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Institut National de la Santé et de la Recherche Médicale (INSERM) U568, Faculty of Medicine, F-06107 Nice, France; and Université de Nice Sophia-Antipolis, UFR Sciences, F-06002 Nice, France
Address all correspondence and requests for reprints to: Jean-François Tanti, Institut National de la Santé et de la Recherche Médicale U 568, Faculty of Medicine, Avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail: tanti{at}unice.fr.
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Introduction |
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, IL-1, and IL-6 is increased in this tissue in the obese state (3, 4). Increased macrophage population in adipose tissue is thought to be responsible for this elevated production of cytokines (5, 6). Proinflammatory cytokines produced in adipose tissue could alter the endocrine function of the tissue and could impinge insulin signaling and action in adipocytes, liver, and muscles leading to the development and/or aggravation of insulin resistance (4).
Insulin regulates blood glucose level through suppression of hepatic endogenous glucose production and stimulation of glucose uptake in muscle and adipocyte. These biological responses require the tyrosine phosphorylation of the insulin receptor substrate (IRS)-1 and/or IRS-2 proteins that in turn bind and activate phosphoinositide 3-kinase (PI 3-kinase). Downstream effectors of PI 3-kinase such as protein kinase B (PKB) are involved in insulin metabolic effects (7). Several reports have shown that TNF- and IL-6 alter insulin signaling by targeting IRS-1 proteins. TNF- increases the serine phosphorylation of IRS-1. This mechanism reduces its tyrosine phosphorylation by the insulin receptor (8, 9, 10) and several kinases including c-Jun NH2-terminal kinase (JNK) (11), mammalian target of rapamycin (mTOR) (12, 13), and ERK (13, 14, 15) have been implicated in the serine phosphorylation of IRS-1. TNF- and IL-6 also enhance the expression of suppressor of cytokine signaling (SOCS) proteins that can attenuate insulin signaling by binding to the insulin receptors and reducing their ability to phosphorylate IRS proteins (16, 17). Alternatively, SOCS proteins can bind directly to IRS proteins, leading to their degradation (18, 19). Finally, TNF- and IL-6 could inhibit IRS-1 expression at the transcriptional level (20, 21, 22). In obesity, alteration of IRS-1 tyrosine phosphorylation in muscle is not linked to a change in its expression. By contrast, a down-regulation of IRS-1 mRNA expression seems to be the major mechanism involved in alteration in IRS-1 tyrosine phosphorylation in adipocytes of obese rodents and adipocytes from type 2 diabetic subjects, obese patients, and relatives of diabetic subjects (23, 24).
Whereas the implication of TNF- and IL-6 in insulin resistance is well documented, little is known about a potential role of IL-1ß. IL-1ß is one of the major proinflammatory cytokines that is produced by monocytes and macrophages (25). IL-1ß exerts its biological function by binding to IL-1 type I receptor and activates the inhibitor-
B kinase/nuclear factor-B (IKK/NF-B) pathway and the three types of MAPKs: ERK, JNK, and p38MAPK (25). Recent studies suggested that IL-1ß could also belong to the network of cytokines involved in insulin resistance. Indeed, in a case/control study, individuals with detectable circulating levels of IL-1ß and elevated levels of IL-6 have an increased risk to develop type 2 diabetes, compared with individuals with increased concentrations of IL-6 but undetectable levels of IL-1ß (26). Furthermore, IL-1ß concentration is elevated in nondiabetic offspring of diabetic individuals and is correlated with the metabolic syndrome (27). Finally, expression of both IL-1ß and its receptor is increased in visceral adipose tissue of obese subjects (28).
However, whether and how this overproduction of IL-1ß could alter the metabolic function of insulin in adipocytes remains unclear. In the present study, we found that IL-1ß markedly inhibits insulin-induced glucose transport in adipocytes by decreasing IRS-1 expression. Furthermore, we demonstrate that activation of the ERK pathway is involved in the inhibitory action of IL-1ß on insulin signaling.
