Insulin resistance: a phosphorylation-based uncoupling of insulin signaling

437 Research News Insulin resistance: a phosphorylation-based uncoupling of insulin signaling Yehiel Zick Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. It is associated with the development of type 2 diabetes an ever-increasing epidemic of the 21st century. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative-feedback control mechanisms, otherwise utilized by insulin itself, to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article describes recent findings that present novel viewpoints of the molecular basis of insulin resistance, focusing on the cardinal role of Ser/Thr protein kinases as emerging key players in this arena. Raf1 Ras SOS SHC Grb2 MEK MAPK Myc Jun Fos Gab-1 SHP-2 pp90 RSK p70 S6K Cbl SHP-2 Fyn CAP CrkII PDK1 PKB C3G TC10 PKCζ PKCλ Insulin, produced by the pancreas, is the major anabolic hormone whose action is essential for growth, development and homeostasis of glucose, fat and protein metabolism 1,2. At the molecular level, insulin binding to its transmembrane receptor () stimulates the intrinsic Tyr kinase activity of the receptor (K), which then phosphorylates target such as Shc and the family of insulin receptor substrate () (-1 to -4) on selective Tyr residues that serve as docking sites for downstream effector molecules. This triggers two major kinase cascades, the phosphoinositide 3-kinase () and the mitogen-activated protein (MAP) kinase pathways, which mediate the metabolic and growth-promoting functions of insulin 1,2 (Fig. 1). Insulin resistance is a common pathological state in which target cells fail to respond to ordinary levels of circulating insulin 3,4. Individuals with insulin resistance are predisposed to developing type 2 diabetes, and insulin resistance is frequently associated with a number of other health disorders, including obesity, hypertension, chronic infection and cardiovascular diseases 5,6. Recent studies have focused on Ser/Thr phosphorylation of the as a key negative-feedback control mechanism DNA / RNA / Protein synthesis Cellular growth S6 that uncouples the from their upstream and downstream effectors and terminates signal transduction in response to insulin, under physiological conditions. Emerging data further suggest that agents such as tumor necrosis factor α (TNFα), free fatty acids (FFA) and cellular stress, which inhibit insulin signaling and induce insulin resistance, take advantage of this mechanism by activating unidentified known as kinases that phosphorylate the and GSK3β Glycogen synthesis Metabolism Glucose transport Fig. 1. Insulin signal transduction. Circulating insulin interacts with its cognate receptor, which is a transmembrane tyrosine kinase, having an α 2 β 2 configuration. Insulin binding to the α subunits leads to a conformational change and stimulation of the receptor kinase activity through autophosphorylation of Tyr residues in the β subunits. The activated insulin receptor kinase (K) then phosphorylates substrate, such as Shc, Gab-1, Cbl/CAP and the family of insulin receptor substrate (), on selective Tyr residues that serve as docking sites for downstream effector molecules. This triggers two major kinase signaling cascades the mitogen-activated protein kinase (M APK) and phosphoinositide 3-kinase () pathways. Recruitment of the Grb-2 and Sos to Tyr-phosphorylated Shc activates the MAPK cascade, whereas association of with the (-1 to -4) results in production of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) that activates PDK1 (dependent kinase 1) and its downstream effector kinases PKB (protein Ser/Thr kinase B, also named Akt),, p70s6 kinase and the atypical isoforms of PKC (PKCζ/PKCλ). Collectively, these kinase cascades mediate the metabolic and growth-promoting functions of insulin, such as translocation of vesicles containing GLUT4 glucose transporters from intracellular pools to the plasma membrane (), stimulation of glycogen and protein synthesis, and initiation of specific gene transcription 1,2. Phosphorylation of Cbl mediates glucose transport in a -independent manner 44. inhibit their function. Thus, while the underlying molecular pathophysiology of insulin resistance is still not well understood, Ser phosphorylation of, which is the focus of this review, represents a new and possibly unifying mechanistic theme. Other potential mechanisms for the induction of insulin resistance, such as increased activity of lipid- or protein-tyr phosphatases (PTPs), or the genetics of insulin resistance, are discussed elsewhere 3,4. 0962-8924/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02129-8

