free controls (Fig. 7B). Further investigations will be required to define the mechanism of association between the insulin receptor and SOCS-3.

We observe that ectopic SOCS-3 expression in HepG2 cells inhibits in vivo insulin receptor autophosphorylation. Subcutaneous IL-6 injections in mice also lead to suppression of hepatic insulin-dependent insulin receptor autophosphorylation. Interestingly, IL-6 does not inhibit insulin receptor autophosphorylation in HepG2 cells (data not shown), although insulin-dependent IRS-1 tyrosine phosphorylation and Akt activation are inhibited. Thus, inhibition of autophosphorylation may not always be a component of IL-6-dependent suppression of insulin receptor signal transduction. Inhibition of autophosphorylation may depend upon the relative concentrations of SOCS-3 and insulin receptor. This is supported by a set of experiments in which HepG2 cells were transfected with increasing levels of SOCS-3 construct. Insulin-dependent activation of Akt was suppressed at lower SOCS-3 levels than were necessary for inhibition of IR autophosphorylation (data not shown). It has also been suggested by Emanuelli et al. (10) that SOCS-3 and insulin receptor substrates compete for association with the activated insulin receptor at Tyr-960. This competitive association would lead to inhibition of insulin receptor substrate phosphorylation. This may be a mechanism of inhibition of insulin receptor signal transduction that is observed at lower SOCS-3/insulin receptor ratios where autophosphorylation is not impaired. At high levels of expression, SOCS-3 may associate with the insulin receptor at an additional site(s) and directly antagonize autophosphorylation. This step probably requires kinase activity and basal levels of autophosphorylation. Perhaps, this interaction involves the kinase inhibitory domain of SOCS-3 acting similarly to this domain in SOCS-1, which has been shown to be essential for mediating inhibition of JAKs and to participate in the association of this SOCS protein to the cytokine receptor complex (18-20). Inhibition is apparently mediated through an N-terminal peptide sequence of SOCS-1 that acts as a pseudosubstrate for JAKs. A similar mechanism with the insulin receptor may be in effect at high SOCS-3 expression in the current studies.

It cannot be ruled out that SOCS-3 also mediates an effect distal to the insulin receptor. Complexing of SOCS-3 with IRS-1/2, for example, is an additional mechanism to explain SOCS-3-dependent inhibition of insulin receptor signal transduction. Kawazoe et al. (12) have reported that SOCS-1 but not SOCS-3 binds to IRS-1 when each is expressed in 293T cells. This group did not observe association of either SOCS-1 or SOCS-3 with the insulin receptor when both were ectopically expressed in these cells. However, we have not observed IRS-1/2 in immunoprecipitates of SOCS-3 under conditions in which the insulin receptor is co-immunoprecipitated. It has been proposed that SOCS proteins play a role in proteosomal targeting of associated proteins by complexing with Elongins B and C (24-27). We can exclude a mechanism by which SOCS-3 promotes IRS-1 degradation in HepG2 cells since IRS-1 levels are unaffected by ectopic expression of SOCS-3 (Fig. 4A). In summary, our current observations, particularly the in vitro kinase assay and the co-immunoprecipitation study, plus those reported by Emanuelli et al. (10, 30) are most consistent with a direct interaction between the insulin receptor and SOCS-3. No stable interaction between SOCS-3 and IRS-1/2 has been observed in the current study, but an association between these proteins cannot be ruled out.

While SOCS-3 has been linked most directly with insulin resistance in insulin responsive tissues, the potential roles of SOCS-1 and SOCS-6 have also been investigated. We have identified these SOCS proteins as binding partners for the insulin receptor and both inhibit insulin receptor signal transduction (11). Interestingly, these SOCS proteins require insulin for maximum association with the insulin receptor but do not inhibit receptor autophosphorylation. As with SOCS-3, SOCS-1 is induced in liver in response to IL-6, though we report here and others have also observed (51) that IL-6 does not induce SOCS-1 in HepG2 cells. It has been suggested that the phenotype of the SOCS-1 knockout mouse indicates a possible role for this protein in glucose homeostasis and insulin action (12). SOCS-1-null mice at postnatal day 7-10 have lower plasma glucose levels than matched controls despite comparable insulin levels. Glucose and insulin tolerance tests were not performed nor were the animals examined using the euglycemic clamp technique. Importantly, SOCS-1-null mice have a 40% decrease in body weight on postnatal day 9 compared with controls and do not survive beyond 3 weeks (52). It is not clear if this growth retardation and brief survival, which accompanies altered lymphopoiesis, is impacting glucose homeostasis independent of insulin action. Interestingly, a SOCS-3 deletion results in embryonic lethality at 12-16 days with marked erythrocytosis (53).

Until very recently, little was known about the physiological significance of SOCS-6. We have reported that ectopically expressed SOCS-6 associates with the insulin receptor and inhibits signal transduction in vivo (11). The insulin receptor kinase is also directly inhibited by SOCS-6 in vitro. Krebs et al. (54) now report that SOCS-6 is ubiquitously expressed and has binding affinity toward IRS-2 and IRS-4. Nonetheless, SOCS-6-null mice do not exhibit defects in glucose homeostasis and the only apparent phenotype is a 10% decrease in body weight. Further investigations will be necessary to determine the role of SOCS-6 and the significance of its association with members of the IRS family.

While considerable evidence points to a role for SOCS proteins in mediating cytokine-dependent insulin resistance, the precise mechanism and relative contribution of these proteins is yet to be determined. The pro-inflammatory cytokines have pleiotropic effects on target tissues. Our current report plus other recent observations suggest that SOCS protein expression may be one of several mechanisms working in concert to mediate cytokine-dependent insulin resistance in the liver and other insulin-responsive tissues.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant R01-38138 (to R. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Member of the Graduate Program in Pharmacology and Physiology, University of Rochester.

§ Member of the Graduate Program in Biochemistry, University of Rochester.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-7811; Fax: 585-273-1101; E-mail: robert_mooney@urmc.rochester.edu.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M210689200

    ABBREVIATIONS

The abbreviations used are: IRS-1, insulin receptor substrate-1; IR, insulin receptor; IL, interleukin; TNF, tumor necrosis factor; SOCS, suppressor of cytokine signaling-3; STAT, signal transducers and activators of transcription; JAK, Janus kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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