Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion

Chronic interleukin-6(IL-6)treatment increased IL-6secretion and induced insulin resistance in adipocyte:prevention by rosiglitazoneClaire Lagathu,Jean-Philippe Bastard,Martine Auclair,Mustapha Maachi,Jacqueline Capeau,and Martine Caron *INSERM U.402and IFR65Saint-Antoine Faculty of Medicine,Paris,FranceReceived 12September 2003AbstractIL-6has emerged as an important cytokine upregulated in states of insulin resistance such as type 2diabetes.We evaluated the chronic effect of IL-6on insulin signaling in 3T3-F442A and 3T3-L1adipocytes.First,cells responded to a chronic treatment with IL-6by initiating an autoactivation process that increased IL-6secretion.Second,IL-6-treated adipocytes showed a decreased protein expression of IR-b subunit and IRS-1but also an inhibition of the insulin-induced activation of IR-b ,Akt/PKB,and ERK1/2.Moreover,IL-6suppressed the insulin-induced lipogenesis and glucose transport consistent with a diminished expression of GLUT4.IL-6-treated adipocytes failed to maintain their adipocyte phenotype as shown by the downregulation of the adipogenic markers FAS,GAPDH,aP2,PPAR-c ,and C/EBP-a .IL-6also induced the expression of SOCS-3,a potential inhibitor of insulin signaling.Finally,the effects of IL-6could be prevented by rosiglitazone,an insulin-sensitizing agent.Thus,IL-6may play an important role in the set-up of insulin resistance in adipose cell.Ó2003Elsevier Inc.All rights reserved.Keywords:Interleukin-6;Insulin resistance;Adipocyte;Thiazolidinedione;CytokineInsulin resistance is a prominent feature of the met-abolic syndrome and type 2diabetes and constitutes a major risk for cardiovascular disease [1].In recent years,evidence has been gathered that type 2diabetes is as-sociated with a cytokine-related acute-phase reaction,part of an overall inflammatory state.Indeed,insulin resistance is correlated with increased blood concentra-tions of several markers of the acute-phase response,including C-reactive protein,the major cytokine medi-ator interleukin-6(IL-6)[2–4],tumor necrosis factor alpha (TNF-a )[5],and interleukin-1-beta (IL-1-b )[6].The blood concentration of IL-6is elevated in subjects with type 2diabetes [2]and obesity [7,8],and a positive correlation was observed between adipose tissue IL-6content and insulin resistance in vivo [7,8].Furthermore,insulin-resistant patients have a high level of soluble IL-6receptor which potentiates IL-6bioactivity,and signsof innate immunity activation are often present before type 2diabetes [9].Studies have indicated that 10–35%of circulating IL-6is derived from adipose tissue [8,10],a production that increases with adiposity [10].Although immune cells,fibroblasts,endothelial cells,and monocytes have traditionally been regarded as the major sources of cir-culating IL-6[8],it has been clearly established that adipocytes themselves can secrete IL-6[8]together with IL-1-b [11]and TNF a [12].TNF a has been demon-strated to negatively interfere with adipocyte metabo-lism at numerous sites including glucose and fatty acid metabolism and insulin receptor signaling [13].Only a few studies have assessed the effect of IL-6on the adi-pocyte metabolism [14].Preliminary studies suggested that a short term treatment with IL-6could affect the adipocyte lipid metabolism [15,16].However,no data are available concerning a long-term treatment with IL-6on insulin signaling in adipocytes.IL-6signal transduction involves the JAK (janus-ac-tivated kinase)/STAT (signal transducers and activators*Corresponding author.Fax:+33-1-40-01-13-52.E-mail address:caron@st-antoine.inserm.fr (M.Caron).0006-291X/$-see front matter Ó2003Elsevier Inc.All rights reserved.doi:10.1016/j.bbrc.2003.10.013Biochemical and Biophysical Research Communications 311(2003)372–379BBRC/locate/ybbrcof transcription)signaling pathway[17]that could cross-react in many points with the insulin signaling network [18].Suppressor of cytokine signaling(SOCS)proteins were