438 Research Update How can the same molecular mechanisms be utilized to terminate insulin signal transduction under physiological or pathological conditions? Which kinases are involved? How does Ser/Thr phosphorylation impact upon protein function and insulin signaling? This article reviews recent experimental data that have begun to shed light upon these questions. Feedback regulation of insulin signaling cascades Control mechanisms are essential for cellular signaling. Control can be achieved by autoregulation, whereby downstream enzymes inhibit upstream elements (homologous desensitization). Alternatively, signals from apparently unrelated receptor pathways can inhibit the signal (heterologous desensitization). Tyr-phosphorylated, which are key players in propagating insulin signaling, are the targets of such feedback regulatory systems. Regulation involves proteasome-mediated degradation 7,8, phosphatase-mediated dephosphorylation 9 and Ser/Thr phosphorylation. The latter is an attractive regulatory mechanism because it enables multilevel control of the activity of kinases, and of the specific targets among nearly 100 potential Ser/Thr-phosphorylation sites in. It is becoming apparent that Ser/Thr phosphorylation of has a dual function in serving either as a positive or a negative modulator of insulin signaling. Phosphorylation of Ser residues within the P-Tyr-binding (PTB) domain of -1 (Fig. 2), by insulin-stimulated protein kinase B (PKB, also known as Akt), protects from the rapid action of PTPs and enables the to maintain their Tyrphosphorylated active conformation, thus implicating PKB as a positive regulator of -1 functions 10. By contrast, Ser/Thr phosphorylation of by other insulin-stimulated serves as a negative-feedback control mechanism that inhibits further Tyr phosphorylation of. Ser/Thr phosphorylation can induce the dissociation of from the insulin receptor () 11,12, hinder Tyr-phosphorylation sites 13, release the from intracellular complexes that maintain them in close proximity to the receptor 14,15, induce degradation of (b) (e) TK 7,16, or turn into inhibitors of the K 17 (Fig. 2). These observations raise the question: which insulin-stimulated kinases act as negative modulators of protein function? A clue was provided when the activity of the insulin-stimulated inhibitory kinases was blocked by inhibitors of the pathway, implicating downstream effectors of (Fig. 1) as negative regulators of protein function 10,12. A potential candidate is the mammalian target of rapamycin (), which Internal complexes (c) TK X TK (a) TK SHP-2 Nck PH PTB Internal complexes (f) TK enhances phosphorylation of Ser residues at the C-terminus of -1. This phosphorylation inhibits insulinstimulated Tyr phosphorylation of -1 and its ability to bind to 18 20. Recent studies provide evidence that PKCζ, which is activated by insulin 21, mediates phosphorylation of protein 12,22. This leads to the dissociation of the complexes 12 (Fig. 2), inhibits the ability of to undergo insulinstimulated Tyr phosphorylation and terminates insulin signaling. -1 serves as a substrate for PKCζ in vitro, and (d) TK SHP-2 Nck Fig. 2. The impact of Ser/Thr phosphorylation on the function of insulin receptor substrate (). (a) The intracellular segment of the insulin receptor () contains a juxtamembrane () region, a Tyr kinase domain (TK) and a C-terminal () region. The four isoforms of (-1 to -4) contain a conserved pleckstrin homology (PH) domain located at their N-terminus that serves to localize the in close proximity to the receptor 45. contain a phosphotyrosine-binding (PTB) domain C-terminal to their PH domain. This domain binds to the NPXY motif of the region of the to promote -1 interactions 46. The C-terminal region of contains multiple Tyr-phosphorylation motifs that serve as docking sites for Src-homology 2 (SH2)- domain-containing that serve as downstream effectors, such as phosphoinositide 3-kinase (), the phosphotyrosine phosphatase (PTP) SHP-2 or the adaptor protein Nck. Ser/Thr phosphorylation of has several effects: it inhibits insulin-stimulated Tyr phosphorylation of the because it releases the from intracellular insoluble multiprotein complexes that include cytoskeletal elements and maintain the in close proximity to the receptor 15 (b), it induces the dissociation of from the domain of the 11 (c), it inhibits the ability of downstream effectors such as to dock and bind to specific Tyr residues at the C-terminal tail of the 13 (d), it turns into inhibitors of the kinase activity of (K) 17 (e), or it induces the degradation of the 16 (f).

