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Endocrinology Vol. 148, No. 6 2994-3003
Copyright © 2007 by The Endocrine Society

Chronic Tumor Necrosis Factor-{alpha} Treatment Causes Insulin Resistance via Insulin Receptor Substrate-1 Serine Phosphorylation and Suppressor of Cytokine Signaling-3 Induction in 3T3-L1 Adipocytes

Ken Ishizuka, Isao Usui, Yukiko Kanatani, Agussalim Bukhari, Jianying He, Shiho Fujisaka, Yu Yamazaki, Hikari Suzuki, Kazuyuki Hiratani, Manabu Ishiki, Minoru Iwata, Masaharu Urakaze, Tetsuro Haruta and Masashi Kobayashi

The First Department of Internal Medicine, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan

Address all correspondence and requests for reprints to: Isao Usui, M.D., Ph.D., The First Department of Internal Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: isaousui-tym{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serine phosphorylation of insulin receptor substrate (IRS)-1 and the induction of suppressor of cytokine signaling 3 (SOCS3) is recently well documented as the mechanisms for the insulin resistance. However, the relationship between these two mechanisms is not fully understood. In this study, we investigated the involvement of SOCS3 and IRS-1 serine phosphorylation in TNF{alpha}-induced insulin resistance in 3T3-L1 adipocytes. TNF{alpha} transiently stimulated serine phosphorylation of IRS-1 from 10 min to 1 h, whereas insulin-stimulated IRS-1 tyrosine phosphorylation was inhibited only after TNF{alpha} treatment longer than 4 h. These results suggest that serine phosphorylation of IRS-1 alone is not the major mechanism for the inhibited insulin signaling by TNF{alpha}. TNF{alpha} stimulation longer than 4 h enhanced the expression of SOCS3 and signal transducer and activator of transcription-3 phosphorylation, concomitantly with the production of IL-6. Anti-IL-6 neutralizing antibody ameliorated suppressed insulin signaling by 24 h TNF{alpha} treatment, when it partially decreased SOCS3 induction and signal transducer and activator of transcription-3 phosphorylation. These results suggest that SOCS3 induction is involved in inhibited insulin signaling by TNF{alpha}. However, low-level expression of SOCS3 by IL-6 or adenovirus vector did not affect insulin-stimulated IRS-1 tyrosine phosphorylation. Interestingly, when IRS-1 serine phosphorylation was enhanced by TNF{alpha} or anisomycin in the presence of low-level SOCS3, IRS-1 degradation was remarkably enhanced. Taken together, both IRS-1 serine phosphorylation and SOCS3 induction are necessary, but one of the pair is not sufficient for the inhibited insulin signaling. Chronic TNF{alpha} may inhibit insulin signaling effectively because it causes both IRS-1 serine phosphorylation and the following SOCS3 induction in 3T3-L1 adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN RESISTANCE IS a predisposing cause for the development of type 2 diabetes. Several proinflammatory cytokines have recently been implicated in the mechanisms for insulin resistance (1, 2). A close relationship between TNF{alpha} and insulin resistance and their molecular mechanisms were mostly examined among such proinflammatory cytokines (3, 4). Serine phosphorylation of insulin receptor substrate (IRS)-1 is a well-known mechanism by which insulin signaling is negatively regulated. TNF{alpha} phosphorylates serine residues on IRS-1 through the activation of several serine kinases including inhibitory-{kappa}B kinase (5), c-Jun N-terminal kinase (6, 7), and mammalian target of rapamycin (8). Serine phosphorylation hinders insulin-stimulated tyrosine phosphorylation of IRS-1 and consequently suppresses the activation of the downstream insulin signaling.