originally described as cytokine-induced molecules involved in negative feedback loop of cytokine[19]and insulin signaling[20].Indeed,SOCS-3can directly inter-act with IR-b and IRS-1and,when overexpressed in adipocytes,inhibit insulin signal transduction[18]. Moreover,SOCS-3is upregulated in adipose tissue of obese mice[20]and IL-6was identified to play a role in insulin resistance in hepatocytes by acting at the insulin signaling pathways through induction of SOCS-3protein expression[21,22].Rosiglitazone belongs to the thiazolidinedione (TZDs)class of antidiabetic drugs that improve meta-bolic control in patients with type2diabetes through the improvement of insulin sensitivity[23].TZDs exert their antidiabetic effects through a mechanism that involves activation of peroxisome proliferator-activated receptor (PPAR cÞ[24].Although PPAR c is predominantly expressed in adipose tissue,the TZDs reduce insulin resistance in adipose tissue,muscle,and the liver.There are still many unknowns about the mechanism of action of TZDs in insulin resistance.Potential signaling factors include free fatty acids and adipocyte-derived cytokines such as TNF a or IL-6,which are overexpressed in obesity and insulin resistance[23].In the current study,we investigated the effect of chronic IL-6treatment(100and200ng/mL,8days)on several functions of cultured3T3-F442A and3T3-L1 adipocytes.We show that adipocytes are target cells for IL-6.IL-6upregulated its own secretion and expression. Furthermore,IL-6induced insulin resistance by inhib-iting the acute phosphorylation of the insulin signaling components(IR-b,IRS-1,ERK1/2,Akt/PKB)and a series of insulin-dependant processes(lipogenesis, glucose transport).IL-6-treated adipocytes failed to maintain their adipocyte phenotype as shown by the downregulation of adipogenic markers(FAS,GAPDH, aP2,PPAR-c,and C/EBP-aÞ.IL-6also increased by 6-fold the expression of SOCS-3,a potential negative regulator of insulin signaling pathway[18].Finally we found that the insulin-sensitizing agent rosiglitazone decreased IL-6secretion and SOCS-3hyperexpression and prevented IL-6adverse effects on insulin signaling. Materials and methodsMaterials.Recombinant human IL-6(rhIL-6)(specific activity, 1Â107U/mg)was provided by PeproTech(NJ,USA)and rosiglitaz-one(BRL49653)was provided by SmithKline Beecham Pharmaceu-ticals.Cell culture and treatment.3T3-F442A preadipocytes were cultured and induced to differentiate as previously described[25].In brief,the preadipocytes were maintained in DMEM containing25mM glucose and10%new born calf serum(NCS)until they reached confluence (day0).They were then grown in DMEM containing10%FCS and 1l M insulin for six days,when more than92%of the cells showed fat accumulation.3T3-L1preadipocytes were subcultured in DMEM containing10%FCS.At confluence(day0),preadipocytes were grown for two days in DMEM containing10%FCS,1l M insulin,0.5mM isobutylmethylxanthine,and1l M dexamethasone and then cultured in DMEM containing10%FCS and1l M insulin.After seven days, more than90%of the cells showed fat accumulation.In some exper-iments,1l M rosiglitazone was added at day0.The effect of IL-6(100 and200ng/mL)was tested on differentiating3T3-F442A cells(from days0–8of differentiation)and on mature3T3-L1adipocytes(from days7–15of differentiation).IL-6measurements.Murine IL-6(mIL-6)concentration was de-termined at day8of differentiation in24h-secretion medium of3T3-F442A cells by the sandwich ELISA Quantikine Mouse test(R&D Systems,USA,MN).This test specifically identifies murine IL-6(de-tection limit:3.1pg/mL)without cross-reaction with the rhIL-6used for cell treatment.Western blotting.Cells were solubilized in a Laemmli buffer with 100mM dithiothreitol.Cell lysates(104cells)were subjected to SDS–PAGE and Western blotting with the indicated antibody:SREBP-1 (K-10),C/EBP a(C-19),PPAR c(H-100),C/EBP b(C-19),and GLUT4 (H-61)(Santa Cruz Biotechnology).Immune complexes were visual-ized with a chemiluminescence method(Enhanced