Tumor necrosis factor-alpha suppresses insulin-induced tyrosine ...

Communication

THE

JOURNAL OF BIOUCICAL CHEMISTRY

Vol. 268,No. 35,Issue of December 15,pp. 26055-26068, 1993 0 1993 by The American Soeiety for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Tumor Necrosis Factor-a Suppresses Insulin-induced mrosine Phosphorylation of Insulin Receptor and Its Substrates*

EXPERIMENTALPROCEDURES Cell Culture and Cell Cytotoxicity Assay-Rat hepatoma Fao cells were grown in RPMI 1640 medium containing 10% fetal calf serum (Biological Industries, Beth Haemek, Israel). For cytotoxicity assays, cells were plated in 35-mm diameter dishes (Nunc, Denmark) at a density of 4 x los cellddish in 2 ml of culture medium. After 24 h the medium was removed, and a serial dilution of recombinant murine TNF (Genentech, San Francisco, CA) was layered in 2 ml of culture medium. Following 24 or 72 h of incubation with the cytokine at 37 "C, the medium was recovered and cells were detached with trypsin. The me(Received for publication, July 26, 1993, and in revised form, dium and thedetached cells were pooled and centrifuged at 1000 x g for October 1, 1993) 10 min. Cell viability was evaluated by trypan blue staining. Insulin and TNF Binding to Intact Cells-Confluent cells in 60-mm Revital FeinsteintB, Hannah Kanety 1, dishes were deprived of serum for 16 h. Cells werethen incubated with Moshe Z. Papall, Bruno LunenfeldS, and 50,000 cpmof A14-m0no-'~~I-insulin (2000 Wmmol, Novo Nordisk, AW&n** Bagsvaerd, Denmark) in 3 ml ofRPMI medium containing 1 mg/ml From the Vnstitute of Endocrinology a n d lpepartment bovine serum albumin (Sigma, radioimmunoassay grade), 25 m of Surgery, Sheba Medical Center, Te1 Hashomer 52621, Hepes, and various concentrations of unlabeled recombinant human Israel and the $Department of Life Sciences, Bar-Zlan insulin (Novo Nordisk). Followingincubation for 2 h at 22 "C, the cells University, Ramat-Gun 52900, Israel were washed twice with phosphate-bufferedsaline and solubilized with 1 ml of phosphate-buffered saline containing 1% Triton X-100.The Tumor necrosisfactor-a (TNF) hasrecentlybeen amount of 1261-insulin bound to the cells wasdetermined in a y counter. shown to induce insulin resistance. We have examined TNF binding was performed essentially in the same manner using 50 the possible effect of TNF on the early events in insulin PM human 1261"l'NF(500 Ci/mmol, Amersham,Aylesbury, Buckinghamshire, UK) and increasing concentrations of unlabeled murine TNF. transmembrane signaling. Incubation of the insulin-sen~ for 1 h led to Nonspecific binding was determined at 1 unlabeled insulin or 10 rm sitive rat hepatoma Fao cells with5 1 1 TNF unlabeled TNF, respectively. a 85% decreaseininsulin-inducedtyrosinephosphoLigand Peatment oflntact Cells-Confluent monolayers of Fao cells, rylationofboththeinsulinreceptorP-subunitand grown in 60-mm dishes, were deprived of serum for 16 h prior to each -1, its major cytosolic substrate. TNF-induced imexperiment. The medium was removed, TNF (0.5-5 m) in serum-free pairment of tyrosine phosphorylation was maximal at medium was added, and the incubation was continued forthe indicated 0.5 MI and was not accompanied by any reduction in time interval. The cells were then incubated without or with 100 rm insulin binding. Sixteen hours TNF of incubation led to human insulin for 1 min at 37 "C. The reaction was terminated by further impairment in insulin-induced tyrosine phos- removing the medium and freezing the cell monolayers with liquid phorylation of these proteins. Our findings suggest that nitrogen. Cells were solubilizedat 4 "C with 0.4 mudish of buffer A (50 m TNF may exert its anti-insulin effectby interrupting the nm Hepes, pH7.6,150 n w sucrose, 2 m~ sodium orthovanadate, 80 x /%glycerophosphate,10 m NaF, 10 m sodium pyrophosphate, 