Tumor necrosis factor a inhibits signaling from the insulin receptor (cy e/lnu non-iul-dependent diabetes mdstus/glucose ranport/tyrine e receptor)

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1 Proc. Nadl. Acad. Sci. USA Vol. 91, pp , May 1994 Biochemlstry Tumor necrosis factor a inhibits signaling from the insulin receptor (cy e/lnu non-iul-dependent diabetes mdstus/glucose ranport/tyrine e receptor) G6KHAN S. HOTAMISLIGIL, DAVID L. MURRAY, LISA N. CHOY, AND BRUCE M. SPIEGELMAN* Dana-Farber Cancer Institute and the Department of Cell Biology, Harvard Medical School, Boston, MA 2115 Communicated by Marc W. Kirschner, February 15, 1994 ABSTRACT Insulin resistance is a common problem associated with Infections and cancer and, most Importantly, is the central component of non-insulin-dependent diabetes mellitus. We have recently shown that tumor necrosis factor (TNF) a Is a key mediator of insulin resistance in animal models of non-insulin-dependent diabetes meilitus. Here, we investigate how TNF-a interferes with insulin action. Chronic exposure of adipocytes to low concentrations of TNF-a strongly inhibits Insulin-stimulated glucose uptake. Concurrently, TNF-a treatment causes a moderate decrease in the insulin-stimulated autophosphoryiation of the Insulin receptor (Ii) and a dramatic decrease in the phosphorylation of IR substrate 1, the major substrate of the IR in vvo. The IR isolated from TNF-a-treated cells is also defective in the ability to autophosphorylate and phosphorylate IR substrate 1 in vitro. These results show that TNF-a directly interferes with the snaling of inui through Its receptor and consequently blocks biological actions of insulin. Insulin resistance, defined as a smaller than expected biological response to a given dose of insulin, is an important component of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to be the underlying mechanism for a cluster of pathologies including dyslipidemias, hypertension, and heart disease (1). Insulin resistance also develops during the course of certain cancers, infections, and trauma, such as burn injuries (2-7). Despite its biological importance, the nature of the defect(s) that leads to insulin resistance has been poorly understood. A role has been suggested for cytokines, especially for tumor necrosis factor (TNF) a, as potential mediators of abnormalities in glucose homeostasis in infection, cancer, and trauma (7-12). Evidence from both whole-animal and cell-culture studies suggests that TNF-a and other cytokines alter glucose metabolism in adipose and skeletal muscle cells and in liver (7, 13-15). Several studies have demonstrated elevated production of TNF-a in naturally occurring and experimentally induced sepsis and burn injury (7-9, 11). Similarly, in certain cancers, a correlation has been observed between TNF-a production and insulin resistance (1). However, a causal relationship between abnormal cytokine production and insulin resistance ofthese pathological states has not been established. Recently, we have demonstrated that the adipose tissues of obese animals with insulin resistance and NIDDM express elevated levels of TNF-a (16-18). In vivo neutralization of TNF-a dramatically improves the sensitivity of these obesediabetic animals to insulin-stimulated peripheral glucose uptake, indicating that TNF-a may be a key mediator of insulin resistance in NIDDM (16, 17). The molecular mechanism(s) by which TNF-a induces insulin resistance in cultured cells or in whole animals is not clear. Previous studies on cultured adipocytes, a commonly The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact used model system to study insulin action, have suggested that the down-regulation of the insulin-regulated glucose transporter (Glut4) mrna and protein might be a mechanism of TNF-a-mediated insulin resistance (13). However, the high levels of human TNF-a used in these studies can induce "dedifferentiation" of adipocytes, which is reflected in a generalized reduction in the expression ofmrnas associated with the adipocyte phenotype, including those of the insulin receptor (IR), Glut4, and other fat-related genes (7, 13, 19). In this context, cells lose sensitivity to insulin, but the interpretation of this result is complicated by the fact that insulin sensitivity is part of the adipocyte phenotype. Here we