Chemiluminescent kit,Amersham Biosciences).Protein expression on Western blot was normalized on the basis of cell number.RNA preparation and real-time RT-PCR.Total RNA was isolated with the RNeasy kit(Qiagen,USA,CA).Complementary-DNA was synthetized using random hexamers and AMV reverse transcriptase (Promega Biosciences,USA,CA).Real-time PCR was performed with the LightCycler system(Roche Molecular Biochemicals).PCRs were performed using the LightCycler SYBR greenfluorophore.The fol-lowing primers were used:aP2,50-AAC ACC GAG ATT TCC TT-30 and50-ACA CAT TCC ACC ACC AG-30;FAS,50-TGC TCC CAG CTG CAG GC-30and50-GCC CGG TAG CTC TGG GTG TA-30; GAPDH,50-CAA GGT CAT CCA TGA CAA CTT-30and50-GGC CAT CCA CAG TCT TCT GG-30;IL-6,50-CTG CAA GAG ACT TCC ATC CAG TT-30and50-GAA GTA GGG AAG GCC TGG-30; SOCS3,50-ACC AGC GCC ACT TCT TCA CG-30and50-GTG GAG CAT CAT ACT GAT CC-30;18S ribosomal RNA,and50-GAG CGA AAG CAT TTG CCA AG-30and50-GGC ATC GTT TAT GGT CGG AA-30.Each sample was normalized on the basis of its RNA18S content.Insulin signaling.The effect of insulin was evaluated on tyrosine phosphorylation of the insulin receptor b-subunit(IR-bÞand IR sub-strate-1(IRS-1),and on the activation of extracellular signal-regulated kinase1/2(ERK-1/2)and protein kinase B(Akt/PKB).At day8,the cells were starved for18h and stimulated for10min by insulin (100nM).Aliquots of cell lysates were immunoblotted with an anti-phosphotyrosine antibody(PY-99,Santa Cruz Biotechnology)or antibodies directed against the activated forms of ERK-1/2(Phospho-ERK(E-4),Santa Cruz Biotechnology)or Akt/PKB(phospho-Akt-ser473,#9271,Cell Signaling Technology).Protein expression was checked by using antibodies directed against IR-b(C-19),IRS-1(C-20),ERK-1/2(C-16)(Santa Cruz Biotechnology)or Akt/PKB(#9272, Cell Signaling Technology).Lipogenesis.Lipogenesis was studied at day8of differentiation in 3T3-F442A adipocytes cultured for24h in serum-free medium.Insulin (100nM)was added for30min.After a further30-min incubation with 0.5l Ci/well of[U-14C]glucose(302mCi/mmol,Amersham Bio-sciences),the cells were beled lipids were extracted with chloroform/methanol(1/2).After30min on ice,the inferior phase was collected,dried by evaporation,and counted in scintillationfluid.The results were expressed as percentÆSEM of above basal value.Glucose transport.Glucose transport was studied on day8of dif-ferentiation in3T3-F442A adipocytes starved for24h.Prior to ex-periment cells were incubated for2h in500l l of glucose transportgathu et al./Biochemical and Biophysical Research Communications311(2003)372–379373solution(pH7.6)containing HEPES12.5mM,NaCl120mM,KCL 5mM,MgSO41.2mM,CaCl21mM,NaHPO41mM,sodium pyru-vate2mM,and BSA2%.Insulin(100nM)and2-DOG mix(0.25l Ci 2-deoxy-D-[1-14C]glucose(60mCi/mmol,Amersham Biosciences)and 0.25mM2-DOG)were successively added for30and5min.To stop transport,cells were washed with PBS,lysed for30min with0.1% SDS,and counted.The results were expressed as percentÆSEM of above basal value.Statistical analysis.Results are meansÆSEM of the indicated number of independently performed experiments.Statistical signifi-cance was determined using the nonparametric Mann–Whitney U test results.The threshold for significance was at P¼0:05.ResultsIL-6activated a regulatory loop in cultured adipocytes resulting in its autocrine/paracrine secretionInitial studies focused on the ability of hrIL-6-treated adipocytes(8days)to secrete murine IL-6.We observed that at concentrations(100and200ng/mL)close to those found in the serum of obese patients[8,10],hrIL-6 induced secretion of IL-6(Fig.1A).IL-6effect was dose-dependent and observed also on IL-6mRNA expression (Fig.1B).IL-6altered insulin signaling in3T3-F442A cells We evaluated the chronic effect of IL-6(8days:from day0to day8)on the insulin signal transduction.As shown in Fig.2,IL-6decreased the protein expression of IRS-1and IR-b(Fig.2A),without modification of the protein expression of ERK1/2and Akt/PKB(Fig.2B). IL-6blunted the ability of