Another important mechanism is induction of signal transducer and activator of transcription (SOCS) proteins. SOCSs were originally cloned as proteins that were induced after the activation of cytokine signaling, such as janus kinase (JAK) and signal transducer and activator of transcription (STAT), and negatively regulate cytokine signaling (9, 10, 11). Recently SOCSs are reported to be induced by not only cytokines but also other stimuli including adipokines, growth factors, or hormones (12, 13, 14, 15). Signal transductions other than cytokine signaling are also suppressed by SOCS. SOCS family consists of eight members (SOCS1-SOCS7 and cytokine-induced SH2 domain-containing protein), and insulin signaling is inhibited mainly by SOCS1 and SOCS3. For example, Ueki et al. (16) reported that SOCS1 and SOCS3 bind to insulin receptor and block signal transduction from the receptor to IRS proteins. Another group has reported that SOCS1 and SOCS3 promote the degradation of IRS-1 and IRS-2, resulting in inhibited insulin signaling (17). However, most of such studies were performed in overexpression of the exogenous SOCS proteins (16, 17). It has not been fully understood whether the induction of endogenous SOCS is really implicated in the mechanisms for insulin resistance.

IL-6 is another proinflammatory cytokine closely related to insulin resistance (18, 19, 20). It is well known that IL-6 induces SOCS3 expression through the activation of the JAK-STAT pathway (21). Because JAK-STAT is not the major signaling pathway for TNF{alpha} (22), it is not clear whether SOCS3 induction may be involved in the mechanisms for TNF{alpha}-induced insulin resistance as well as serine phosphorylation of IRS-1. We conducted the present study to clarify the involvement of SOCS3 induction as a mediator of TNF{alpha}-induced insulin resistance, especially in its relationship to IRS-1 serine phosphorylation in 3T3-L1 adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM and fetal calf serum were purchased from Gibco BRL Life Technologies (Rockville, MD). Monoclonal antiphosphotyrosine antibody was from Transduction Laboratories (Lexington, KY). Anti-IRS-1 was from Upstate Biotechnology (Lake Placid, NY). Antiphospho-IRS-1 (Ser636), antiphospho-NF{kappa}B (p65) (Ser536), antiphospho-STAT3 (Tyr705), anti-STAT3, antiphospho-Akt (Ser473), and anti-Akt were from Cell Signaling Technology (Beverly, MA). Anti-IL-6 antibody was from R&D Systems (Minneapolis, MN). Horseradish peroxidase-conjugated antimouse and antirabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Lactacystin was from Calbiochem (La Jolla, CA). Recombinant human TNF{alpha} was kindly provided by Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). Endogen mouse IL-6 ELISA kit was from Pierce Biotechnology, Inc. (Rockford, IL). All other reagents were from standard supplies.

Cell culture and treatment
Murine 3T3-L1 cells obtained from American Type Culture Collection (Manassas, VA) were cultured, maintained, and differentiated essentially as previously described (23, 24). Briefly, cells were plated and grown for 2 d after confluence in DMEM/high glucose supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum in a 10% CO2 environment. Differentiation was then induced by changing the medium to the same one containing 0.5 mmol/liter 3-isobuty-1-methylxanthine, 1 µmol/liter dexamethasone, and 1 µmol/liter insulin for 3 d and then to the medium containing 0.8 µmol/liter insulin for another 3 d. On 14–16 d after the induction of differentiation, when more than 95% of the cells had the morphological and biochemical properties of adipocytes, the cells were used for experiments. TNF{alpha} was dissolved in PBS and added to the cell culture medium.

Adenovirus vectors
Adenovirus vectors encoding SOCS1 and SOCS3 were kindly provided by Dr. Tetsuji Naka (Osaka University, Osaka, Japan). A virus vector encoding ß-galactosidase (LacZ) was used as a control. Differentiated 3T3-L1 adipocytes were infected with adenovirus 48 h before the experiments.

Real-time RT-PCR
Total RNA was isolated from 3T3-L1 adipocytes using ISOGEN according to the manufacturers’ instruction (Nippon Gene, Tokyo, Japan). It was reverse transcribed with standard reagents (Applied Biosystems, Foster City, CA). A volume of 3.85 µl of each reverse-transcribed reaction was amplified in a 10-µl PCR mixture with the TaqMan PCR kits according to the manufacturer’s instruction (TaqMan; Applied Biosystems). Briefly, samples were incubated at 95 C for 10 min for an initial denaturation, followed by 40 PCR cycles. Each cycle consists of 92 C for 15 sec and 60 C for 1 min.