2 m early insulin-stimulated tyrosine phosphorylation events, which are crucial to insulin transmembrane sig- sodium EGTA, 2 nm sodium EDTA, 1%Triton X-100,0.1%SDS, 1 m phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin, 10 pg/ml leupeptin, naling. and 10 pg/ml trypsin inhibitor); all reagents were obtained from Sigma. The solubilized cells were scraped and sedimented by centrihgation for 15 min at 4 "C a t 12,000 x g. Aliquots of the supernatants were norInsulin resistance has been demonstrated in sepsis and ad- malized for protein, mixed with concentrated (5 x) Laemmli sample vanced cancer (1,2). Recently, it has been suggested that el- buffer (lo),boiled for5 min, and resolved on7.5 or 6%SDS-PAGE under reducing conditions. For immunoprecipitation cellsweresolubilized evation of tumor necrosis factor-a (TNF)' in severe infection may play a role in causing this phenomenon (3, 4). Adminis- with 0.4 mudish of buffer B (buffer A without SDS). Immunoprecipitation from Fa0 Cell Extracts-Rabbit polyclonal intration of TNF to rats alters lipid and protein metabolism in sulin receptor antibodies were generated against a synthetic peptide liver, adipose tissue, and skeletal muscle (5,6) and impairs corresponding to positions 1309-1324 of the human insulin receptor. insulin action on peripheral glucose disposal and hepaticglu- Rabbit polyclonal IRS-1antibodies (11) were raised against a synthetic cose output(3). Chronic exposure of 3T3-Ll adipocytes to5 m peptide segment of IRS-1 (pep 80) ( g i f t from P. Rothenberg, University TNF'in culture blocks insulin-stimulated hexose uptake (7). of Pennsylvania, Philadelphia, PA). Antibodies were added to 60 pl of This has been explained by suppression of the glucose trans- 50%Protein A-Sepharose (Pharmacia, Uppsala, Sweden) in 0.1 M Tris, pH 7.6, and incubated for 1 h at 4 "C. The complex was pelleted at porters GLUT-4 a n d GLUT-1. To further investigate the mecha12,000 x g (5min), washed three times with 0.1 M Tris, pH 7.6, and once nisms of TNF-induced insulin resistance, we have studiedthe with buffer B. Cell extracts (1.2-1.5 ml) were incubated for 4 h at 4 "C early steps of the insulin transmembrane signaling in intact with the antibody-Protein A-Sepharosecomplex.Theimmunocomhepatoma cells (Fao) treated with TNF. This cell line is highly plexes were pelletedby centrifugation at 12,000 x g and washed twice insulin-sensitive and served in the study of insulin receptor with buffer B and twice with buffer B containing 0.1%Triton X-100. The pellets were then suspended in Laemmli sample buffer and resolved on tyrosine kinase (8, 9). 7.5 or 6% SDS-PAGE. Western Immunoblotting-Electrophoretic transfer of phosphopro* This work was supported in part by a grant from the Revson Foun- teins from the gels to nitrocellulosepapers was carried out as previously dation of the Israeli Academy of Sciences (toA. K.).The costs of publication of this article were defrayed in part by the payment of page described (12). Blots were incubated with affinity-purified (3 pg/ml) charges. This article must therefore be hereby marked "aduertisement" phosphotyrosine antibodies (gififrom M. White, Joslin Research Laboratories, Boston, MA), followed by incubation with anti-rabbit immuin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. noglobulin G peroxidase-linked F(ab'), fragment (Amersham). A che5 This work is Dart of this author's MSc. thesis. ** To whom correspondence shouldbe addressed. Tel.: 972-3-5302802; miluminescent peroxidase substrate (ECL,Amersham) was applied according to the manufacturer's instructions, and the membranes were Fax: 972-3-5302803. * The abbreviations used are: tumor necrosis factor-a: PAGE. exposed briefly to x-ray film. polyacrylamide gel electrophoresis. Intracellular ATP Content-Intracellular content of ATP was deter-

TNF.