insulin to increase the tyro-sine phosphorylation of its receptor b-subunit(Fig.2A) and decreased the insulin-induced activation of ERK1/2 and Akt/PKB,three key enzymes of the insulinsignaling Fig.2.IL-6alters insulin signaling in3T3-F442A cells.3T3-F442A preadipocytes were differentiated in the presence of the indicated concentration of hrIL-6.At day8of differentiation,IL-6-treated or untreated cells were stimulated by insulin(100nM).Aliquots were immunoblotted with antibodies that recognized the b subunit of the insulin receptor(IR-b),its substrate IRS-1,the phosphorylated tyro-sines of IR-b(A),the downstream insulin signaling enzymes ERK1/2 and Akt/PKB,and their phosphorylated forms(B).A representative immunoblot from3to5separate experiments isshown.Fig.3.IL-6alters insulin-stimulated lipogenesis and glucose transport. 3T3-F442A preadipocytes were differentiated in the presence of the indicated concentration of IL-6.(A)At day8of differentiation, IL-6-treated or untreated cells were stimulated by insulin(100nM)and lipogenesis was evaluated by incorporation of[U-14C]glucose and ex-pressed as percentage above basal(control:105Æ15pmol/h/106cells). Results are meansÆSEM of3–6experiments performed in duplicate.(B)IL-6-treated or untreated cells were stimulated by insulin(100nM) and glucose transport was evaluated by incorporation of2-deoxy-D-[1-14C]glucose and expressed as percentage above basal(control: 0.44Æ0.05nmol/min/106cells).Results are meansÆSEM of3–4 experiments performed in duplicate.(C)IL-6-treated or untreated cells were solubilized and aliquots were immunoblotted with anti-GLUT4 antibody.A representative immunoblot from3separate experiments is shown.***P<0:001.374 gathu et al./Biochemical and Biophysical Research Communications311(2003)372–379pathways (Fig.2B).Although a chronic IL-6treatment did not modify the range of lipolysis (data not shown),it totally suppressed the insulin-induced lipogenesis (Fig.3A)and glucose transport (Fig.3B),consistent with the decreased GLUT4protein expression (Fig.3C).Thus,IL-6could directly decrease adipose cell response to insulin.IL-6alters the adipocyte phenotypeIn 3T3-F442A cells,IL-6(8days:from day 0to day 8)did not modify the early steps of the differentiation program.This was indicated by the lack of IL-6effect on clonal expansion (not shown)and C/EBP b and SREBP-1protein expression measured at days 2and 8of dif-ferentiation,respectively (Fig.4A).By contrast,at day 8,IL-6decreased protein expression of PPAR c and C/EBP a (Fig.4A)and the mRNA expression of FAS,GAPDH,and aP2(Fig.4B),all markers of the adipo-cyte phenotype and insulin sensitivity.The defective expression of FAS and GAPDH,two enzymes of lipo-genesis,may explain the lack of insulin activation of this pathway (Fig.3A).The decreased expression of adipo-genic markers was observed without significant decrease of the number of differentiated adipocytes evaluated by counting the cells with lipid droplets (88.5%Æ1.3%and 92.5%Æ2.2%of total cells in IL-6-treated and untreated cells,respectively,at day 8of differentiation).In fully differentiated 3T3-L1adipocytes,IL-6(8days:from day 7to day 15)also altered insulin response and the ex-pression of adipogenic markers.We observed a de-creased expression of IR-b and IRS-1protein,a blunted insulin-stimulated phosphorylation of ERK1/2and Akt/PKB,and an altered expression of FAS,GAPDH,and aP2(mRNA)and of C/EBP a and PPAR c (protein)(Data not shown).These results indicated that IL-6had an effect on adipocytes whether the treatment occurred during (3T3-F442A)or after completion of differentia-tion (3T3-L1).Rosiglitazone reverted all the adverse effects of IL-6on adipose cellCo-treatment of 3T3-F442A cells with IL-6(100ng/ml)and rosiglitazone (1l M)prevented IL-6from in-creasing its own secretion (Fig.5A).Interestingly,ros-iglitazone also decreased the basal IL-6secretion,suggesting that one possible mechanism forinsulinFig.4.IL-6alters the adipocyte phenotype.3T3-F442A preadipocytes were differentiated in the presence of the indicated concentration of IL-6.