439 Insulin receptor Insulin PDK-1 Agents inducing insulin resistance (e.g. TNFα, FFA oxidative stress) Activation of MAPK PKB JNK PKC IKKβ endogenous -1 forms complexes with PKCζ in an insulin-dependent manner 22. These findings suggest that PKCζ can function as an insulin-stimulated kinase, although downstream effectors of PKCζ could also fulfill this role. A potential candidate effector is the IκB kinase β (IKKβ). Although there is no evidence that IKKβ is activated by insulin, IKKβ binds to PKCζ both in vitro and in vivo, serves as an in vitro substrate for PKCζ and is activated by a functional PKCζ 23. PKB, and PKCζ are downstream effectors of in the insulin signaling pathway. This suggests that their action should be orchestrated to allow phosphorylation by PKB and sustained activation of -1, prior to the activation of or PKCζ the actions of which are expected to terminate insulin signal transduction. Of note, the negativefeedback control mechanism induced by PKCζ (or ) includes a selfattenuation mode, whereby mediated activation of PKCζ inhibits -1 function, reduces complex formation between -1 and and thereby inhibits further activation of PKCζ itself. PKCζ IKKβ Positive-feedback control Negative-feedback control (physiological) Insulin resistance (pathological) Fig. 3. Ser/Thr-phosphorylated insulin receptor substrate () as modulators of insulin action and insulin resistance. Ser/Thr phosphorylation of has a dual role, either to enhance or to terminate signaling by insulin. Ser residues of the phosphotyrosine-binding (PTB) domain of -1, located within consensus protein kinase B (PKB) phosphorylation sites, presumably function as positive effectors of insulin signaling by protecting from the action of protein tyrosine phosphatases (PTPs) 10. Hence, PKB propagates insulin signaling by phosphorylating downstream effectors (Fig. 1) and by phosphorylating, thus generating a positive-feedback loop for insulin action. Insulin also activates and PKCζ, which mediate phosphorylation of Ser/Thr residues within the protein either directly or through activation of downstream effectors such as IκB kinase β (IKKβ). Phosphorylation of these sites is part of the negative-feedback control mechanism induced by insulin that results in the termination of insulin signaling. Agents that abrogate insulin action, such as free fatty acids (FFA) and tumor necrosis factor α (TNFα), take advantage of this negativefeedback control mechanism by activating (e.g. JNK, PKCζ, IKKβ,, MAPK) that, by mediating phosphorylation of, inhibit the function of (cf. Fig. 2), terminate insulin action and induce insulin resistance. Other aspects of insulin signaling are also subjected to homologous desensitization. Chronic stimulation with insulin results in persistent phosphorylation of the GDP GTP exchange factor msos, which keeps it dissociated from the adaptor Grb2 and allows the GTPase Ras to return to its GDP-bound, inactive, phase 24. This process is apparently mediated by a MAPK that phosphorylates msos 25. Hence, two major insulin signaling pathways, mediated by and Shc, are subjected to homologous desensitization in the form of insulin-induced Ser/Thr phosphorylation. This conclusion suggests that there might be value in pharmacological interventions aimed at disease states in which this mechanism is the underlying cause of insulin resistance. Ser/Thr phosphorylation of and insulin resistance A contributing role to the induction of insulin resistance is attributed to agents such as phorbol esters and TNFα 26, whose common feature is their ability to enhance the Ser/Thr phosphorylation that inhibits insulin-stimulated Tyr phosphorylation of 27. Since Ser/Thr phosphorylation of is stimulated by insulin treatment and by inducers of insulin resistance, the question arises as to whether the same kinases and signaling pathways are being activated under both physiological and pathological conditions. Recent studies have attempted to address this question. Role of PKCζ and IKKβ The idea that TNFα and insulin might stimulate the same kinases emerged when it was