examined the effects of low-dose chronic TNF-a treatment on insulin action in detail and showed that TNF-ainduced insulin resistance involves inhibition of insulin signaling in cultured adipocytes. MATERIALS AND METHODS Reagents. Cytokines were purchased from Genzyme, except interleukin (IL) 11 (R&D Systems, Minneapolis). All cytokines were murine except TNF-.3 and IL-il, which were human. Anti-IR monoclonal antibody was purchased from Oncogene Science, anti-ir polyclonal antibody was from C. Ronald Kahn (Harvard Medical School), anti-irs-i antibodies and recombinant IRS-1 were from Morris F. White (Harvard Medical School), anti-phosphotyrosine monoclonal antibody was from Thomas Roberts (Harvard Medical School), anti-glut4 polyclonal antibody was from Morris J. Birnbaum (Harvard Medical School), and platelet-derived growth factor (PDGF) (B3( and anti-pdgf receptor antibodies were from Charles D. Stiles (Harvard Medical School). Cell Culture, Cytokine Treatments, and Glucose Transport. Murine 3T3-L1 or -F442A cells were grown and differentiated, as described (16, 2). After maximal adipocyte conversion (1 days after confluence) cytokine treatments were started at indicated doses and continued for 5 days in Dulbecco's modified Eagle's medium (DMEM)/1Q% fetal calf serum/insulin at 1 ng/ml. For glucose transport, cells were washed twice with phosphate-buffered saline on day 5 and incubated in serum-free DMEM for 2 h before assay. Glucose transport assays (using insulin at 5 ng/ml to stimulate glucose uptake) were done as described (2). Insulinstimulated glucose transport is presented as percent stimulation over basal (for every TNF-a dose) compared with control cells. Analysis of IR Tyrosine Kinase Activity in Cells. Fully differentiated adipocytes were treated for 4 days with cytokines at the indicated doses in DMEM/1% fetal calf serum/ insulin at 1 ng/ml. On day 4 cells were washed twice with phosphate-buffered saline to remove serum and maintained overnight in serum-free medium/.5% bovine serum albumin Abbreviations: TNF, tumor necrosis factor; NIDDM, non-insulindependent diabetes mellitus; Glut4, insulin-regulated glucose transporter; IL, interleukin; PDGF, platelet-derived growth factor; IR, insulin receptor; IRS-1, IR substrate 1. *To whom reprint requests should be addressed.

2 Biochemistry: Hotamisfigil et al. fraction V (ICN)/murine TNF-a (Genzyme). Insulin (bovine; Sigma) was added directly to this medium to a final concentration of 1 ng/ml. One hundred seconds after insulin addition, medium was rapidly withdrawn, and cells were frozen by floating the dishes on liquid nitrogen. Control adipocytes were cultured and treated identically to the TNFa-treated cells, except for TNF-a addition. Protein extracts were prepared as described (21). One milligram of extract protein was immunoprecipitated overnight at 4C by adding monoclonal anti-ir or anti-ir substrate 1 (IRS-1) antibodies at 1 pg/ml. Immune complexes were collected on protein A-Sepharose beads (Pharmacia), washed three times in RIPA buffer (.15 M NaCl/1 mm phosphate buffer, ph 7./1% Nonidet P-4/1% sodium deoxycholate/.1% SDS), boiled in loading buffer, and loaded onto SDS/7.5% PAGE gels (21, 22). The proteins were transferred to nitrocellulose, and immunoblot analysis was performed (Promega), using a 1:2 dilution of a monoclonal anti-phosphotyrosine, 1:1 dilution of a polyclonal anti-ir, or 1:5 dilution of a polyclonal anti-irs-i, as the primary antibody, followed by alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega) for detection. Analysis of IR Tyrosine Kinase Activity in Viltn. IRs were purified from control and TNF-a-treated cells by wheat germ agglutinin-affinity chromatography, as described (21). IRs were quantitated by insulin-binding activity and were further confirmed by immunoblotting with anti-ir antibodies (as described above). For IR autophosphorylation, aliquots of wheat germ agglutinin-purifled receptors were incubated in 5 mm Hepes, ph 7.6/.25% Triton X-1/5 mm MnCl2/ 1 nm insulin for 3 min at 29C. Phosphorylation was initiated by adding 5 pm ATP containing 4 pci (1 Ci = 37 GBq) of [t-32p]atp, for each time point, as described (21). Reactions were stopped at the indicated times, and the IRs were immunoprecipitated by using anti-ir antibody at 1 pg/ml, electrophoresed on SDS/7.5% PAGE gels, dried, and