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Materials and Methods |
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Antibodies against IRS-2 and phosphotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal IRS-1 antibody used in immunoprecipitation experiments was raised against a peptide corresponding to the last 14 amino acids of IRS-1 (Eurogentec, Seraing, Belgium). Monoclonal anti-IRS-1 antibody used in immunoblotting experiments was purchased from BD Biosciences (PharMingen, San Diego, CA). Antibodies against the ß-subunit of insulin receptor, Glut 4, Glut 1, and inhibitor-B (IB) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-PKB (Thr308), PKB, phospho-ERK, ERK, phospho-JNK1/2, JNK1/2, phospho-p38MAPK, and p38MAPK were purchased from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated and fluorescein isothiocyanate (FITC)-coupled secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Animals
ob/ob, db/db mice and their lean control littermates were purchased from Charles River Laboratories (St. Aubin les Elbeuf, France) and housed at the animal facility of the Faculty of Medicine (Nice, France). Mice were maintained on a 12-h light, 12-h dark cycle and provided free access to water and standard rodent show. Mice were killed by cervical dislocation, and epididymal fat pads were removed and freeze clamped in liquid nitrogen. Principles of laboratory animals care were followed and the Ethical Committee of the Faculty of Medicine approved the animal experiments.
Cells culture
3T3-L1 fibroblasts were grown at 7% CO2 and 37 C in 35- or 100-mm dishes in DMEM, 25 mM glucose, and 10% calf serum and induced to differentiate in adipocytes as previously described (13). Briefly, 2 d after confluence, medium was changed for DMEM, 25 mM glucose, and 10% FCS supplemented with isobutylmethylxanthine (0.25 mM), dexamethasone (0.25 µM), insulin (5 µg/ml), and pioglitazone (10 µM). The medium was removed after 2 d and replaced with DMEM, 25 mM glucose, and 10% FCS supplemented with insulin (5 µg/ml) and pioglitazone (10 µM) for 2 d. Then the cells were fed every 2 d with DMEM, 25 mM glucose, and 10% FCS. 3T3-L1 adipocytes were used 8–15 d after the beginning of the differentiation protocol.
Human preadipocytes (Biopredic, Rennes, France) were grown at 5% CO2 and 37 C in 12-well collagen-coated plates in DMEM Ham’s F12 containing 15 mM HEPES, 2 mM L-glutamine, 5% FCS, 1% antimycotic solution, ECGS/H-2, hEGF-5, and HC-500 from supplement pack preadipocyte growth medium (Promocell, Heidelberg, Germany). Differentiation in adipocytes was induced after confluence by changing the medium for DMEM Ham’s F12 15 mM HEPES, 2 mM L-glutamine, and 3% FCS supplemented with biotin (33 µM), insulin (100 nM), pantothenate (17 µM), isobutylmethylxanthine (0.2 mM), dexamethasone (1 µM), and rosiglitazone (10 µM). The medium was removed after 3 d and replaced with Ham’s F12 containing 15 mM HEPES, 2 mM L-glutamine, and 10% FCS supplemented with biotin (33 µM), insulin (100 nM), pantothenate (17 µM), and dexamethasone (1 µM). Then the cells were fed every 2 d with the same medium. Human adipocytes were used 15 d after the beginning of the differentiation protocol.