26055

TNF Suppresses Insulin-induced Phosphorylation

26056

mined as described (13). Briefly, Fao cells grown to confluency in 35-mm plates were incubated in serum-free medium and then incubated for 1 h with the same medium in the absence or presence of ThT (0.5-5 m). Following treatment, cells were lysed in 0.2 ml of buffer A, and the ATP content of the 12,000 x g supernatant was determined by the luciferin/ luciferase assay (13). Chemiluminescence was monitored using a Lumad3M luminometer (model M 2010A). Results are means of duplicate measurements that did not vary by more than 10%.

RESULTS and CytotoxicEffect on Fa0 Hepatoma Cells-To establish the presence of TNF receptors on the rat hepatoma cell line, Fao cells were incubated with 1251-TNFand an increasing concentration of unlabeled TNF. When incubated for 2 h with 50 PM 1251-TNF,cells bound 4.4% of the input radioactivity, >90% of which could be competed for by excess unlabeled ligand (Fig. 1,inset). Scatchard analysisof the TNF binding to Fao cells resulted in a linear plot with a K d value of 7 x 10"O M and a B,of 100 fmoV2 x lo7 cells (3000 receptors/ cell). TNF effect on cellviability was examined following incubation with increasing concentrations of TNF for 24 or 72 h.As seen in Fig. 1,a gradual decrease in cell viability was observed with increasing TNF Concentration already after 24 h of incubation and was more pronouncedafter 72 h. A 50% decrease in cell viability was detected at 1n~ TNF after 24 hof incubation and with 0.1 n~ TNF at 72 h (Fig. 1). Inhibition by TNF of Insulin-stimulated ?vrosine Phosphorylation in Intact Fa0 Cells-Insulin receptor is a protein tyrosine kinase that upon stimulation by insulin phosphorylates the receptor itself and several intracellular proteins on tyrosine residues (8, 9, 14).To examine the effect of TNF on insulinstimulated tyrosine phosphorylation, TNF-treated cells were stimulated for 1min by insulin. Cell extracts were analyzed by SDS-PAGE, blotted onto nitrocellulose filters, and probed with phosphotyrosine antibodies. Addition of insulin to cells caused phosphorylation of a 180-185-kDa band (Fig. 2, lane 6 ) .PreinI "for 1h resulted in a 65% decrease in cubation with 5 n~ ' the insulin-stimulated phosphorylation of this broad band, as well as alteration in itselectrophoretic mobility (Fig.2, lane c). Longer exposure of the same gels to autoradiography revealed an additional 95-kDa band that underwent insulin-stimulated phosphorylation (Fig. 2, lane e). Insulin-induced phosphoryla-

a b c d e f TNF

Insulin

-

TNF (nM)

1.TNF effect on cell viability. For cytotoxicityassay cells were plated in 35-mm diameter dishes a t a density of 4 x lo6 cellddish in 2 ml of culture medium. After24 h the medium was removed and a serial dilution of TNF was layered in 2 ml of the same medium for24 or 72 h a t 37 "C. Relative cell viability after TNF exposure (empty bars, for 24 h; hatched bars, for 72 h) is given as a percentage of viable cells with the indicated concentration of TNF out of viable cells without TNF. Inset, competition curve for TNF binding to intact Fao cells. Cells were plated in 60-mm diameter dishes and incubated with lZ5I-labeledTNF and the indicated concentrations of unlabeled TNF. Binding was expressed as percent of radioactivity bound to 2 x lo7 cells out of total added radiolabeled TNF.

j

Mrx10'~

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- + ++ .c

-200-

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-

TNF Binding to

FIG.