Immunoblotting
Immunoblotting was performed as described previously (23, 24). Briefly, 3T3-L1 adipocytes were lysed in a cell-solubilizing buffer containing 30 mmol/liter Tris (pH 7.4), 150 mmol/liter NaCl, 10 mmol/liter EDTA, 1% Nonidet-P40, 1 mmol/liter phenylmethylsulfonylfluoride, 10 µmol/liter leupeptin, 1 mmol/liter Na3VO4, and 50 mmol/liter NaF. The cell lysates were boiled with Laemmli sample buffer for 5 min, resolved by 7.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) in the Trans-Blot cell apparatus (Bio-Rad, Hercules, CA). The membranes were blocked with dried milk and incubated with the indicated antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized with chemiluminescence reagents according to the manufacturer’s protocol (Amersham, Piscataway, NJ). The intensities of blots were quantified by scanning the film using Scion Image (Frederick, MD).

Quantification of cytokine production
The concentration of IL-6 in the culture medium was measured using an endogen mouse IL-6 ELISA kit according to the manufacturer’s instruction (Pierce Biotechnology). The absorbance was measured by iEMS Reader MF (Labsystems, Helsinki, Finland).

Statistical analysis
All data are presented as means ± SEM. The statistical comparison among the groups was carried out using Student’s t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Short-term TNF{alpha} treatment enhances serine phosphorylation but does not inhibit tyrosine phosphorylation of IRS-1
The effect of TNF{alpha} treatment on insulin signaling was first examined. Insulin-stimulated tyrosine phosphorylation of IRS-1 was not inhibited during the early time period (Fig. 1AGo). TNF{alpha} treatment for 4 h slightly and for 24 h dramatically decreased both protein levels and tyrosine phosphorylation of IRS-1. The ratio of IRS-1 tyrosine phosphorylation to IRS-1 protein tended to be reduced after TNF{alpha} exposure longer than 4 h, compared with the ratio without TNF{alpha} stimulation, but the reduction was not statistically significant (Fig. 1BGo). Pretreatment with lactacystin, a proteasomal inhibitor, prevented the loss of IRS-1 level with long-term TNF{alpha} treatment (Fig. 1CGo). These results suggest that reduced insulin signal at IRS-1 level is mainly due to the decreased expression of this protein, which may be, at least in part, through its proteasomal degradation. To further test the mechanisms for the inhibited insulin signaling at IRS-1 level by TNF{alpha} treatment, we next examined serine phosphorylation of IRS-1 using specific antibodies, which recognize phosphorylated IRS-1 on Ser307, Ser612, or Ser636. TNF{alpha} transiently enhanced serine phosphorylation of IRS-1 with the maximal response at 30 min to 1 h on Ser307 and 10–15 min on Ser612/636 (Fig. 1DGo). Because of the different time courses, serine phosphorylation alone does not explain the inhibited tyrosine phosphorylation of IRS-1 observed after TNF{alpha} treatment longer than 4 h in 3T3-L1 adipocytes.


Figure 1
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  		FIG. 1. Effect of TNF{alpha} treatment on IRS-1. A and B, Differentiated 3T3-L1 adipocytes were pretreated with 10 ng/ml TNF{alpha} for the indicated time periods and then stimulated by insulin for 5 min. C, Differentiated 3T3-L1 adipocytes were incubated with or without 10 µM lactasystin for 30 min and then stimulated by 10 ng/ml TNF{alpha} for 24 h. D, Differentiated 3T3-L1 adipocytes were stimulated by 10 ng/ml TNF{alpha} for the indicated time periods. Total cell lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies as described in Materials and Methods. The representative results from three or four independent experiments are shown (A, C, and D). The intensity of each band was quantified by Scion Image and is shown as mean ± SEM (B). IB, Immunoblotting.