Immunoprecipitation and immunoblotting
3T3-L1 adipocytes were treated as indicated in figure legends for different periods of time at 37 C, 7% CO2 in DMEM 25 mM glucose, 10% FCS. The cells were washed with ice-cold buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 10 mM pyrophosphate, 2 mM sodium orthovanadate] before solubilization for 1 h at 4 C in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 10 mM pyrophosphate, 2 mM sodium orthovanadate, 100 nM okadaic acid, protease inhibitors, and 1% Triton X-100 (vol/vol)]. After centrifugation at 14,000 x g for 10 min at 4 C, the supernatant (cells lysates) was incubated for 4 h at 4 C with antibody of interest (5–10 µg/sample) preadsorbed on protein-A-Sepharose beads. The beads were washed three times with the lysis buffer and boiled for 5 min in Laemmli buffer. The proteins were separated by SDS-PAGE using a 7.5 or 10% resolving gel. Proteins were transferred to polyvinylidene difluoride membrane, and the membrane was blocked with saline buffer [10 mM Tris (pH 7.4), 320 mM NaCl, 0.1% Tween 20] containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature and incubated overnight at 4 C with the indicated antibody. After incubation with horseradish-peroxydase conjugated secondary antibodies, proteins were detected by enhanced chemiluminescence. Some membranes were subsequently incubated at 55 C for 30 min in stripping buffer [62 mM Tris (pH 6.7), 100 mM 2-mercaptoethanol, and 2% sodium dodecyl sulfate] and reprobed with the indicated antibody.
Glucose transport
3T3-L1 adipocytes were incubated or not with IL-1ß at 20 ng/ml for 48 h as indicated in the figure legends. Cells were then washed twice with Krebs-Ringer phosphate buffer [10 mM phosphate buffer (pH 7.4), 1.25 mM MgSO4, 1.25 mM CaCl2, 136 mM NaCl, 4.7 mM KCl] and incubated without or with 0.5 or 100 nM insulin for 20 min in Krebs-Ringer phosphate buffer supplemented with 0.2% BSA. Glucose transport was determined by the addition of 2-[3H]deoxyglucose (0.1 mM, 0.5 µCi/ml) as described previously (29). The reaction was stopped after 3 min at 37 C by washing the cells four times with ice-cold PBS. Cells were lysed in lysis buffer, and glucose uptake was assessed by scintillation counting. Results were normalized for protein content measured by BCA assay.
Real-time quantitative PCR
Total RNAs from 3T3-L1 adipocytes or white adipose tissues were prepared using Trizol reagent (Life Technologies Inc., Paisley, UK). The integrity of the RNA was confirmed by electrophoresis in ethidium bromide containing agarose gels, and the RNA concentration was determined spectrophotometrically. cDNA was synthesized using Moloney murine leukemia virus transcriptase (Invitrogen, Carlsbad, CA) from 1 µg of total RNA. PCRs were performed using an AbiPrism 7000 sequence detection system instrument and software (Applied Biosystems, Foster City, CA). The PCR conditions were: 2 min at 50 C, 10 min at 95 C, followed by 40 cycles of a two-step PCR denaturation at 95 C for 15 sec and annealing extension at 60 C for 60 sec. Each sample contained 0.5–5.0 ng cDNA in 1x SYBRGreen PCR master mix (Eurogentec) and 200 or 400 nM of each primer (Invitrogen) in a final volume of 25 µl. A control without cDNA was performed for each experiment. The number of cycles required for the fluorescence to reach a threshold limit was determined in duplicate for each sample. For each target an efficiency of the PCR method between 95 and 100%, a reproducibility of cycle threshold values with a SE less than 3% and a linear range of covering more at least 7 log units were obtained. To exclude the contamination of nonspecific PCR products such as primer dimers, melting curve analysis was applied to all final PCR products after the cycling protocols. The relative amounts of the different mRNAs were quantified by using the second derivative maximum method. 36B4 was used as an invariant control, and the relative quantification for a given gene was corrected to 36B4 mRNA values. The results were expressed relative to the control condition, which was arbitrarily assigned a value of 1.
Primers used were: IRS-1 (sense) 5'-GTGAACCTCAGTCCCAACCATAAC-3', (antisense) 5'-CCGGCACCCTTGAGTGTCT-3'; IRS-2 (sense) 5'-TCCCACATCGGGCTTGAA-3', (antisense) 5'-CTGCACGGATGACCTTAGCA-3'; peroxisome proliferator-activated receptor (PPAR)-
(sense) 5'-CTGTTTTATGCTGTTATGGGTGAAA-3', (antisense) 5'-CGACCATGCTCTGGGTCAA-3; CCAAT/enhancer binding protein (C/EBP)- (sense) 5'-GACCATTAGCCTTGTGTGTACTGTATG-3', (antisense) 5'-TGGATCGATTGTGCTTCAAGTT-3'; 36B4 (sense) 5'-TCCAGGCTTTGGGCATCA-3', (antisense) 5'-CTTTATCAGCTGCACATCACTCAGA-3.