autoradiographed. For IRS-1 phosphorylation, aliquots were incubated with 1 nm insulin for 15 min at 2"C, as described (23). Two micrograms of baculovirus-produced IRS-1 was added, and the reaction was stopped at the indicated times. IRS-1 was immunoprecipitated and analyzed, as described for IR. RESULTS AND DISCUSSION To examine the effects of TNF-a on insulin action specifically, murine 3T3-Li or 3T3-F442A adipocytes were treated chronically with low amounts of murine TNF-a that cause no morphological changes in cells and no general reduction of mrnas associated with the adipocyte phenotype (16). As shown in Fig. 1 for Li cells, treatment with TNF-a at 1 ng/ml caused a 35% reduction in insulin-stimulated uptake of 2-deoxy[3H]glucose. With TNF-a at 2.5 ng/ml, insulinstimulated 2-deoxy[3H]glucose uptake was inhibited by 8%6. Thus, low TNF-a concentrations can induce significant resistance to the effects of insulin on glucose uptake. However, when Glut4 protein content of these cells was examined (Fig. 1), a significant decrease was not observed until cells were treated with TNF-a at 7.5 ng/ml. These results suggest that insulin-responsive cells can be a direct target for TNF-a and that the TNF-a-mediated insulin resistance in adipocytes involves an additional and more proximal step than the gross quantitative control of insulin-regulated glucose transporters. Essentially identical results were also obtained with another line of murine adipocytes, 3T3-F442A, although these cells have a much greater background of non-insulindependent glucose uptake (data not shown). Because 3T3- F442A adipocytes differentiate more readily and more consistently, they were used in the experiments described below. A Proc. Nati. Acad. Sci. USA 91 (1994) 4855 GlIuV~tlu -.> B -" - 12 TIC 1I O O 8 ~ I ;; 2f ThEF. FIG. 1. Effects of TNF-a on total membrane Glut4 protein (A) and insulin-stimulated glucose transport (B) in cultured adipocytes. (A) Lanes 1-4 display the total membrane Glut4 protein levels from cells treated 5 days with TNF-a at, 1, 2.5, and 7.5 ng/ml, respectively. Adipocytes were >95% differentiated and morphologically intact; further characterization of these cells has been described (16). The half-maximal inhibition of insulin-stimulated glucose transport was achieved at a TNF-a concentration of 1.5 ±.5 ng/ml. The values (mean ± SEM of two independent experiments) of insulin-stimulated glucose transport at TNF-a of, 1, 2.5, and 7.5 ng/ml are 1, 64.7 ± 4.3, 16.6 ± 5.4, and 18.8 ± 5.3, respectively. Upon binding to its receptor, insulin activates the tyrosine kinase activity in the ( subunit (24); this leads to autophosphorylation on multiple tyrosine residues which, in turn, activates receptor kinase activity toward other cellular substrates such as IRS-1 (25, 26). IRS-1 is believed to be an important mediator of IR tyrosine kinase activity, which is essential for many, if not all, ofthe biological effects ofinsulin (25, 26). To determine whether TNF-a interferes with this part of the IR signal-transduction pathway, we immunoprecipitated the IR and IRS-1 from extracts of 3T3-F442A adipocytes treated with TNF-a and analyzed the extent of insulin-stimulated tyrosine phosphorylation by immunoblotting with anti-phosphotyrosine antibodies. TNF-a treatment results in a dramatic, dose-dependent reduction in IRS-1 phosphorylation, first evident at.5 ng/ml (Fig. 2). Significantly, IRS-1 protein levels were unaffected at all doses. A more modest reduction in the tyrosine phosphorylation of the IR is observed at a dose of 1 ng/ml (3%6) and is further decreased at increased TNF-a concentrations. Some reduction in the IR protein is also observed, but only at doses of TNF >5 ng/ml. In a large series of experiments (Fig. 2), treatment of cells with TNF-a at.5-5 ng/ml caused a 15-5% decrease in autophosphorylation of the IR without a significant change in the quantity of IR protein (8 experiments), whereas the same treatment caused a 2-85% decrease in IRS-1 tyrosine phosphorylation without any change in the amount of IRS-1 protein (11 experiments). Experiments done by using 3T3-L1 adipocytes also demonstrated a similar range of inhibition of the insulin-stimulated IR and IRS-1 phosphorylations with doses of TNF-a between.5 and 5 ng/ml. These results indicate that TNF-a can have direct effects on insulin-stimulated tyrosine phosphorylation cascades. Interestingly, no change in these insulin-stimulated phosphorylations was seen before the third day of TNF-a treatment (Fig. 3A). Likewise, inhibition of insulin-stimulated glucose transport did not occur before this time (data not shown). Thus, chronic exposure to TNF-a was necessary for this effect, in contrast to many other acute effects of TNF-a (7, 8). The insulin-resistant state induced by TNF-a in \\..--4 I a