realized that TNFα activates PKCζ and its downstream target ΙΚΚβ 23,28. Potential mechanisms could involve TNFα-mediated activation of sphingomyelinase 29 and production of ceramide, which stimulates PKCζ activity 30. Indeed, the effects of TNFα are mimicked by sphingomyelinase and ceramide analogs 11,31, suggesting that TNFα triggers a ceramide-activated kinase such as PKCζ. Alternatively, TNFα can induce complex formation between PKCζ, p62 and RIP that serve as adaptors of the TNF receptor and link PKCζ to TNFα signaling 32. More recent studies shifted the spotlight to IKKβ, a downstream target of PKCζ. Activation or overexpression of IKKβ attenuated insulin signaling, whereas IKKβ inhibition by high doses of salicylates 33 or by a reduction in IKKβ gene dose 34 reversed obesity- and dietinduced insulin resistance 34, implicating IKKβ as a potential mediator of insulin resistance. At the molecular level, inhibition of IKKβ prevented Ser/Thr phosphorylation of induced by high-fat diet, TNFα or phosphatase inhibitors, whereas it improved insulinstimulated Tyr phosphorylation of, indicating that IKKβ or its downstream effectors serve as kinases (Fig. 3). The effects of salicylates on protein function were in part secondary to the enhanced K activity induced by salicylate treatment of insulinresistant animals 34. These results suggest that IKKβ can negatively regulate the activity of both and 34, making it a target for insulin sensitization. c-jun N-terminal kinase (JNK) The c-jun N-terminal kinase (JNK) promotes insulin resistance by associating with -1 and phosphorylating Ser307, which inhibits insulin-stimulated Tyrphosphorylation of -1 35. Since Ser307

440 Research Update is adjacent to the PTB domain of -1, its phosphorylation might disrupt the interaction between the juxtamembrane domain of the and the PTB domain of -1 (Fig. 2). Interestingly, insulin and TNFα stimulate phosphorylation of -1 at Ser307 through distinct pathways. While insulin stimulates downstream of, TNFα effects are mediated by members of the MAPK pathway 36. Of note, JNK itself is unlikely to serve as an insulin- or TNFαstimulated kinase because its activity is insensitive to inhibitors that block phosphorylation of Ser307 in response to these stimuli 36. Ser307, phosphorylated by currently unknown kinases, might therefore integrate feedback and heterologous signals to attenuate -1- mediated signals and contribute to insulin resistance. Conventional PKCs and M AP kinases The kinases described above PKCζ, IKKβ and JNK are newcomers to the game and join a respected list of Ser/Thr kinases already implicated in phosphorylating when triggered by agents that induce insulin resistance. These include conventional members of the PKC family, such as PKCα, activated by phorbol esters or endothelin-1 18, the activity of which is mediated, at least partially, by members of the MAPK pathway 37. These kinases phosphorylate -1 at Ser612 (located in a consensus MAPK phosphorylation site) and at additional sites in its C-terminal tail 37. Such phosphorylation prevents the association of -1 with the juxtamembrane domain of, impairs the ability of -1 to undergo insulinstimulated Tyr phosphorylation and inhibits recruitment of downstream effectors such as 18. Obesity, insulin resistance and the kinase connection Elevated levels of FFA are characteristics of obesity, insulin resistance and type 2 diabetes, and increasing evidence supports the contention that FFA inhibit insulin action at peripheral target tissues 38,39. A recent study 40 combined an observation made 120 years ago, indicating that high doses of salicilates lower blood glucose concentrations in diabetic patients 41, with contemporary knowledge regarding obesity and activation of IKKβ induced by high-fat diets, to show that salicilates prevent fatinduced muscle insulin resistance by inhibiting the activity of IKKβ and its ability to mediate phosphorylation and inactivation of -1 function 40. Lipid infusion failed to alter insulin signaling in skeletal muscle of IKKβ knockout mice, further implicating a protective role