Preparation of plasma membrane sheets and immunofluorescence labeling
3T3-L1 cells were grown on glass coverslips and differentiated into adipocytes as described above. Cells were treated without or with IL-1ß at 20 ng/ml for 48 h and stimulated or not with insulin (100 nM) for 20 min. Plasma membrane sheets were prepared as previously described (30). Cells were washed twice with ice-cold PBS and fixed with 0.55 mg/ml poly-L-lysine for 1 min at 4 C and then swollen by three successive rinses with an hypotonic buffer [30 mM HEPES (pH 7.5), 70 mM KCl, 5 mM MgCl2, 3 mM EGTA]. The swollen cells were sonicated in hypotonic buffer containing 1 mM dithiothreitol and proteases inhibitors, and the bound membrane sheets were fixed with 4% paraformaldehyde and blocked with PBS containing 1% BSA and 4% calf serum. Plasma membrane sheets were then incubated with anti-Glut 4 or anti-Glut 1 antibodies (5 µg/ml in blocking buffer) for 1 h at room temperature and washed 3 x 10 min with blocking buffer. After washes, lawns were incubated for 1 h at room temperature with FITC-conjugated antigoat antibodies and WGA-Texas Red to normalize and then rinsed out with three 10-min washes with blocking buffer. The coverslips were mounted in Mowiol onto glass slides. Plasma membrane sheets were analyzed with an Axiovert 200 microscope using a Plan-Neofluar x40 1.3 numeral aperture oil objective (Carl Zeiss, Göttingen, Germany). Images were acquired using a cooled digital camera (Coolsnap HQ; Roper Scientific Princeton Instruments, Evry, France), and quantification was made using Metamorph image analysis software (Universal Imaging Corp., Downington, PA) with autothreshold detection of pixels as previously described (30).
Statistical analysis
Statistical analysis was performed by Student’s t test or Mann-Whitney test. P < 0.05 was considered significant.
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B and the phosphorylation of the MAPKs, ERK, JNK, and p38MAPK (Fig. 1A
). These data indicated that 3T3-L1 adipocytes are responsive to IL-1ß stimulation.
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, upper panel). Importantly, IL-1ß treatment of 3T3-L1 adipocytes for 48 h reduced the absolute response at both insulin concentrations (Fig. 1B, lower panel). Thus, the increment in glucose uptake at 0.5 nM insulin was 3.91 ± 0.21 and 1.71 ± 0.12 nmol.mg–1. 3 min–1 for control or IL-1ß-treated cells, respectively, and at 100 nM insulin, this increment was 6.90 ± 0.48 and 4.01 ± 0.42 nmol.mg–1. 3 min–1 for the same conditions. Furthermore, 0.5 nM insulin induced 56.6 ± 0.8 vs. 42.6 ± 0.4% of the maximal insulin effect in the absence or presence of IL-1ß, respectively, indicating that the insulin sensitivity was decreased.
Effect of IL-1ß on glucose transporters expression and insulin-induced glucose transporters translocation
We first examined whether the inhibitory effect of IL-1ß on glucose uptake was due to a change in glucose transporters expression. 3T3-L1 adipocytes were treated without or with IL-1ß for 48 h, and the amount of Glut 1 and Glut 4 in cell lysate was quantified by immunoblotting with specific antibodies. As shown in Fig. 2A, IL-1ß treatment increased the amount of Glut 1 and slightly decreased Glut 4 expression but did not modify the differentiation state of adipocyte because C/EBP, PPAR, and adipocyte lipid binding protein 2 mRNAs expression was not altered (Fig. 2B). The increased Glut 1 expression induced by IL-1ß was associated with an increase in the amount of the transporter at the plasma membrane (Fig. 2C, left panel) and could explain the enhancement of glucose uptake induced by IL-1ß alone. Importantly, prolonged IL-1ß treatment did not modify the basal amount of Glut 4 at the plasma membrane but inhibited by 50% the amount of Glut 4 at the plasma membrane after insulin stimulation (Fig. 2C, right panel). These results indicated that IL-1ß can trigger an insulin-resistant state for glucose transport by altering insulin-induced Glut 4 translocation.
