Cellular mechanism of insulin resistance: potential links with inflammation

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1 (2003) 27, S6 S11 & 2003 Nature Publishing Group All rights reserved /03 $25.00 PAPER Cellular mechanism of insulin resistance: potential links with inflammation G Perseghin 1, K Petersen 2 and GI Shulman 2,3,4 * 1 Internal MedicineFSection of Nutrition/Metabolism and Unit of Clinical Spectroscopy, Istituto Scientifico H San Raffaele via Olgettina 60, Milan, Italy; 2 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; 3 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA; and 4 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Insulin resistance is a pivotal feature in the pathogenesis of type 2 diabetes, and it may be detected y before the clinical onset of hyperglycemia. Insulin resistance is due to the reduced ability of peripheral target tissues to respond properly to insulin stimulation. In particular, impaired insulin-stimulated muscle glycogen synthesis plays a significant role in insulin resistance. Glucose transport (GLUT4), phosphorylation (hexokinase) and storage (glycogen synthase) are the three potential ratecontrolling steps regulating insulin-stimulated muscle glucose metabolism, and all three have been implicated as being the major defects responsible for causing insulin resistance in patients with type 2 diabetes. Using 13 C/ 31 P magnetic resonance spectroscopy (MRS), we demonstrate that a defect in insulin-stimulated muscle glucose transport activity is the rate-controlling defect. Using a similar 13 C/ 31 P MRS approach, we have also demonstrated that fatty acids cause insulin resistance in humans due to a decrease in insulin-stimulated muscle glucose transport activity, which could be attributed to reduced insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. Furthermore, we have recently proposed that this defect in insulin-stimulated muscle glucose transport activity may be due to the activation of a serine kinase cascade involving protein kinase Cy and IKK-b, which are key downstream mediators of tissue inflammation. Finally, we propose that any perturbation that leads to an increase in intramyocellular lipid (fatty acid metabolites) content such as acquired or inherited defects in mitochondrial fatty acid oxidation, defects in adipocyte fat metabolism or simply increased fat delivery to muscle/liver due to increased energy intake will lead to insulin resistance through this final common pathway. Understanding these key cellular mechanisms of insulin resistance should help elucidate new targets for treating type 2 diabetes. (2003) 27, S6 S11. doi: /sj.ijo Keywords: insulin resistance; fatty acids; glycogen; glucose transport; magnetic resonance spectroscopy; salicylate; protein kinase C; IKK-b Introduction The primary factors causing type 2 diabetes mellitus are still unknown, but it is clear that insulin resistance plays a pivotal role in its development. Evidence for this comes from studies demonstrating that insulin resistance is a consistent finding in patients with type 2 diabetes; 1 it may be detected y before the onset of overt hyperglycemia 2 and prospective studies have demonstrated that insulin resistance is the best predictor of whether or not an individual will later become diabetic. 3 It is estimated that by the year 2020, there will be *Correspondence: GI Shulman, Investigator, Professor of Internal Medicine and Cellular and Molecular Physiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Congress Avenue, S269 New Haven, CT 06520, USA. gerald.shulman@yale.edu approximately 250 million people affected by type 2 diabetes mellitus worldwide. 4 It is therefore important to understand the pathogenic mechanisms of insulin resistance in order to identify novel targets for primary and secondary prevention. In this short review, we will focus on some recent advances in our understanding of insulin resistance in man. In particular, we will discuss the following: (1) potential ratecontrolling steps involved in insulin-stimulated muscle glucose metabolism and (2) new insights into the mechanism of fatty acid-induced insulin resistance in skeletal muscle. Insulin resistance and muscle glucose metabolism Using 13 C NMR spectroscopy to monitor the rate of [1-13 C] glucose incorporation into muscle glycogen we found that,

2 under steady-state plasma concentrations of insulin and glucose that mimic postprandial conditions, muscle glycogen synthesis was approximately 50% lower in diabetic subjects than in normal volunteers. 5 When the mean rate of muscle glycogen synthesis was extrapolated to the whole body, the synthesis of muscle glycogen accounted for most of the whole-body glucose uptake, and virtually all of the nonoxidative glucose metabolisms in both normal and diabetic subjects. These studies demonstrate that under hyperglycemic, hyperinsulinemic conditions, muscle glycogen synthesis is the major pathway for glucose metabolism in both normal and diabetic individuals, and that defective muscle glycogen synthesis plays a major role in causing insulin resistance in patients with type 2 diabetes. In a subsequent study, we performed 13 C and 31 P NMR studies to measure intramuscular glycogen synthetic rates and concentrations of glucose-6-phosphate in the muscles of patients with type 2 diabetes and muscle of age- and weight-matched control subjects. 