3 4856 Biochemistry: Hotamisligil et al. adipocytes appeared to be a reversible phenomenon, and complete recovery occurred 3 days after the termination of TNF-a treatments (data not shown). These effects of TNF-a treatment on IR signaling have some specificity. In parallel experiments, we observe no effect oftnf-a on tyrosine phosphorylation of the adipocyte PDGF receptor (a and f3 chains) stimulated by PDGF-pP (data not shown). Whether the inhibitory effect oftnf on the IR is completely specific in the tyrosine kinase family of receptors remains to be determined. Insulin resistance is usually a quantitative phenomenon; higher insulin doses can often result in some improvement in glucose homeostasis, even in animals or humans with extensive insulin resistance (1). It is therefore of interest to know whether TNF-a inhibition of insulin signaling could be affected by escalating insulin levels. Fig. 3B shows that TNFa-mediated inhibition of insulin-stimulated tyrosine kinase activity could be partially compensated by increased insulin doses. However, even at the highest insulin concentration used (1 ng/ml), the insulin-stimulated tyrosine phosphorylation of IR and IRS-1 from TNF-a-treated adipocytes was still significantly lower than that of the control cells (4% and 2% of controls, respectively). Cytokines such as TNF-f3, IL-1, IL-6, IL-11, and interferon share several biological activities with TNF-a (27). In addition, the signal-transduction pathways of these cytokines also exhibit considerable overlap (27). We therefore examined several cytokines to determine whether any of these molecules could change insulin-stimulated phosphorylation similar to that seen with TNF-a. Dose-dependent decreases in insulin-stimulated tyrosine phosphorylation of IRS-1 and IR were also seen after TNF-P, IL-la, IL-6, and interferon 'y (Fig. 4). None of these treatments caused significant changes in the levels of IRS-1 or IR protein at the concentrations used (data not shown). No change in insulinstimulated phosphorylations was seen with IL-183, IL-3, IL-4, IL-1, IL-11, and leukemia inhibitory factor treatments (Fig. 4). These results suggest that several members of the cytokine network have the potential to affect insulin action, possibly through a common molecular mechanism. Decreased tyrosine phosphorylation of the IR and IRS-1 could be explained by a defect in the IR, a defect in both the IR and IRS-1, or a molecule that modified both, such as a tyrosine phosphatase. To approach this problem, we asked 2 12 C~~~~~~~~ o 1,,, 8 CIO 6 4 C,, = 2 H TNF-ca dose, ng/ml FIG. 2. Effect oftnf-a on insulin-stimulated tyrosine phosphorylation of the IR (i) and IRS-1 (o). Murine 3T3-F442A adipocytes were cultured and differentiated in 1%6 fetal calf serum and insulin at 1 ng/ml and treated with TNF-a for a total of 5 days at the indicated doses. The extent of tyrosine phosphorylation or protein quantity was determined by immunoprecipitation of IR and IRS-1 from cellular extracts followed by immunoblotting with antiphosphotyrosine, anti-ir, or anti-irs-1 antibodies. The halfmaximal inhibition of insulin-stimulated tyrosine phosphorylation of IR and IRS-1 was achieved with TNF-a at 2. ±.5 ng/ml. Immunoblots were scanned in an Abaton Scan 3/GS digital scanner and quantitated by using the image 1.33 image-analysis program. The values (mean ± SEM) represent tyrosine phosphorylation (as percent of control) corrected for protein quantity. whether TNF-a treatment of cells reduced the kinase activity of the isolated IR, as measured in vitro. The IR was partially purified from TNF-a-treated adipocytes (4 ng/ml) by wheat germ agglutinin affinity chromatography and subjected to an in vitro kinase assay designed to measure autophosphorylation of the IR and phosphorylation of recombinant IRS-1 in response to insulin (21, 23, 25, 26). In these preparations the receptors were