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). We then determined whether IL-1ß could alter the insulin-induced IRS tyrosine phosphorylation. IRS-1 or IRS-2 was immunoprecipitated from 3T3-L1 adipocytes with specific antibodies, and their tyrosine phosphorylation was quantified by immunoblotting with antiphosphotyrosine antibody. As shown in Fig. 3, IL-1ß alone did not induce IRS-1 or IRS-2 tyrosine phosphorylation. Importantly, IL-1ß inhibited insulin-induced IRS-1 tyrosine phosphorylation by 60% but did not modify insulin-induced IRS-2 tyrosine phosphorylation. In parallel to the decrease in IRS-1 tyrosine phosphorylation, less IRS-1 was immunoprecipitated in IL-1ß-treated cells, suggesting that IL-1ß could alter IRS-1 expression. To directly assess this possibility, we analyzed IRS-1 expression in homogenates prepared from 3T3-L1 adipocytes treated for 24 or 48 h with IL-1ß. IL-1ß decreased IRS-1 protein expression by 50 and 60% at 24 and 48 h, respectively (Fig. 4A), whereas IRS-2 amount was unchanged (data not shown).
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, IL-1ß markedly reduced the IRS-1 amount in human adipocytes, suggesting that IL-1ß could participate in the negative regulation of IRS-1 in adipocytes of obese subjects.
We then found, by using real-time PCR, that IL-1ß treatment decreased IRS-1 mRNA amount by 40% (Fig 4B) indicating that the decrease in IRS-1 protein was partly linked to a reduced gene expression. In agreement with these in vitro findings, IL-1ß mRNA expression was increased in two models of obese mice and IRS-1 mRNA was decreased, whereas the IRS-2 mRNA level was not markedly reduced (Fig. 5).
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IL-1ß is more potent than IL-6 to inhibit IRS-1 expression
Because IL-1ß increases IL-6 production in fat cells (31) and because IL-6 was shown to down-regulate IRS-1 expression (20), we compared the ability of IL-1ß and IL-6 to regulate IRS-1 expression. Treatment of 3T3-L1 adipocytes with IL-6 (20 ng/ml) for 24 h slightly decreased IRS-1 amount, whereas in the same conditions, IL-1ß induced a 50% inhibition in IRS-1 expression (Fig. 4C). These data indicate that IL-1ß is more potent than IL-6 to down-regulate IRS-1 expression, and from these data, it is unlikely that the observed effect on IRS-1 expression is mediated by an increase in IL-6 production.
Insulin-induced PKB activation and AS160 phosphorylation are reduced in IL-1ß-treated 3T3-L1 adipocytes
Activation of PKB after tyrosine phosphorylation of IRS-1 or IRS-2 is a critical step for insulin-induced glucose transport and Glut 4 translocation (7). Because IL-1ß differentially regulated the expression and the tyrosine phosphorylation of these proteins, we aimed at determining the effect of IL-1ß treatment on insulin-induced PKB activation. PKB activation was monitored by immunoblotting with a phosphospecific antibody against threonine 308 located in the activation loop of PKB, phosphorylation that correlates with PKB activation. Importantly, after IL-1ß treatment, the insulin-induced phosphorylation of PKB was reduced by 50% without any change in the total amount of PKB (Fig. 6). We then assessed the effect of IL-1ß on phosphorylation of the PKB substrate AS160 (Akt substrate of 160 kDa). Indeed, PKB-induced phosphorylation of AS160, a protein containing a Rab GTPase activating protein domain promotes Glut 4 translocation to the plasma membrane (32, 33). Using a phosphospecific antibody against the PKB phosphorylation site on AS160, we found that IL-1ß treatment markedly reduced insulin-induced AS160 phosphorylation (Fig. 6). These data confirmed that PKB activity was reduced after IL-1ß treatment, and the reduced AS160 phosphorylation may provide a mechanism for IL-1ß-induced insulin resistance on Glut 4 translocation and glucose uptake.