(A)At day 2(C/EBP b )or 8(C/EBP a ,SREBP-1and PPAR c )of differentiation,IL-6-treated or untreated cells were solubilized and aliquots were immunoblotted with the antibodies against the indicated transcription factor.A representative immunoblot from three separate experiments is shown.(B)At day 8of differentiation,total RNA was extracted and subjected to real-time PCR.FAS,GAPDH,and aP2mRNA levels normalized to 18S expression were determined relative to untreated cells (1.0).Results are means ÆSEM of 4–8experiments performed in triplicate.**P <0:01,***P <0:001.sensitization may be mediated by an inhibition of IL-6secretion and mRNA expression (Fig.5B).Rosiglitaz-one (1l M)prevented IL-6from decreasing insulin re-sponse:the insulin-dependent phosphorylation of ERK1/2and Akt/PKB was reestablished (Fig.6A)as well as insulin-induced lipogenesis (Fig.6B).Rosiglit-azone also impaired the downregulation by IL-6of mRNA expression of FAS,GAPDH,and aP2(Fig.6C).Rosiglitazone inhibited SOCS-3expression and its induc-tion by IL-6We then tested whether a chronic IL-6treatment could increase the expression of SOCS-3,a potentialinhibitor of insulin signaling.As shown in Fig.7A,IL-6upregulated the mRNA expression of SOCS-3up to 6-fold at 200ng/ml.Rosiglitazone exerted a negative effect on the basal and IL-6-induced SOCS-3expression (Fig.7B).In accordance with a decreased basal IL-6secretion,rosiglitazone also decreased the basal SOCS-3expression,suggesting that it can prevent IL-6effect through a direct or indirect effect on IL-6signaling.DiscussionThe adipose tissue can play an important role in modulating whole body insulin sensitivity through its endocrine functions,such as cytokine and adiponectin release.Each of the proinflammatory cytokines,TNF a [5],IL-1-b [6],and IL-6[2–4]has been recently impli-cated in the development of insulin resistance and type 2diabetes.In insulin-resistant patients the degree of cor-relation between the IL-6concentration and the severity of insulin resistance was actually found to be higher than that found with TNF a [2,26].The adipose tissue plays a key role in IL-6production [14]and is a major target of the cytokine.The objective of the present study was to evaluate the chronic effect of IL-6(8days)on adipocytes during differentiation (3T3-F442A)or on fully mature adipocytes (3T3-L1)and to approach the mechanism whereby IL-6induced insulin resistance.We initially demonstrated that adipose cells re-sponded to IL-6by increasing the production and se-cretion of IL-6.This autocrine stimulation loop,that has been observed in vascular smooth muscle cells [27],probably reinforces the adverse effects of IL-6on adi-pose cell.This autoactivation by IL-6is almost totally prevented by the antidiabetic agent,rosiglitazone [23].Interestingly,rosiglitazone decreased basal secretion of IL-6(Fig.5)and TNF a [28]which reinforcestheFig.6.Rosiglitazone prevented the IL-6-induced insulin resistance.3T3-F442A preadipocytes were differentiated in the presence of 100ng/ml IL-6(open bars)and/or 1l M of rosiglitazone (dark bars).At day 8of differentiation,IL-6-treated or untreated cells were stimulated by insulin (100nM).Aliquots were immunoblotted with antibodies that recognized ERK1/2and Akt/PKB and their phosphorylated forms.A representa-tive immunoblot from three separate experiments is shown.(A)Lipo-genesis was evaluated as described in Fig.3.Results are means ÆSEM of 3–6experiments performed in duplicate.(B)Total RNA was extracted and subjected to real-time PCR.FAS,GAPDH,and aP2mRNA levels normalized to 18S were determined relative to untreated cells (1.0).(C)Results are means ÆSEM of 4–8experiments performed in triplicate.