- - +-

g h i

--+--+--+ ++ -++ -++

IR. AbJ

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IRS-1

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L IRS-1 A b J

FIG.2. Effect of TNF on insulin-stimulated protein tyrosine phosphorylation in intactFao cells. Fao cells wereincubated with 5 m TNF in serum-free medium for 1 h and stimulated with 100 m insulin for 1 min a t 37 "C. Cell extracts were subjected to SDS-PAGE under reducing conditions (lams a-fandm ) or to immunoprecipitation. Immunoprecipitates with insulin receptor ( I R )or IRS-1 antibodies (Ab) were also analyzed by SDS-PAGE (lanes g-i andj-l, respectively). Proteins were transferred to nitrocellulose papers, and phosphotyrosinecontaining proteins wereprobed with phosphotyrosine antibodies. These proteins were visualized using a chemiluminescent peroxidase substrate and autoradiography.

A

-TNF

W +TNF

0-

I

-1 0

FIG.3. Competition curvesfor insulin bindingto control and TNF-treatedcells. Insulin binding was determined essentially as described in Fig. l in cells preincubated with and without TNF for l h a t 37 "C. Binding was expressed as percent of radioactivity bound to 2 x lo7 cells out of total added radiolabeled insulin.

tion of this band was also markedly reduced in TNF-treated cells (Fig. 2, lane f ) . In Fao rat hepatoma cells the insulinstimulated broad band at M, 180,000-185,000 was shown to include a t least two tyrosine-phosphorylated proteins, IRS-1 and a high molecular weight 185-kDa phosphoprotein (15). To identify the 180-185- and 95-kDa phosphoproteins, cellextracts were subjected to immunoprecipitation with either insulin receptor- or IRS-1-specific antibodies. These experiments verified that the95-kDa phosphoprotein, whose insulin-stimulated phosphorylation was inhibited by "NF, is the insulin receptor P-subunit (Fig. 2, lanes g-i) and that TNF inhibited the insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 2, lanes j-Z). IRS-1 co-migrated with the lower part of the 180185-kDa tyrosine-phosphorylated broad band (Fig. 2, lanes k-m). Incubation of Fao cells with 5 n~ TNF for 1h had no significant effect on insulin binding (Fig. 3). Thus, the effect observed on tyrosine phosphorylation of the insulin receptor P-subunit and IRS-1 was not due to changes in the concentration of the receptor in thesecells. The decrease was also not secondary to the TNF effect on ATP concentration; intracellular ATP content of Fao cells after 1h of incubation with 0,0.5,1, and5 n~ TNF was 8.1 0.5, 7.7 0.7, 10.8 1.2, and 10.0 1.1 nmoVmg protein, respectively. To further characterize TNF effect on insulin-stimulated tyrosine phosphorylation, the same experiments were repeated with different time and dose exposure to TNF. Whereas prein-

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TNF Suppresses Insulin-induced Phosphorylation