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), whereas phosphorylated JNK was not detected (data not shown). For directly assessing the role of ERK and p38MAPK in IL-1ß-induced down-regulation of IRS-1 expression and PKB activation, 3T3-L1 adipocytes were treated with 10 µM U0126 or PD169316, potent inhibitors of ERK or p38MAPK pathways, respectively, and incubated without or with IL-1ß for 48 h. Incubation with PD169316 did not prevent the down-regulation of IRS-1 expression induced by IL-1ß (data not shown). By contrast, U0126 that totally inhibited ERK activation (Fig. 8A) fully prevented the decrease in IRS-1 mRNA amount induced by IL-1ß treatment (Fig. 8B) but only partially prevented the decrease in IRS-1 protein expression (Fig. 8C). These data indicate that ERK activation plays a major role in the negative regulation of IRS-1 expression induced by IL-1ß. To determine whether insulin-induced PKB activation could be rescued by blocking ERK pathway, adipocytes were treated as described above and stimulated without or with insulin for 5 min. Inhibition of ERK with U0126 partially overcame the inhibitory effect of IL-1ß (Fig. 9, A and B) on PKB and AS160 phosphorylation. Furthermore, inhibition of ERK with U0126 also partially prevented the inhibitory effect of IL-1ß on insulin-induced Glut 4 translocation to the plasma membrane (Fig. 9C). These data are in favor of the importance of ERK activation by IL-1ß in the inhibition of insulin signaling and Glut 4 translocation.
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Discussion |
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and IL-6 in insulin resistance is well documented, the potential role of IL-1ß in the alteration of insulin signaling and metabolic effects is poorly documented.
In the present study, no acute inhibitory effect of IL-1ß was observed on insulin-induced glucose uptake or on insulin signaling in accordance with a recent report with IL-1 (34). By contrast, in the ß-pancreatic cell line RINm5F, IL-1ß was shown to rapidly induce SOCS-3 expression that evoked a decrease in insulin receptor phosphorylation and IRS phosphorylation (35). Moreover, the effect of IL-1ß is clearly different from the TNF- effect that promotes a rapid inhibition of IRS-1 tyrosine phosphorylation through its serine phosphorylation (11, 36).
We found that prolonged IL-1ß treatment induces an inhibition of insulin effect on glucose uptake as also recently published (37) during the revision process of this manuscript. A sustained increase in the expression of IL-1ß in adipose tissue during obesity could thus participate in the development of the insulin resistance. This inhibitory effect was mainly due to the down-regulation of the expression of IRS-1 and to a lesser degree Glut 4. TNF- and IL-6 have also been shown to negatively regulate the expression of these two proteins (20, 21, 22). However, whereas TNF- markedly suppressed Glut 4 expression (20), we found that IL-1ß had only a modest effect. Furthermore, upon prolonged exposure to TNF-, mature adipocytes lose their terminally differentiated phenotype, whereas we found no effect of IL-1ß on PPAR or C/EBP mRNAs expression in our experimental conditions, indicating that IL-1ß did not induce a change in the differentiation state of the 3T3-L1 adipocytes. In agreement with these findings, it was recently reported that treatment of mature 3T3-L1 adipocytes with IL-1ß for 6 d did not modify their differentiation state (37), whereas addition of the cytokine during the differentiation process markedly altered the adipocyte phenotype (37, 38).