quantitated by insulin-binding activity, and these values were further confirmed by immunoblotting; no changes were seen in insulin binding per unit of IR protein (data not shown). When equal amounts of IRs were assayed for their tyrosine kinase activity, a 4-5% overall decrease in insulin-stimulated autophosphorylation was observed at all time points (.5-1 min) in material from TNF-a-treated cells compared with controls (Fig. SB). There was a similar reduction in the ability of IRs from TNF-a-treated cells to phosphorylate recombinant IRS-1 (Fig. SA). In several experiments using two different cellular preparations, maximal phosphorylation of IRS-1 by IR from treated cells was 2-5% of that seen by IR from control cells. These data demonstrate defective action of the IR purified from TNFa-treated cells. The insulin resistance of cancer, infection, and NIDDM involves a postinsulin-binding abnormality, which leads to decreases in insulin responsiveness (1-3, 5, 28). Many studies have focused on glucose transporter systems as potential targets for such a postreceptor defect, especially in NIDDM (1). Although defects in the quantity and function of Glut4 have been demonstrated in insulin-resistant rodents and humans with NIDDM, the impact of these alterations on overall insulin resistance has been unclear (29-32). In addition, some studies have failed to confirm this correlation in human NIDDM, especially in muscle (33, 34). Likewise, a decrease has been observed in the Glut4 content of adipocytes in experimental animal models of endotoxemia and sepsis; however, this decrease does not correlate with the in vivo insulin responsiveness of adipose tissue (35). Several pieces of data showing that TNF-a can specifically reduce the expression ofglut4 mrna in cultured adipocytes (13, 14, 16, 17) suggested that this mechanism could be at least partly A INS TNF IRS-1 ;>Tvr so - B IRS-1 IR DTvr - DAYS ; INS -TN F PTyr Proc. Natl. Acad Sci. USA 91 (1994) IR DTyr --. we -..m,i m4i.- OWNWOM _'; - '- t-a FIG. 3. (A) Time course of inhibition of the IR tyrosine kinase. Murine 3T3-F442A adipocytes were treated with TNF-a at 2 ng/ml for the indicated periods and stimulated by insulin (INS) at 1 ng/ml. (B) Effect of increased insulin (INS) on TNF-a inhibition of insulin-stimulated tyrosine phosphorylations of IR and IRS-1. Lanes: 1-5, no TNF-a; 6-1, TNF-a (2 ng/ml)-treated cells; 1 and 6, no insulin; 2 and 7, insulin at 1 ng/ml; 3 and 8, insulin at 3 ng/ml; 4 and 9, insulin at 6 ng/ml; 5 and 1, insulin at 1 ng/ml. ptyr, phosphotyrosine.

4 Biochemistry: Hotamisligil et al. Proc. Natl. Acad. Sci. USA 91 (1994) o 1 2 o 1 O 8 Q 4 z 2 o ~ U) - _C cr Control TNF-ctTNF- 1L1-a 1L1-11L4 1L6 IL1 IL11 IFN-Y LIF Cytokines responsible for the in vivo effects of TNF-a neutralization on insulin action in animal models of NIDDM (16). However, neutralization of TNF-a in vivo improved insulin-stimulated glucose uptake in Zucker fatty rats (1), without affecting expression of Glut4 mrna in fat or muscle (data not shown). Moreover, gross Glut4 down-regulation alone does not account for TNF-a-mediated inhibition of insulin-stimulated glucose transport in adipocytes (Fig. 1). Hence, TNF-a must affect more proximal steps in insulin signaling. Defects in proximal steps of insulin signaling have been demonstrated in NIDDM, such as a reduction in the tyrosine kinase activity of the IR, as measured by insulin-stimulated autophosphorylation (1, 36). It is well known that the IR tyrosine kinase activity is an absolute requirement for the biological activities of insulin (1, 37, 38). Recent studies have also demonstrated that tyrosine phosphorylation of IRS-1 is decreased in fat and muscle in insulin-resistant states (39). IRS-1, the major substrate for the IR tyrosine kinase (25, 26), is a cytoplasmic protein with multiple phosphorylation sites and is believed to play a pivotal role in transducing signals from the IR to a number of different targets through its structural capability of interacting with proteins containing Src homology region 2 domains (25, 4). We demonstrate here that TNF-a directly interferes with insulin action by decreasing the tyrosine kinase activity of the IR toward itself and by reducing the subsequent phosphorylation of IRS-1 in cultured adipocytes. Although the precise role of IRS-1 in various actions of