 
Long-term TNF{alpha} stimulation enhances IL-6 secretion, STAT3 phosphorylation, and SOCS3 expression
Recently it has been well documented that SOCS3 inhibits insulin signaling (15, 25). But it was not clear whether SOCS3 induction was involved in the mechanisms for chronic TNF{alpha}-induced insulin resistance. We examined the induction of SOCS3 mRNA after the stimulation with TNF{alpha} up to 24 h, and compared the results with IL-6 stimulation, a well-known inducer of SOCS3. The expression of SOCS3 started to increase at 2 h and reached the peak level at 24 h after TNF{alpha} treatment (Fig. 2AGo). In contrast, the expression of SOCS3 showed a transient peak 30 min after IL-6 treatment and the low-level expression (~20% of the peak) continued thereafter until 24 h (Fig. 2BGo).


Figure 2
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  		FIG. 2. Effects of TNF{alpha} or IL-6 on SOCS3 induction and STAT3 phosphorylation. Differentiated 3T3-L1 adipocytes were stimulated by either 10 ng/ml TNF{alpha} or 10 ng/ml IL-6 for the indicated time periods. A and B, Induction of SOCS3 mRNA was measured by real-time RT-PCR as described in Materials and Methods. C and D, Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three independent experiments are shown. E, Differentiated 3T3-L1 adipocytes were stimulated by 10 ng/ml TNF{alpha} for the indicated time periods. Concentration of IL-6 in the culture media was measured by ELISA kit as described in Materials and Methods. Data are shown as mean ± SEM from three independent experiments.

 
To know which signaling was involved in SOCS3 induction after TNF{alpha} or IL-6 stimulation, we examined the phosphorylation of nuclear factor-{kappa}B (NF{kappa}B), a major signaling molecule for TNF{alpha}, and STAT3, a major molecule for IL-6 signaling (Fig. 2Go, C and D). After TNF{alpha} treatment, NF{kappa}B phosphorylation transiently enhanced at 5 min and returned to the lowest phosphorylation level (~15% of the peak) at 30 min, followed by the second enhancement (~30% of the peak) at 24 h. Phosphorylation of STAT3 started at 4 h and continued until 24 h (Fig. 2CGo). In contrast, IL-6 treatment phosphorylated STAT3 with the maximum level from 10 to 30 min transiently and kept the lower phosphorylation level (~20% of the maximum) until 24 h. IL-6 did not enhance the phosphorylation of NF{kappa}B (Fig. 2DGo). Time course of STAT3 phosphorylation was similar to that of SOCS3 induction after the treatment with either TNF{alpha} or IL-6, but time course of NF{kappa}B phosphorylation was quite different.

These results suggest that the induction of SOCS3 after TNF{alpha} treatment is not dependent on the activation of NF{kappa}B but is possibly dependent on STAT3 activation. Because STAT3 is not the major signaling molecule for TNF{alpha}, we hypothesized that TNF{alpha} might stimulate STAT3 phosphorylation via the production of other cytokines. The production of IL-6 was measured as one of such candidate cytokines. Concentration of IL-6 in the cell culture media increased continuously from 2 to 24 h after TNF{alpha} treatment (Fig. 2EGo). The time course was not quite similar to that of SOCS3 induction or STAT3 phosphorylation (Fig. 2Go, A and C). We examined the dose dependency of IL-6 for SOCS3 induction and STAT3 phosphorylation. The maximal responses of SOCS3 expression and STAT3 phosphorylation were induced by 2 ng/ml IL-6, which was the concentration secreted 4 h after TNF{alpha} treatment. IL-6 concentration higher than 2 ng/ml did not have a stronger effect on SOCS3 expression and STAT3 phosphorylation (data not shown). We theorized that TNF{alpha} treatment for 4 h was sufficient for IL-6 secretion much enough for the maximal STAT3 phosphorylation and SOCS3 induction.