IL-1ß increases the expression of IL-6 in fat cells (31), but it seems unlikely that the inhibition of IRS-1 expression was totally mediated by IL-6. Indeed, we found that a high level of IL-6 modestly decreased IRS-1 expression, compared with IL-1ß. By contrast, IL-6 was more potent than IL-1ß to decrease Glut 4 and PPAR expression (20, 21), suggesting that IL-1ß could regulate IRS-1 expression independently of its effect on IL-6 expression.
Whereas the IRS-1 amount was decreased after IL-1ß treatment, the expression of IRS-2 was unchanged. Despite the normal IRS-2 amount and tyrosine phosphorylation, we found that insulin-induced PKB activation was markedly altered, leading to a decreased phosphorylation of its substrate AS160, a Rab GTPase-activating protein recently described to play a role in insulin-stimulated Glut 4 translocation (32, 33). These data support that IRS-1 rather IRS-2 is involved in insulin-induced glucose uptake. In agreement with this finding, previous studies have shown that insulin-induced glucose uptake is altered in adipocytes from IRS-1-deficient mice (39). Furthermore, in L6 myotube, knockdown of IRS-1 by small interfering RNA strategy markedly impaired glucose transport, and knockdown of IRS-2 was without effect (40).
Elevated activities of the MAPKs, ERK, JNK, and p38MAPK, are found in adipocytes or muscles of obese and insulin-resistant rodent and humans (8), and IL-1ß is known to activate these protein kinases (25). We found that prolonged IL-1ß treatment induced a sustained activation of ERK and p38MAPK but not JNK, suggesting that an increase in IL-1ß expression in adipose tissue could thus participate in the elevated activity of these kinases in obesity. Such activation of these kinases could be involved in the inhibitory effect of IL-1ß on insulin-induced glucose transport. Indeed, activation of p38MAPK was shown to be involved in the down-regulation of Glut 4 expression (41). Thus, the small decrease in Glut 4 amount in IL-1ß-treated cells could be due to the sustained activation of p38MAPK. On the other hand, we found that pharmacological inhibition of ERK pathway totally prevented the decrease in IRS-1 mRNA and partially prevented the inhibition of IRS-1 protein expression induced by IL-1ß treatment. In parallel, the insulin-induced activation of PKB, phosphorylation of AS160, and Glut 4 translocation were improved. These results indicate that activation of ERK pathway by IL-1ß negatively regulates IRS-1 mRNA transcription and underscore an important role of ERK pathway activation in IL-1ß-induced down-regulation of the IRS-1/PI 3-kinase/PKB signaling pathway necessary for insulin-induced Glut 4 translocation and glucose transport. A negative cross talk between these two pathways was evidenced by different studies. For example, activation of ERK through expression of constitutively active MEK inhibits IRS-1 expression to a greater extent than activation of p38MAPK or JNK (42). Other studies, using pharmacological inhibitors, demonstrated that ERK activation altered IRS-1 function by promoting its serine phosphorylation and that inhibition of ERK pathway could improve insulin resistance (13, 14, 15, 43, 44, 45). In agreement with an important role of ERK in insulin resistance, a recent report demonstrated that ERK-1-deficient mice are more sensitive to insulin on a high-fat diet regimen (46). Thus, ERK activation in response to different stimuli could be one important event to impair normal insulin metabolic effect by altering IRS-1 function.
The fact that ERK inhibition only partially prevents the decrease in IRS-1 expression induced by IL-1ß indicates that another ERK-independent pathway is involved at a posttranscriptional level to regulate IRS-1 protein amount. Such a mechanism could be a regulated degradation of IRS-1 that has been shown to be a long term inhibitory mechanism involved in insulin resistance (8). For example, prolonged insulin treatment reduces the level of IRS-1 through proteasome-dependent process (47, 48), whereas osmotic (49) or oxidative (50) stresses induce IRS-1 degradation through a proteasome-independent pathway. Some studies have proposed that SOCS-3 proteins by binding to IRS proteins could promote their ubiquitin-mediated degradation (18), but this finding is controversial (51). We found that IL-1ß was able to induce SOCS-3 in 3T3-L1 adipocytes (data not shown) as previously shown in some other cell types (35, 52). Thus, it is plausible that SOCS-3 is involved, at a posttranscriptional level, in IL-1ß-induced down-regulation of IRS-1 protein amount in adipocytes.