insulin remains to be determined, the ability of TNF-a to interfere with IRS-1 phosphorylation is indicative of reduced signaling downstream of the IR. It will be important to investigate whether insulin phosphorylation cascades are also disrupted by TNF-a in muscle cells because muscle is a central tissue in whole-body glucose disposal (1). The nature of the defect in the IR induced by TNF-a treatment is not yet apparent. Because this defect can be observed in partially purified preparations of the IR, a covalent modification of the receptor itself may be suggested. Interestingly, covalent modification of the IR by both protein kinases A and C has been shown to reduce the intrinsic kinase activity of the IR (23, 41), whereas TNF-a has been shown in FIG. 4. Effect of different cytokines on insulin-stimulated tyrosine phosphorylation of IR and IRS-1. IRS-1 (A) and IR (B). Murine 3T3-F442A adipocytes were cultured and differentiated in 1o fetal calf serum and insulin at 1 ng/ml and treated with the indicated cytokines at 2 and 5 ng/ml for a total of 5 days. At doses used in these experiments none of the cytokines caused significant changes in adipocyte morphology. The extent of tyrosine phosphorylation was determined by immunoprecipitation of IR and IRS-1 from cellular extracts followed by immunoblotting with antiphosphotyrosine antibodies. Immunoblots are scanned and quantitated as described in Fig. 2 legend. Extent of phosphorylation (mean ± SEM) is presented as the percentage of the control that received insulin but no cytokine treatment. IFN-y, interferon fy; LIF, leukemia inhibitory factor. some systems to elevate protein kinase A and C levels (42, 43). Alternatively, there could be a tight but noncovalent Or._ >1 CZ) or Q- an Cl) U- - Cl CZ an cc i / TNF.* Control Time, hr 12 - B 1 a 8 4 Ax 2 -)- TNF.O - Control r Time, min FIG. 5. In vitro protein kinase activity of the IR purified from control and TNF-a-treated (4 ng/ml) murine 3T3-F442A adipocytes. (A) Insulin-stimulated phosphorylation of IRS-1. (B) Insulinstimulated autophosphorylation of the IR. Wheat germ agglutinin fractions derived from TNF-treated and control cells that contained equal insulin binding were used for determining in vitro kinase activity of the IR. Quantitations were done on a Phosphorlmager (Molecular Dynamics).

5 4858 Biochemistry: Hotamisligil et al. association of an inhibitory molecule with the IR. The fact that at least 3 days oftnf-a treatment are needed to observe alterations in insulin-stimulated phosphorylations suggests that alterations in gene expression are involved in these effects. The ability of TNF-a and several other cytokines to interfere with the most proximal part of the insulin signaltransduction pathway is likely to have physiological relevance for obesity-linked insulin resistance and NIDDM because of the overexpression of TNF-a seen in obesity (16-18). However, these data may also have relevance for other peripheral insulin resistance states, such as cancer and infection (2-7), where a potential role for TNF-a and other cytokines has been suggested. The link between the cytokine network and insulin action may explain the molecular mechanisms of insulin resistance in these pathological states and defines a potentially important target for therapeutic intervention. Because many anticytokine therapeutics are under development, a general role for TNF-a or other cytokines in insulin resistance may offer additional opportunities to treat these severe metabolic disorders. Note. While this paper was being reviewed, it was reported that TNF-a acutely (within 1 hr) suppresses insulin-stimulated tyrosine phosphorylation of IR and its cellular substrates in Fao hepatoma cells (44). Whether or not the effects of TNF-a on hepatoma cells develop through similar mechanisms and are relevant in the physiological context described in this paper remains to be determined. We thank Martin M. Myers, Morris F. White, Bentley Cheatham, C. Ronald Kahn, and MorrisJ. Birnbaum for their helpful advice. We are grateful to Adrianne Budavari for her technical assistance. This work was supported by a grant from the National Institutes of Health (DK 42539). 1. Moller, D. E. (1993) Insulin Resistance (Wiley, London). 2. Copeland, G. P., Leinster, S. J., Davis, J. 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