IL-6 stimulation or low-level expression of SOCS3 alone does not affect insulin signaling
Because the data in Fig. 2Go suggest the involvement of IL-6 production and SOCS3 induction in the mechanisms for insulin resistance by long-term TNF{alpha} treatment, we examined the direct effects of IL-6 stimulation or SOCS3 expression on insulin signaling. First, the effects of IL-6 treatment on insulin signaling were examined. As shown in Fig. 3AGo, IL-6 treatment up to 24 h did not alter IRS-1 protein, insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. These results suggest that the IL-6 production alone does not explain the mechanism for insulin resistance induced by long-term TNF{alpha} treatment. Then the effects of SOCS3 expression on IRS-1 level were examined by using adenovirus vectors encoding SOCS3 (Fig. 3BGo). Infection of the highest titer of SOCS3 adenovirus vector [30 multiplicity of infection (m.o.i.)] increased SOCS3 mRNA level to approximately 1000-fold of the basal (Fig. 3Go, lane 5). Less titer of the virus vector (3 m.o.i.) or 30 min IL-6 treatment increased SOCS3 mRNA to the lower level (~100-fold of basal) (Fig. 3BGo, lanes 3 and 6). Western blot analysis using anti-SOCS3 antibody detected the proteins only when the high amount of SOCS3 was expressed by virus vectors (lanes 4 and 5). IRS-1 protein was decreased only by the highest expression of SOCS3 (lane 5), probably due to the degradation of the protein as reported recently (17). In contrast, less expressions of SOCS3 by adenovirus vectors (lanes 2–4) or IL-6 (lane 6) did not change IRS-1 level, suggesting that a large amount of SOCS3 is necessary to degrade IRS-1 by itself. Interestingly, 24 h TNF{alpha} treatment decreased IRS-1 protein (lane 7), although it induced SOCS3 mRNA to the lower level (3- to 4-fold of basal) than 30 min IL-6 treatment (~100-fold of basal). These results suggest that other mechanisms exist, which help the insufficient ability of less expression of SOCS3 to cause insulin resistance in long term TNF{alpha} treatment.


Figure 3
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  		FIG. 3. Effects of IL-6 stimulation or SOCS3 expression on insulin signaling. A, Differentiated 3T3-L1 adipocytes were pretreated with IL-6 for the indicated time periods and then stimulated by insulin for 5 min. B, Differentiated 3T3-L1 adipocytes were infected with adenovirus vectors encoding SOCS3 or LacZ of the indicated m.o.i. Two days after the infection, some cells were stimulated by TNF{alpha} or IL-6 for the indicated time periods. Expression of SOCS3 mRNA was measured by real-time RT-PCR as described in Materials and Methods. Data are shown as mean ± SEM of three independent experiments. Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three or four independent experiments are shown.

 
Anti-IL-6 neutralizing antibody inhibits SOCS3 induction and ameliorates suppressed insulin signaling by TNF{alpha}
To further clarify the involvement of IL-6 signaling in the mechanisms for insulin resistance by long-term TNF{alpha} treatment, we examined the effects of anti-IL-6 neutralizing antibody on insulin signaling. To know the efficacy of the antibody before testing its effects on insulin signaling, we first examined the dose dependency of the antibody on IL-6-stimulated STAT3 phosphorylation. The final concentration of 0.5 µg/ml completely inhibited the STAT3 phosphorylation, indicating that it can be used as a neutralizing antibody for IL-6 signaling (data not shown). The anti-IL-6 antibody decreased STAT3 phosphorylation and SOCS3 expression induced by 24 h TNF{alpha} stimulation by 57 and by 38%, respectively (Fig. 4Go, A, C, and D). The phosphorylation of NF{kappa}B was not affected (Fig. 4Go, A and B). These results indicate that the activation of IL-6 signal may not be sufficient but is necessary for the maximal activation of STAT3 and induction of SOCS3 by the long-term TNF{alpha} stimulation. Partial inhibition by the IL-6 antibody may be explained as follows: some mechanisms other than IL-6 secretion may also be required, such as concomitant secretion of other cytokines. Another possibility is that the antibody’s efficiency may be limited by accessibility to membrane-bound or intracellular IL-6.


Figure 4
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  		FIG. 4. Effects of anti-IL-6 neutralizing antibody on TNF{alpha}-induced STAT3 phosphorylation and SOCS3 induction. A, Anti-IL-6 antibody (5 µg/ml) was added to the cell culture media 10 min before the treatment with 10 ng/ml TNF{alpha} for 5 min or 24 h. Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three or four independent experiments are shown. B and C, The intensity of each band was quantified by Scion Image and is shown as mean ± SEM. *, P < 0.05. D, Anti-IL-6 antibody was added to the cell culture media 10 min before the stimulation with 10 ng/ml TNF{alpha} for 24 h. Induction of SOCS3 mRNA was measured by real-time RT-PCR as described in Materials and Methods. Data are shown as mean ± SEM of three independent experiments. *, P < 0.05.