6 Intracellular glucose-6- phosphate is an intermediary metabolite between glucose transport/hexokinase and glycogen synthesis, so its intracellular concentration will respond to the relative activities of these two steps. In the event of decreased activity of glycogen synthase in diabetes, glucose-6-phosphate concentrations in diabetic patients would be expected to increase relative to that of normal individuals. In that series of experiment, we found that incremental changes in glucose- 6-phosphate in the type 2 diabetic patients in response to insulin stimulation were significantly blunted, suggesting either decreased glucose transport activity or decreased hexokinase II activity as the cause of muscle insulin resistance. To examine whether this defect was a primary defect or an acquired defect secondary to other factors, we also studied lean, normoglycemic, insulin-resistant offspring of parents with type 2 diabetes (IR offspring), examining the rate of muscle glycogen synthesis and the muscle glucose-6- phosphate concentration under the same clamp conditions. 7 These individuals have a high likelihood of developing diabetes later in life. 2 Using this approach, we found that these IR offspring had a 50% reduction in the rate of insulinstimulated whole-body glucose metabolism, which could be attributed to a decrease in the rate of muscle glycogen synthesis. This reduction in insulin-stimulated muscle glycogen synthesis was associated with a blunted insulinstimulated increment of intramuscular glucose-6-phosphate concentration. The data suggest that defects in glucose transport/phosphorylation activity are a very early event in the pathogenesis of type 2 diabetes. We next examined whether we could reverse this defect in glucose transport/ phosphorylation activity with chronic exercise training. A similar cohort of IR offspring was recruited to exercise four times a week at 65% VO 2 max for 40 min on a stairmaster for 6 weeks. We found that following this exercise regimen, the IR offspring normalized their rates of insulin-stimulated muscle glycogen synthesis, which could be attributed to correction of their defect in glucose transport/phosphoryla- tion activity. 8 These data suggest that aerobic exercise might be useful in reversing the insulin resistance in these prediabetic individuals and thus prevent the development of type 2 diabetes. Finally, to delineate whether glucose transport or hexokinase II activity was rate controlling for insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes, we developed a novel 13 C NMR method to assess intracellular free-glucose concentrations noninvasively in muscles. 9 Intracellular glucose is an intermediary metabolite between glucose transport and glucose phosphorylation, and its concentration reflects the relative activities of glucose transporters (particularly GLUT4) and of hexokinase II. If hexokinase II activity is reduced relative to glucose transport activity in diabetes, one would predict a substantial increase in the intracellular glucose concentration, whereas if glucose transport is primarily responsible for maintaining intracellular glucose metabolism, intracellular glucose and glucose- 6-phosphate should change proportionately. We found that the intracellular glucose concentration was far lower in the diabetic subjects than the concentration expected if hexokinase II was the primary rate-controlling enzyme for glycogen synthesis. These data suggested a predominant role for glucose transport control of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes, but they do not rule out the possibility that there are other downstream abnormalities in the pathway of glycogen synthesis that do not exert rate-controlling effects under these conditions. Fatty acid-induced muscle insulin resistance Increased plasma free fatty acid concentrations are typically associated with many insulin-resistant states, including obesity and type 2 diabetes mellitus. 10 In a cross-sectional study of young, normal-weight offspring of type 2 diabetic patients, we found an inverse relationship between fasting plasma fatty acid concentrations and insulin sensitivity, consistent with the hypothesis that altered fatty acid metabolism contributes to insulin resistance in patients with type 2 diabetes. 11 Furthermore, recent studies measuring intramyocellular triglyceride content by 1 H NMR 12,13 have shown an even stronger relationship between the accumulation of intramyocellular triglyceride and insulin resistance. In a classic series of studies, Randle et al demonstrated that fatty acids compete with glucose for substrate oxidation in isolated rat heart muscle and rat diaphragm muscle. They speculated that increased fat oxidation causes the insulin resistance associated with obesity. 14 The mechanism that they proposed to explain the insulin resistance was that an increase in fatty acids caused an increase in the intramitochondrial acetyl CoA/CoA and NADH/NAD þ ratios, with subsequent inactivation of pyruvate dehydrogenase. This in turn would cause intracellular citrate concentrations to increase, leading to inhibition of phosphofructokinase, a key rate-controlling enzyme in glycolysis. Subsequent accu- S7