for IKKβ inactivation in fat-induced development of insulin resistance 40. Although the key data in this study are correlational, they place IKKβ as a potential mediator of Ser phosphorylation of. The mechanism by which lipids might activate IKKβ presumably involves an increase in FFA-derived metabolites, such as diacylglycerol and ceramide, which are potent activators of PKCθ 42 and PKCζ 30, both known to activate IKKβ. Obesity-induced insulin resistance is not limited to the effects of increased levels of FFA or TNFα. Other adipokines secreted by fat cells, such as resistin 43, might also contribute to the development of insulin resistance through Ser/Thr phosphorylation of, but further studies are required to address this possibility. Conclusions and future directions Our understanding at the molecular level of insulin signal transduction, insulin resistance, and the connection between the two, is evolving extremely rapidly. Current findings implicate as major targets for insulin-induced, phosphorylation-based, negative-feedback control mechanisms that uncouple the insulin receptor from its downstream effectors and terminate insulin signaling under physiological conditions. The kinases involved are still under investigation, with current focus on PKCζ, IKKβ and as potential candidates. Recent studies further strengthen the concept that the varied agents and conditions that induce insulin resistance, such as TNFα, FFA and obesity, also activate kinases, with IKKβ and its downstream effectors being key candidates. Still other inducers of insulin resistance, such as endothelin-1, presumably utilize additional kinases to phosphorylate (Fig. 3). These findings raise several pertinent questions: which kinases are indeed the kinases? At present, PKB, PKCζ, IKKβ, MAPK, JNK and appear as potential candidates, but additional kinases are likely to emerge. Even in the case of IKKβ, its role as an kinase is presently unresolved. The facts that inducers of insulin resistance activate IKKβ, while salicylates, which selectively inhibit IKKβ activity, prevent Ser/Thr phosphorylation of and insulin resistance, implicate IKKβ as a potential kinase. Still, there is no direct evidence to indicate that IKKβ indeed phosphorylates, and it might well be that downstream effectors of IKKβ play this role. The rapid phosphorylation of, which occurs upon activation of IKKβ, argues against the possibility that IKKβmediated activation of NFκB leads to de novo synthesis of inducers of insulin resistance. Nevertheless, further studies are required to address this possibility. As each of the potential kinases has a unique substrate specificity, the question remains as to which Ser sites are being modified by each kinase and what the consequences are of such phosphorylation. Present studies indicate that negative-regulatory sites are found both in close proximity to the PTB domain and at the C-terminal end of -1. How does each phosphorylation site affect the structure and consequently the function of the? Ser phosphorylation might impair interactions, or might induce degradation of, and, in so doing, inhibit altogether subsequent -mediated signaling. Alternatively, phosphorylation of a Ser site might selectively interfere with binding of a specific effector of (Fig. 2), thus impeding only selected aspects of insulin signaling. Are the same or different mechanisms being utilized by insulin or agents that induce resistance to regulate the activity of kinases? While recent studies indicate that the same kinases (PKCζ, IKKβ) are being activated (e.g. by insulin, FFA and TNFα), there is no evidence that these kinases are being activated along the same pathways. Indeed, kinases localized along different pathways are activated by insulin and TNFα to induce phosphorylation of the inhibitory Ser307 site. Given the large number of stimuli, pathways, kinases and potential sites involved, it appears that Ser/Thr phosphorylation of represents a combinatorial consequence of several kinases, activated by different pathways, acting in concert to phosphorylate multiple sites.