 
Then the effect of this antibody on TNF{alpha}-induced insulin resistance was examined. TNF{alpha} treatment for 24 h decreased tyrosine phosphorylation of IRS-1, expression of IRS-1, and phosphorylation of Akt by 66, 65, and 47%, respectively (Fig. 5Go, A–D). An addition of the anti-IL-6 antibody 30 min before TNF{alpha} stimulation clearly but partially inhibited TNF{alpha}-induced STAT3 phosphorylation in both the presence and absence of insulin (Fig. 5Go, A and E). The anti-IL-6 antibody increased tyrosine phosphorylation of IRS-1, expression of IRS-1, and Akt phosphorylation by 39, 40, and 28%, respectively (Fig. 5Go, A–D). The ratio of tyrosine phosphorylation to expression level of IRS-1 was not altered significantly with or without the IL-6 antibody, suggesting that the enhanced signal is mainly due to the increased expression level of IRS-1 (data not shown). These results suggest that the activation of IL-6 signaling and after SOCS3 induction are not sufficient but at least a part of the mechanisms for the suppressed insulin signal by long-term TNF{alpha} treatment.


Figure 5
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  		FIG. 5. Effect of anti-IL-6 antibody on the suppressed insulin signaling by TNF{alpha}. A, Anti-IL-6 antibody was added to the cell culture media 10 min before the treatment with 10 ng/ml TNF{alpha} for 24 h. Differentiated 3T3-L1 adipocytes were stimulated by 20 nM insulin for 5 min. Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three independent experiments are shown. B–E, The intensity of each band was quantified Scion Image and is shown as mean ± SEM. *, P < 0.05.

 
IRS-1 degradation by TNF{alpha} is enhanced with low-level expression of SOCS3 or IL-6 stimulation
To know whether the low-level expression of SOCS3 observed after long-term TNF{alpha} treatment was associated with insulin resistance, we next examined the effects of low-level expression of SOCS3 on insulin signaling together with TNF{alpha} treatment. As observed in Fig. 3BGo, the expression of SOCS3 by 3 m.o.i. adenovirus vectors did not affect IRS-1 level by itself (Fig. 6AGo). Interestingly, the low-level expression of SOCS3 enhanced IRS-1 degradation in the presence of 4–24 h TNF{alpha} treatment. Furthermore, the combination treatment of low level SOCS3 expression and short-term (1 h) TNF{alpha} treatment suppressed insulin-stimulated Akt phosphorylation (Fig. 6BGo). We next used IL-6 treatment to induce endogenous expression of SOCS3 instead of adenovirus infection, and the effect of combination treatment with TNF{alpha} on insulin signaling was examined (Fig. 6Go, C and D). IL-6 treatment for 30 min does not affect IRS-1 level, as shown in Fig. 3Go; it enhanced the degradation of IRS-1 by 4 h TNF{alpha} treatment (Fig. 6CGo). Insulin-stimulated Akt phosphorylation was also suppressed by 4 h TNF{alpha} treatment only in the presence of the concomitant IL-6 treatment (Fig. 6DGo). These results suggest that low-level expression of either exogenous or endogenous SOCS3 is not sufficient to induce IRS-1 degradation by itself but enhances TNF{alpha}-induced IRS-1 degradation, leading to the inhibited insulin signaling.


Figure 6
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  		FIG. 6. Effects of TNF{alpha} on insulin signaling in the presence or absence of low-level SOCS3 expression. A and B, Differentiated 3T3-L1 adipocytes were infected with or without adenovirus vectors encoding SOCS3 of 3 m.o.i. Two days after the infection, cells were stimulated by TNF{alpha} for the indicated time periods (A). Two days after the infection, cells were treated with TNF{alpha} for 1 h and then stimulated by insulin for 10 min (B). C, Differentiated 3T3-L1 adipocytes were treated with TNF{alpha} for the indicated time periods and then stimulated by IL-6 for the last 30 min. D, Differentiated 3T3-L1 adipocytes were treated with TNF{alpha} for 4 h and/or IL-6 for 30 min and then stimulated by insulin for 10 min. Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three independent experiments are shown. The intensity of each band was quantified Scion Image and is shown as mean ± SEM. *, P < 0.05.