26057

gardless of the mechanism, TNF effects seem specific for the insulin receptor and not secondary to a generalized toxic metabolic effect.In our system, short-term incubation with TNF did c7 pp185 0“2 not alter intracellular ATP content and did not change the X phosphorylation pattern of cytosolic proteins (not shown). In 5116 other systems, incubation of A431 cells with TNF increased tyrosine phosphorylation of the epidermal growth factor receptor (24). In the U937 human monoblastoid cell line TNF induced serine phosphorylation of a 26-kDa protein without modifying the phosphorylation pattern of all other cytosolic or membranal proteins (25). Previous inquiry into the mechanism of TNF suppressive Time Dose effect on insulin action was performed in 3T3-Ll adipocytes. F’IG. 4. TNF effects on insulin-stimulated m i n e phosphe rylation in Fao cells at different timesand doses. Confluent mono- Stephens and Pekala (7) found in these cells a decrease in cell content of layers of Fao cells were treated as described in Fig. 2 with TNFfor the membrane GLUT-4 content and intracellular time and doses indicated, prior to stimulation with insulin. Tyrosine- GLUT-1 after 12 days of exposure to TNF. A decrease in insulin containing proteins were detected as described in Fig. 2. receptor and GLUT-4 mRNA levels was noticed in these cells after 3-6 days of TNF treatment. In contrast, TNF effects on cubation with TNF for 1h caused a 65% decrease in phospho- insulin-stimulated hexose transport were already evident after rylation of the 180-185-kDa broad band, 16-h TNF exposure 24 h of exposure. We therefore suggest that though late effects completely abolished phosphorylation of these proteins. TNF of TNF may be due to alterations in glucose transporters and alone did not induce tyrosine phosphorylation of this band (Fig. insulin receptor number, short termeffects (1-24 h) resultfrom 4, left panel). A maximal decrease in phosphorylation of the alterations in the insulin-induced receptor-mediated tyrosine 180-185-kDa proteins was seen already a t 0.5 n~ TNF after 1 phosphorylation. h of incubation (Fig. 4, right panel). Beyond its potential role as a regulator of insulin action in severe infection and emaciation, it has recently been specuDISCUSSION lated that inrodent models of obesity TNF is the cause and link Insulin exerts its effects by binding to a specific receptor on of obesity-induced insulin resistance (26, 27). Several animal the cell surface (14). Upon binding of the insulin to the extramodels of diabetes manifest a form of disease characterized by cellular a-subunit of the receptor, the tyrosine-specific protein kinase of the intracellular P-subunit of the receptor is acti- obesity, hyperinsulinemia, and impaired glucose tolerance (28). vated. This activation results in autophosphorylation of the In several of these models a decrease in phosphorylation of the P-subunit followed by tyrosine phosphorylation of several in- insulin receptor in insulin-dependent tissues was observed in tracellular substrates (8,9,14). The tyrosine kinase activity of parallel to development of obesity and hyperinsulinemia (29, the receptor is crucial for insulin action, as abolishing the ki- 30). Moreover, in one such model (ob/ob mice) a reduction in nase activity by either antibodies or point mutations abrogates phosphorylation of I N - 1 in both muscle and liver was also animal model neutralization of insulin signaling (14). Of the insulin receptor substrates the demonstrated (30). In the same first and best characterized is a 185-kDa protein recently TNF abolished insulin resistance (26). Thus in intactanimals, cloned and termed IRS-1(11,16). Recent research intothe role like in cell culture, the mechanism for the alleged role of TNF of this protein describes it as a “docking protein,” which in its on insulin action may be through suppression of insulin-intyrosine-phosphorylated state associates with high affinity cel- duced tyrosine phosphorylation. lular proteins that contain src homology-2 (SH-2) domains (14). Acknowledgments-We thank Dr. Yehiel Zick, Yael Biener, and Yaron This association activates several intracellular processes cul- Hadari for helpful discussions, and Irit Weiss and Ruth Dror for techminating in thevarious effects of insulin. In our studywe have nical assistance. shown that in theFao rat hepatoma cell line TNF suppresses REFERENCES insulin-induced receptor and IRS-1 tyrosine phosphorylation. 1. Bennegard, K., Lundgren, F., and Lundholm, K. (1986) Clin. Physiol. 6,539This profound effect onthe initial insulin transmembranesig547 naling steps was evident after a short exposure of these cells to 2. Lang, C.H., and Dobrescu, C. (1989)Am. J. Physiol. 257, E301-E308 mF(h) Insulin 200 -

1

1 16

TNF(nM) hsulin

0.5 2 5

I -

-+ + + +

n

TNF.

TNF acts through specific membrane receptors whose mechanism of signal transduction has not yet been fully elucidated (17). TNF-induced activation of protein kinase C has been demonstrated in several cell types (18).Protein kinase A (19) and multiple other serindthreonine protein kinases (20) were also implicated in TNF signal transduction in other cells. Interestingly, in Fao cells activation of protein kinase C by phorbol esters has been shown to decrease the tyrosine kinase activity of the insulin receptor through multisite phosphorylation on serindthreonine residues (21, 22). Such TNF-induced serindthreonine phosphorylation of the insulin receptor may well be the mechanism by which the observed reduced tyrosine phosphorylation of the receptor occurs. Alternatively, the TNF effect may be mediated through the activation of specific tyrosine phosphatases. Activation of these phosphatases by TNF has been suggested based on experiments in humanfibroblasts where use of orthovanadate, a classic tyrosine phosphatase inhibitor, suppressed the proliferative effects of TNF (23). Re-

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