441 While many questions await answers, the new paradigms and emerging target kinases described above give a novel viewpoint of the molecular basis for insulin resistance. This should enable rational drug design to selectively inhibit the activity of the relevant enzymes and generate a novel class of therapeutic agents for type 2 diabetes. Acknowledgements I am grateful to Steven E. Shoelson and Gerald I. Shulman for communicating their results before publication, and to Ronit Sagi-Eisenberg for most helpful comments and a critical review of this manuscript. I apologize to those authors whose work could not be cited owing to space limitations. References 1 Virkamaki, A. et al. (1999) Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J. Clin. Invest. 103, 931 943 2 LeRoith, D. and Zick, Y. (2001) Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care 24, 588 597 3 Kahn, B.B. and Flier, J.S. (2000) Obesity and insulin resistance. J. Clin. Invest. 106, 473 481 4 Matthaei, S. et al. (2000) Pathophysiology and pharmacological treatment of insulin resistance. Endocrin. Rev. 21, 585 618 5 Taylor, S.I. (1999) Deconstructing type 2 diabetes. Cell 97, 9 12 6 Saltiel, A.R. (2001) New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell 104, 517 529 7 Haruta, T. et al. (2000) A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. Mol. Endocrinol. 14, 783 794 8 Takano, A. et al. (2001) Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Mol. Cell. Biol. 21, 5050 5062 9 Elchebly, M. et al. (1999) Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1b gene. Science 283, 1544 1548 10 Paz, K. et al. (1999) Phosphorylation of insulin receptor substrate-1 (-1) by PKB positively regulates -1 function. J. Biol. Chem. 274, 28816 28822 11 Paz, K. et al. (1997) A molecular basis for insulin resistance: elevated Serine/Threonine phosphorylation of -1 and -2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation. J. Biol. Chem. 272, 29911 29918 12 Liu, Y.F. et al. (2001) Insulin stimulates PKCζmediated phosphorylation of insulin receptor substrate-1 (-1). A self-attenuated mechanism to negatively regulate the function of. J. Biol. Chem. 276, 14459 14465 13 Mothe, I. and Van Obberghen, E. (1996) Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. J. Biol. Chem. 271, 11222 11227 14 Tirosh, A. et al. (1999) Oxidative stress disrupts insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3- kinase in 3T3-L1 adipocytes. A putative cellular mechanism for impaired protein kinase B activation and GLUT4 translocation. J. Biol. Chem. 274, 10595 10602 15 Clark, S.F. et al. (2000) Release of insulin receptor substrate from an intracellular complex coincides with the development of insulin resistance. J. Biol. Chem. 275, 3819 3826 16 Pederson, T.M. et al. (2001) Serine/threonine phosphorylation of -1 triggers its degradation: possible regulation by tyrosine phosphorylation Diabetes 50, 24 31 17 Hotamisligil, G.S. et al. (1996) -1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-α- and obesity-induced insulin resistance. Science 271, 665 668 18 Li, J. et al. (1999) Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway. J. Biol. Chem. 274, 9351 9356 19 Ozes, O.N. et al. (2001) A phosphatidylinositol 3- kinase/akt/ pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1. Proc. Natl. Acad. Sci. U. S. A. 98, 4640 4645 20 Tremblay, F. and Marette, A. Amino acids and insulin signaling via the /p70 S6 kinase pathway: A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J. Biol. Chem. (in press) 21 Standaert, M.L. et al. (1997) Protein kinase C-ζ as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport. J. Biol. Chem. 272, 30075 30082 22 Ravichandran, L.V. et al. (2001) PKC-ζ phosphorylates -1 and