**P <0:01,***P <0:001,and NS,notsignificant.potential role of these cytokines as mediators of insulin resistance.We next observed that IL-6altered insulin signaling after a chronic exposure(8days)to differentiating (3T3-F442A)or fully differentiated(3T3-L1)adipo-cytes.In fact IL-6had dual effects on insulin action.It acutely(30–180min)mimicked insulin action on ERK 1/2and Akt activation(not shown)and chronically induced insulin resistance.Indeed,the inhibition of ERK1/2and Akt/PKB phosphorylation appeared after a24-h exposure and aggravated after4days of expo-sure.IL-6had adverse effects at both the receptor (IR-b and IRS-1tyrosine phosphorylation)and post-receptor levels(ERK1/2,Akt/PKB,glucose transport, and lipogenesis).Finally,we observed that IL-6interfered with the last step of adipocyte differentiation.Indeed,IL-6did not affect the transcription factors SREBP-1and C/EBP b that are expressed early in the differentiation program [29]but decreased the expression of C/EBP a and PPAR c,which play a role in both terminal differentia-tion,maintenance of the adipocyte phenotype and in-sulin sensitivity[29,30].This was confirmed by the adverse effects of IL-6on the markers of the mature adipocyte involved in glucose and lipid metabolism (FAS,GAPDH,GLUT4,and aP2)and insulin sensi-tivity(IRS-1and IR-bÞ.Surprisingly,when compared to the other adipogenic markers,aP2is weakly decreased by IL-6(by15–20%).This could be explained by the fact that unlike FAS,GAPDH,and GLUT4,aP2expression is not controlled by insulin[31,32].The regulation of aP2expression remains unclear[33],even if PPAR c and fatty acids were shown to be responsible for the high expression level of aP2in adipocytes in vitro[34,35].In our study,the low incidence of IL-6on aP2expression may also be explained by the fact that IL-6poorly af-fected the differentiation program of3T3-F442A cells (88.5%Æ1.3%and92.5%Æ2.2%of IL-6-treated and untreated cells contained lipid droplets,respectively) and did not induce dedifferentiation of3T3-L1adipo-cytes(89–95%of cells contained lipid droplets).Thus, we conclude that IL-6preferentially altered the set-up and maintenance of insulin response(C/EBP a, GAPDH,FAS,GLUT4,IR-b,and IRS-1)rather than the adipocyte differentiation program(SREBP-1,C/ EBP b,and aP2).Interestingly,rosiglitazone prevented all the adverse effects of IL-6on both differentiating(3T3-F442A)and fully differentiated(3T3-L1)adipocytes.Rosiglitazone increases whole body insulin sensitivity[23]and de-creases insulin resistance in muscle and liver probably via endocrine signaling from adipocytes.This hypothesis is supported by the fact that rosiglitazone decreased IL-6secretion.Suppressor of cytokine signaling(SOCS)proteins have been recently implicated in insulin signaling network[18].Indeed,SOCS-3can bind to phosphory-lated tyrosine of IR-b and inhibit insulin signaling[36]. Here,we show that the negative effect of IL-6on insulin signaling is linked to the upregulation of SOCS-3 mRNA expression.Rosiglitazone-treated cells had a decreased SOCS-3and IL-6mRNA expression that could result from the decreased IL-6secretion or from a direct effect of rosiglitazone on IL-6signaling.The group of Spiegelman[37]provided evidence that TNF a can directly blunt insulin action in vitro and in animal studies via an increased serine/threonine-phos-phorylation of IRS-1.In the present study we failed to detect increased IRS-1serine phosphorylation in re-sponse to IL-6treatment(not shown).Alternatively, recent evidence suggests that some of the effects of TNF a may be mediated by its ability to induce IL-6and IL-6receptor expression in tissues such as the liver and muscle[38,39].Interestingly,in accordance with in vivo studies[40]we have found that a chronic treatment with TNF a(20ng/mL,8days)of3T3-F442A adipocytes in-duced upregulation of IL-6mRNA level(4.5-fold)(not shown).The upregulation of IL-6gene expression might contribute to the impairment of insulin sensitivity found in states of increased TNF a levels.Other mechanisms such as the downregulation of adiponectin expression by IL-6[41]probably also con-tribute to insulin resistance induced by IL-6.We con-clude that IL-6may act in concert with other cytokines which are upregulated or downregulated