 
Low-level expression of SOCS3 efficiently induces IRS-1 degradation after its serine phosphorylation
For the reason that long-term TNF{alpha} treatment caused IRS-1 degradation, although it induced just a low-level expression of SOCS3, we hypothesized that serine phosphorylation of IRS-1 might help the function of SOCS3 to degrade IRS-1. Anisomycin is well known to stimulate IRS-1 serine phosphorylation as TNF{alpha} is (26). To clarify this hypothesis, the effects of anisomycin on IRS-1 degradation were examined with or without low-level expression of SOCS3. Anisomycin treatment for 4 h did not affect the expression level of IRS-1 by itself, but it enhanced IRS-1 degradation with the low-level expression of SOCS3 by adenovirus (Fig 7AGo) or with 30 min IL-6 treatment (Fig. 7BGo). These results suggest that IRS-1 serine phosphorylation and SOCS3 induction synergistically enhance IRS-1 degradation. Accordingly, TNF{alpha} may effectively induce IRS-1 degradation by enhancing both IRS-1 serine phosphorylation after short-term stimulation and then SOCS3 induction after long-term stimulation.


Figure 7
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  		FIG. 7. Effect of anisomycin on IRS-1 degradation in the presence or absence of low-level SOCS3 expression. A, Differentiated 3T3-L1 adipocytes were infected with adenovirus vectors encoding SOCS3 of 3 m.o.i. Two days after the infection, cells were stimulated by 1 µg/ml anisomycin for 4 h. B, Differentiated 3T3-L1 adipocytes were treated with IL-6 for 30 min and then stimulated by 1 µg/ml anisomycin for 4 h. Total cell lysates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies as described in Materials and Methods. The representative results from three independent experiments are shown. The intensity of each band was quantified Scion Image and is shown as mean ± SEM. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of recent studies have revealed that IRS-1 is one of the most important targets when TNF{alpha} suppresses insulin signaling. The involvement of serine phosphorylation of IRS-1 has been documented (5, 6, 8, 27, 28). However, the time course of suppressed tyrosine phosphorylation and serine phosphorylation of IRS-1 were quite different, as shown in Fig. 1Go, suggesting that serine phosphorylation of IRS-1 alone may be a partial, but not a sufficient, mechanism for TNF{alpha}-induced insulin resistance at least in 3T3-L1 adipocytes. In this study, we first examined the involvement of IL-6 production and SOCS3 induction as the mechanisms for TNF{alpha}-induced insulin resistance. Long-term TNF{alpha} actually promoted the production and secretion of IL-6 in the culture media, the concentration of which was high enough to stimulate SOCS3 induction (Fig. 2Go). The experiments using anti-IL-6 antibody revealed that activation of IL-6 signaling was necessary for STAT3 phosphorylation, SOCS3 induction (Fig. 4Go, A–D), and inhibition of insulin signaling observed after long-term TNF{alpha} treatment (Fig. 5AGo). These results suggest that IL-6 production and the following STAT3 phosphorylation and SOCS3 induction play important roles in the mechanisms for TNF{alpha}-induced insulin resistance in 3T3-L1 adipocytes. Recent studies reported the close relationship between IL-6 and insulin resistance. For example, expression levels of IL-6 as well as TNF{alpha} in adipose tissue from obese people are higher than lean control (29). However, the relationship between IL-6 and TNF{alpha} is not fully understood, when they induce insulin resistance. The current results are meaningful in the sense that a function of IL-6 in the process of TNF{alpha}-induced insulin resistance was clarified.