In conclusion, our data show that IL-1ß decreases insulin-induced glucose transport in adipocytes mainly by inhibiting IRS-1 expression through a reduction in IRS-1 mRNA amount that is dependent on ERK pathway activation and by a posttranscriptional mechanism that is independent of ERK. Thus, IL-1ß that is produced by resident macrophages in adipose tissue could act in synergy with TNF- and IL-6 to impair adipocytes biology that could be an important event for the development of the insulin resistance. Indeed, an attenuation of insulin signaling in adipocytes can impair the lipid buffering capacity of the adipocytes that could favor accumulation of lipids in muscle and liver with deleterious effects on insulin action. Moreover, modification of glucose transport in adipocytes could alter the secretory function of adipocyte in a way that is detrimental to insulin action in muscle and liver.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 12, 2006
Abbreviations: AS160, Akt substrate of 160 kDa; C/EBP, CCAAT/enhancer binding protein; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; IB, inhibitor-B; IRS, insulin receptor substrate; JNK, c-Jun NH2-terminal kinase; PKB, protein kinase B; PI 3-kinase, phosphoinositide 3-kinase; PPAR, peroxisome proliferator-activated receptor; SOCS, suppressor of cytokine signaling.
Received May 23, 2006.
Accepted for publication October 5, 2006.
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S. Weise, S. Kralisch, G. Sommer, U. Lossner, M. Bluher, M. Stumvoll, and M. Fasshauer Tissue inhibitor of metalloproteinase-1 mRNA production and protein secretion are induced by interleukin-1{beta} in 3T3-L1 adipocytes J. Endocrinol., July 1, 2008; 198(1): 169 - 174. [Abstract] [Full Text] [PDF]
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M. Li, D. H. Kim, P. L. Tsenovoy, S. J. Peterson, R. Rezzani, L. F. Rodella, W. S. Aronow, S. Ikehara, and N. G. Abraham Treatment of Obese Diabetic Mice With a Heme Oxygenase Inducer Reduces Visceral and Subcutaneous Adiposity, Increases Adiponectin Levels, and Improves Insulin Sensitivity and Glucose Tolerance Diabetes, June 1, 2008; 57(6): 1526 - 1535. [Abstract] [Full Text] [PDF]
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 F. W. Kiefer, M. Zeyda, J. Todoric, J. Huber, R. Geyeregger, T. Weichhart, O. Aszmann, B. Ludvik, G. R. Silberhumer, G. Prager, et al. Osteopontin Expression in Human and Murine Obesity: Extensive Local Up-Regulation in Adipose Tissue but Minimal Systemic Alterations Endocrinology, March 1, 2008; 149(3): 1350 - 1357. [Abstract] [Full Text] [PDF]
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E. Park, V. Wong, X. Guan, A. I Oprescu, and A. Giacca Salicylate prevents hepatic insulin resistance caused by short-term elevation of free fatty acids in vivo J. Endocrinol., November 1, 2007; 195(2): 323 - 331. [Abstract] [Full Text] [PDF]
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 J. Shen, D. K. Arnett, J. M. Peacock, L. D. Parnell, A. Kraja, J. E. Hixson, M. Y. Tsai, C.-Q. Lai, E. K. Kabagambe, R. J. Straka, et al. Interleukin1{beta} Genetic Polymorphisms Interact with Polyunsaturated Fatty Acids to Modulate Risk of the Metabolic Syndrome J. Nutr., August 1, 2007; 137(8): 1846 - 1851. [Abstract] [Full Text] [PDF]
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 M. Sanchez-Alavez, I. Klein, S. E. Brownell, I. V. Tabarean, C. N. Davis, B. Conti, and T. Bartfai Night eating and obesity in the EP3R-deficient mouse PNAS, February 20, 2007; 104(8): 3009 - 3014. [Abstract] [Full Text] [PDF]
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