in adipocytes in insulin resistance.In conclusion,these data provide clear evidence that IL-6is not only produced by the fat cells but it is also capable of inducing insulin resistance in these cells.A chronic exposure to IL-6inhibits insulin signal trans-duction in both differentiating(3T3-F442A)and differ-entiated(3T3-L1)adipocytes.In accordance with other studies[14–16],IL-6altered the adipocyte phenotype. Rosiglitazone had a powerful preventive effect on the ability of IL-6to alter insulin signaling.Whether this effect of rosiglitazone operated through an improvement of the adipocyte phenotype is still unclear,but the effect of rosiglitazone on basal IL-6secretion and on SOCS-3 expression suggests that one mechanism for insulin-sensitizing by rosiglitazone may be through a direct ef-fect on IL-6signaling.While these data showed that one site of action of IL-6is IR-b autophosphorylation,the precise mechanism for IL-6-dependent insulin resistance in adipocytes has to be pursued.AcknowledgmentsThis work was supported by grants from INSERM.Claire Lagathu is a recipient of a fellowship from the French Ministere de la Recherche.The authors are grateful to Laurent Yvan-Charvet and Dr.Claude Forest for providing the3T3-L1and3T3-F442A cell lines.gathu et al./Biochemical and Biophysical Research Communications311(2003)372–379377References[1]J.S.Yudkin,M.Kumari,S.E.Humphries,V.Mohamed-Ali,Inflammation,obesity,stress and coronary heart disease:is interleukin-6the link?Atherosclerosis148(2000)209–214. [2]J.P.Bastard,M.Maachi,J.Tran Van Nhieu, C.Jardel, E.Bruckert, A.Grimaldi,J.J.Robert,J.Capeau, B.Hainque, Adipose tissue IL-6content correlates with resistance to insulin activation of glucose uptake both in vivo and in vitro,J.Clin.Endocrinol.Metab.87(2002)2084–2089.[3]J.M.Fernandez-Real,M.Vayreda,C.Richart,C.Gutierrez,M.Broch,J.Vendrell,W.Ricart,Circulating interleukin6levels, blood pressure,and insulin sensitivity in apparently healthy men and women,J.Clin.Endocrinol.Metab.86(2001)1154–1159. [4]B.Vozarova,C.Weyer,K.Hanson,P.Tataranni,C.Bogardus,R.Pratley,Circulating interleukin-6in relation to adiposity, insulin action,and insulin secretion,Obes.Res.9(2001)414–417.[5]G.Hotamisligil,N.Shargill,B.Spiegelman,Adipose expression oftumor necrosis factor-alpha:direct role in obesity-linked insulin resistance,Science259(1993)87–91.[6]H.O.Besedovsky,A.Del Rey,Metabolic and endocrine actions ofinterleukin-1.Effects on insulin-resistant animals,Ann.N.Y.Acad.Sci.594(1990)214–221.[7]J.P.Bastard,C.Jardel,E.Bruckert,P.Blondy,J.Capeau,M.Laville,H.Vidal,B.Hainque,Elevated levels of interleukin6are reduced in serum and subcutaneous adipose tissue of obese women after weight loss,J.Clin.Endocrinol.Metab.85(2000) 3338–3342.[8]S.K.Fried,D.A.Bunkin,A.S.Greenberg,Omental and subcu-taneous adipose tissues of obese subjects release interleukin-6: depot difference and regulation by glucocorticoid,J.Clin.Endo-crinol.Metab.83(1998)847–850.[9]S.Muller,S.Martin,W.Koenig,P.Hanifi-Moghaddam,W.Rathmann,B.Haastert,G.Giani,T.Illig,B.Thorand,H.Kolb, Impaired glucose tolerance is associated with increased serum concentrations of interleukin6and co-regulated acute-phase proteins but not TNF-alpha or its receptors,Diabetologia45 (2002)805–812.[10]V.Mohamed-Ali,S.Goodrick, A.Rawesh, D.R.Katz,J.M.Miles,J.S.Yudkin,S.Klein,S.W.Coppack,Subcutaneous adipose tissue releases interleukin-6,but not tumor necrosis factor-alpha,in vivo,J.Clin.Endocrinol.Metab.82(1997)4196–4200.[11]L.Flower,R.Gray,J.Pinkney,V.Mohamed-Ali,Stimulation ofinterleukin-6release by interleukin-1beta from isolated human adipocytes,Cytokine21(2003)32–37.[12]G.S.Hotamisligil,P.Arner,J.F.Caro,R.L.Atkinson, B.M.Spiegelman,Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance,J.Clin.Invest.95(1995)2409–2415.[13]J.Sethi,G.Hotamisligil,The role of TNF alpha in adipocytemetabolism,Semin.Cell.Dev.Biol.10(1999)19–29.