impairs its ability to activate PI 3-kinase in response to insulin. J. Biol. Chem. 276, 3543 3549 23 Lallena, M-J. et al. (1999) Activation of IκB kinase β by protein kinase C isoforms. Mol. Cell. Biol. 19, 2180 2188 24 Langlois, W.J. et al. (1995) Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation. J. Biol. Chem. 270, 25320 25323 25 Fucini, R.V. et al. (1999) Insulin-induced desensitization of extracellular signal-regulated kinase activation results from an inhibition of Raf activity independent of Ras activation and dissociation of the Grb2-SOS complex. J. Biol. Chem. 274, 18651 18658 26 Moller, D.E. (2000) Potential role of TNF-α in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol. Metab. 11, 212 217 27 Feinstein, R. et al. (1993) Tumor necrosis factor-α suppresses insulin-induced tyrosine phosphorylation of insulin receptor and its substrates. J. Biol. Chem. 268, 26055 26058 28 Martin, A.G. et al. (2001) Regulation of nuclear factor κ B transactivation. Implication of phosphatidylinositol 3-kinase and protein kinase C ζ in c-rel activation by tumor necrosis factor α. J. Biol. Chem. 276, 15840 15849 29 Adam-Klages, S. et al. (1996) FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86, 937 947 30 Muller, G. et al. (1995) PKC ζ is a molecular switch in signal transduction of TNF-α, bifunctionally regulated by ceramide and arachidonic acid. EMBO J. 14, 1961 1969 31 Kanety, H. et al. (1996) Sphingomyelinase and ceramide suppress insulin-induced tyrosine phosphorylation of the insulin receptor substrate- 1. J. Biol. Chem. 271, 9895 9897 32 Sanz, L. et al. (1999) The interaction of p62 with RIP links the atypical PKCs to NF-κB activation. EMBO J. 18, 3044 3053 33 Yin, M.J. et al. (1998) The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(κ)B kinase-β. Nature 396, 77 80 34 Yuan, M. et al. (2001) Reversal of obesity and diet induced insulin resistance with salicylates or targeted disruption of IKK β. Science 293, 1673 1677 35 Aguirre, V. et al. (2000) The c-jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J. Biol. Chem. 275, 9047 9054 36 Rui, L. et al. (2001) Insulin/IGF-1 and TNF-α stimulate phosphorylation of -1 at inhibitory Ser307 via distinct pathways. J. Clin. Invest. 107, 181 189 37 De Fea, K. and Roth, R. A. (1997) Modulation of insulin receptor substrate-1 tyrosine phosphorylation and function by mitogenactivated protein kinase. J. Biol. Chem. 272, 31400 31406 38 Shulman, G.I. (2000) Cellular mechanisms of insulin resistance. J. Clin. Invest. 106, 171 176 39 Spiegelman, B.M. and Flier, J.S. (2001) Obesity and the regulation of energy balance. Cell 104, 531 543 40 Kim, J.K. et al. (2001) Prevention of fat-induced insulin resistance by salicylate J. Clin. Invest. 108, 437 446 41 Ebstein, W. (1876) Berliner Klinische Wochnschrift 13, 337 340 42 Schmitz-Peiffer, C. et al. (1997) Alterations in the expression and cellular localization of protein kinase C isozymes ε and β are associated with insulin resistance in skeletal muscle of the highfat-fed rat. Diabetes 46, 169 178 43 Steppan, C.M. et al. (2001) The hormone resistin links obesity to diabetes. Nature 409, 307 312 44 Pessin, J.E. and Saltiel, A.R. (2000) Signaling pathways in insulin action: molecular targets of insulin resistance. J. Clin. Invest. 106, 165 169 45 Voliovitch, H. et al. (1995) The pleckstrinhomology (PH) domain of insulin receptor substrate-1 (-1) is required for proper interaction of -1 with the insulin receptor. J. Biol. Chem. 270, 18083 18087 46 Eck, M.J. et al. (1996) Structure of the -1 PTB domain bound to the juxtamembrane region of the insulin receptor. Cell 85, 695 705 Yehiel Zick Dept of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel. e-mail: Yehiel.zick @ weizmann.ac.il