A recent study reported that SOCS3 induced insulin resistance by directly binding to IRS-1 and promoting the ubiquitination and subsequent degradation of IRS-1 (17). In the current study, we also confirmed that adenovirus-mediated overexpression of exogenous SOCS3 (~1000-fold of the basal mRNA level) decreased IRS-1 expression by itself. But the maximum induction of endogenous SOCS3 by IL-6 or TNF{alpha} stimulation was only approximately 100- or approximately 4-fold of basal expression, respectively. The dose dependency experiment using different m.o.i. of adenovirus vector revealed that low-level expression of SOCS3 (~100-fold of basal) induced by 3 m.o.i. adenovirus vector or by short-term IL-6 stimulation alone could not cause IRS-1 degradation or inhibition of the downstream insulin signaling (Fig. 3Go). Interestingly, not only TNF{alpha} stimulation but also anisomycin enhanced IRS-1 degradation in the presence of the low level expression of SOCS3 by adenovirus vector or IL-6 treatment (Figs. 6Go and 7Go). Furthermore, our previous study has revealed that the combination treatment of IL-1{alpha} and IL-6 efficiently inhibits insulin signaling, although separate treatment with either IL-1{alpha} or IL-6 does not (30). Because either anisomycin or IL-1{alpha} stimulates IRS-1 serine phosphorylation as TNF{alpha} does, these results suggest that the existence of both IRS-1 serine phosphorylation and SOCS3 induction are necessary for IRS-1 degradation. In case of TNF{alpha} stimulation, we speculate that short-term TNF{alpha} first enhanced serine phosphorylation of IRS-1 by activating inhibitory-{kappa}B kinase or c-Jun N-terminal kinase and then long term TNF{alpha}-induced SOCS3 expression through the production of IL-6, leading to the degradation of IRS-1. This may explain why short-term IL-6 stimulation or short-term TNF{alpha} stimulation failed to cause IRS-1 degradation and insulin resistance because the former induces SOCS expression but does not enhance IRS-1 serine phosphorylation, and the latter stimulates serine phosphorylation but does not induce SOCS expression (30). We further speculate that the endogenous SOCS3 recognizes IRS-1 protein only when IRS-1 is serine phosphorylated, whereas the overexpression of SOCS3 over the physiological limit (~1000-fold of basal) may cause IRS-1 degradation by recognizing IRS-1 nonspecifically, even if IRS-1 is not serine phosphorylated.

As shown in Fig. 5Go, anti-IL-6 antibody recovered insulin signaling inhibited by TNF{alpha} partially, suggesting that other mechanisms exist, in addition to IRS-1 degradation via SOCS3 induction. IRS-1 serine phosphorylation is one of such mechanisms as mentioned above. And the altered localization of IRS-1 may also be a part of the inhibited activation of IRS-1, as we reported recently (24, 26). Furthermore, the induction of SOCS1 may also be another important mediator to suppress insulin signaling by TNF{alpha}. Ueki et al. (16) recently reported that SOCS1 binds to the kinase domain of insulin receptor and suppresses tyrosine phosphorylation of IRS-1. We confirmed that TNF{alpha} induced the expression of SOCS1 mRNA, which was not affected by adding anti-IL-6 antibody in 3T3-L1 adipocytes (data not shown). This result indicates that SOCS1 induction after long-term TNF{alpha} stimulation may be independent of IL-6-stimulated STAT3 activation. All these possible mechanisms may explain the reason for the partial recovery of insulin signaling by anti-IL-6 neutralizing antibody in cells treated with TNF{alpha} (Fig. 4Go).

In conclusion, the current study has revealed that IRS-1 serine phosphorylation or SOCS3 induction may not cause insulin resistance separately, but the concomitant existence of these two mechanisms is necessary for the effective suppression of insulin signaling after chronic TNF{alpha} treatment in 3T3-L1 adipocytes. IL-6 production and the activation of the IL-6 signaling play important roles during this process.


   Footnotes
 
First Published Online March 22, 2007

Abbreviations: IRS, Insulin receptor substrate; JAK, janus kinase; m.o.i., multiplicity of infection; NF{kappa}B, nuclear factor-{kappa}B; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

This work was supported in part by the Grant-in-Aid for Scientific Research 17590918 from the Ministry of Education, Science, Sports, and Culture of Japan (to M.K.).

Disclosure Summary: The authors have nothing to disclose.

Received December 18, 2006.

Accepted for publication March 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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