[14]G.Path,S.R.Bornstein,M.Gurniak,G.P.Chrousos,W.A.Scherbaum,H.Hauner,Human breast adipocytes express inter-leukin-6(IL-6)and its receptor system:increased IL-6production by beta-adrenergic activation and effects of IL-6on adipocyte function,J.Clin.Endocrinol.Metab.86(2001)2281–2288. [15]A.S.Greenberg,R.P.Nordan,J.McIntosh,J.C.Calvo,R.O.Scow,D.Jablons,Interleukin6reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in3T3-L1adipocytes:a possible role for interleukin6in cancer cachexia,Cancer Res.52 (1992)4113–4116.[16]T.Tanaka,H.Itoh,K.Doi,Y.Fukunaga,K.Hosoda,M.Shintani,J.Yamashita,T.H.Chun,M.Inoue,K.Masatsugu,N.Sawada,T.Saito,G.Inoue,H.Nishimura,Y.Yoshimasa,K.Nakao,Down regulation of peroxisome proliferator-activatedreceptorgamma expression by inflammatory cytokines and its reversal by thiazolidinediones,Diabetologia42(1999)702–710.[17]T.Taga,M.Hibi,Y.Hirata,K.Yamasaki,K.Yasukawa,T.Matsuda,T.Hirano,T.Kishimoto,Interleukin-6triggers the association of its receptor with a possible signal transducer,gp130, Cell58(1989)573–581.[18]B.Emanuelli,P.Peraldi, C.Filloux, D.Sawka-Verhelle, D.Hilton,E.Van Obberghen,SOCS-3is an insulin-induced negative regulator of insulin signaling,J.Biol.Chem.275(2000)15985–15991.[19]R.Starr,T.Willson,E.Viney,L.Murray,J.Rayner,B.Jenkins,T.Gonda,W.Alexander,D.Metcalf,N.Nicola,D.Hilton,A family of cytokine-inducible inhibitors of signalling,Nature387 (1997)917–921.[20]B.Emanuelli,P.Peraldi,C.Filloux,C.Chavey,K.Freidinger,D.J.Hilton,G.S.Hotamisligil,E.Van Obberghen,SOCS-3inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice,J.Biol.Chem.276(2001)47944–47949.[21]J.Senn,P.Klover,I.Nowak,T.Zimmers,L.Koniaris,R.Furlanetto,R.Mooney,Suppressor of cytokine signaling-3 (SOCS-3),a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes,J.Biol.Chem.278(2003)13740–13746.[22]J.J.Senn,P.J.Klover,I.A.Nowak,R.A.Mooney,Interleukin-6induces cellular insulin resistance in hepatocytes,Diabetes51 (2002)3391–3399.[23]H.Hauner,The mode of action of thiazolidinediones,DiabetesMetab.Res.Rev.18(2002)S10-5.[24]B.M.Spiegelman,PPAR-gamma:adipogenic regulator and thia-zolidinedione receptor,Diabetes47(1998)507–514.[25]M.Caron,M.Auclair,C.Vigouroux,M.Glorian,C.Forest,J.Capeau,The HIV protease inhibitor indinavir impairs sterol regulatory element-binding protein-1intranuclear localization, inhibits preadipocyte differentiation,and induces insulin resis-tance,Diabetes50(2001)1378–1388.[26]P.A.Kern,S.Ranganathan,C.Li,L.Wood,G.Ranganathan,Adipose tissue tumor necrosis factor and interleukin-6expression in human obesity and insulin resistance,Am.J.Physiol.Endo-crinol.Metab.280(2001)E745–E751.[27]M.Klouche,S.Bhakdi,M.Hemmes,S.Rose-John,Novel path toactivation of vascular smooth muscle cells:up-regulation of gp130 creates an autocrine activation loop by IL-6and its soluble receptor,J.Immunol.163(1999)4583–4589.[28]P.G.McTernan, A.L.Harte,L.A.Anderson, A.Green,S.A.Smith,J.C.Holder,A.H.Barnett,M.C.Eggo,S.Kumar,Insulin and rosiglitazone regulation of lipolysis and lipogenesis in human adipose tissue in vitro,Diabetes51(2002)1493–1498.[29]R.M.Cowherd,R.E.Lyle,R.E.McGehee Jr.,Molecular regulationof adipocyte differentiation,Semin.Cell Dev.Biol.10(1999)3–10.[30]Z.Wu,E.D.Rosen,R.Brun,S.Hauser,G.Adelmant,A.E.Troy,C.McKeon,G.J.Darlington,B.M.Spiegelman,Cross-regulationof C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity,Mol.Cell3(1999) 151–158.[31]K.H.Kaestner,R.J.Christy,ne,Mouse insulin-respon-sive glucose transporter gene:characterization of the gene and trans-activation by the CCAAT/enhancer binding protein,Proc.A87(1990)251–255.[32]S.Le Lay,I.Lefrere,C.Trautwein,I.Dugail,S.Krief,Insulin andsterol-regulatory element-binding protein-1c(SREBP-1C)regula-tion of gene expression in3T3-L1adipocytes.Identification of CCAAT/enhancer-binding protein beta as an SREBP-1C target, J.Biol.Chem.277(2002)35625–35634.[33]A.Soukas,N.D.Socci,B.D.Saatkamp,S.Novelli,J.M.Fried-man,Distinct transcriptional profiles of adipogenesis in vivo and in vitro,J.Biol.Chem.276(2001)34167–34174.378 gathu et al./Biochemical and Biophysical Research Communications311(2003)372–379。