3 S8 mulation of glucose-6-phosphate would inhibit hexokinase II activity, resulting in an increase in intracellular glucose concentrations and decreased glucose uptake. A recent series of studies by our group has challenged this conventional hypothesis. 15 We used 13 C and 31 P MR spectroscopy to measure skeletal muscle glycogen and glucose-6-phosphate concentrations in healthy subjects. The subjects were maintained at euglycemic, hyperinsulinemic conditions with either low or high levels of plasma fatty acids. The increment of the plasma fatty acid concentration for 5 h caused a reduction of approximately 50% in insulinstimulated rates of muscle glycogen synthesis and wholebody glucose oxidation compared to controls. In contrast to the results from the model of Randle and co-workers, which predicted that fat-induced insulin resistance would result in an increase in intramuscular glucose-6-phosphate, we found that the drop in muscle glycogen synthesis was preceded by a fall in intramuscular glucose-6-phosphate, suggesting that increases in plasma fatty acid concentrations initially induce insulin resistance by inhibiting glucose transport or phosphorylation activity, and that the reduction in muscle glycogen synthesis and glucose oxidation follows. The reduction in insulin-activated glucose transport and phosphorylation activity in normal subjects maintained at high plasma fatty acid levels is similar to that seen in obese individuals, 16 patients with type 2 diabetes, 5,6 and lean, normoglycemic insulin-resistant offspring of type 2 diabetic individuals. 7,8 Hence, accumulation of intramuscular fatty acids (or fatty acid metabolites) appears to play an important role in the pathogenesis of insulin resistance seen in obese patients and patients with type 2 diabetes. To distinguish between the possible effects of fatty acids on glucose transport activity and on hexokinase II activity, we measured intracellular concentrations of glucose in muscle using 13 C magnetic resonance spectroscopy (MRS). 17 The logic of this experiment was similar to that described above, in which we followed intramyocellular concentrations of glucose-6-phosphate to determine the relative activity of glucose transport and glycogen synthesis. As intracellular glucose is an intermediary metabolite between glucose transport and hexokinase II, its concentration reflects the relative activities of these two steps. If a decrease in hexokinase II activity was responsible for the lower rate of insulin-stimulated muscle glycogen synthesis, intracellular glucose concentrations should increase. However, if the impairment was at the level of glucose transport, there should be no difference or a decrease in the intracellular glucose concentration. We found that elevated plasma fatty acid concentrations caused a significant reduction in intracellular glucose concentration in the lipid infusion studies compared to control studies in which glycerol (the other metabolite released by lipolysis) was infused in the absence of any exogenous fatty acid. These data imply that the rate-controlling step for fatty acidinduced insulin resistance in humans is glucose transport, and offer further evidence against the Randle mechanism, which predicts an increase in both intracellular glucose-6- phosphate and glucose concentrations. This reduced glucose transport activity could be the result of fatty acid effects on the GLUT4 transporter directlyfalterations in the trafficking, budding, fusion or activity of GLUT4For it could result from fatty acid-induced alterations in upstream insulin signaling events, resulting in decreased GLUT4 translocation to the plasma membrane. To explore the latter possibility, we examined IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in muscle biopsy samples, using the identical lipid infusion protocol described above for the study of fatty acid effects. We found that elevations in plasma fatty acid concentrations similar to the previous MRS studies abolished insulin-stimulated IRS-1-associated PI 3- kinase activity compared with a four-fold insulin stimulation observed in the glycerol-control infusion studies. 17 The reduced insulin-stimulated PI 3-kinase activity may be due to a direct effect of intracellular free fatty acids (or some fatty acid metabolite) on PI 3-kinase, or may be secondary to alterations in upstream insulin signaling events. Consistent with an indirect effect, we found that a similar lipid infusion protocol in rats resulted in a reduction of insulin-stimulated IRS-1 tyrosine phosphorylation, which was associated with the activation of protein kinase Cy, 18 a known serine kinase that has been shown to be activated by diacylglycerol. An attractive hypothesis to account for the effects of fatty acids in muscle cells may be that increasing intracellular fatty acid metabolites, such as diacylglycerol or fatty acyl CoAs, activates a serine/threonine kinase cascade or protein kinase C, leading to phosphorylation of serine/threonine sites on insulin receptor substrates. 19 Serine-phosphorylated forms of these proteins fail to associate with and activate PI 3-kinase, resulting in decreased activation of glucose transport and other downstream-associated events. If this hypothesis is correct, any perturbation that results in accumulation of intracellular fatty acyl CoAs or other fatty acid metabolites in muscle and liver, either through increased delivery and/or decreased metabolism, might be expected to induce insulin resistance. Prevention of fatty acid-induced muscle insulin resistance by salicylate-induced inhibition of IKK-b In 1876, Ebstein 20 and again in 1901 Williamson and Lond 21 found that high doses of sodium salicylate dramatically reduced glucosuria in diabetic patients. In the 1950s, more detailed prospective analyses suggested that glucose homeostasis might be improved by aspirin therapy in diabetic subjects; in the 1980s, conflicting results on the effects of aspirin in normal and diabetic subjects were subsequently reported. The mechanism by which salicylate may affect whole-body glucose metabolism remained unknown until Yin et al 22 discovered that salicylate inhibits the activity of a known serine kinase called IkB kinase-b (IKK-Eb). In our laboratory, we hypothesized that salicylate might prevent fatty acid-induced insulin resistance in skeletal muscle by inhibiting the activity of IKK-b, and subsequent

4 serine phosphorylation of IRS-1 by IKK-b and decreased activation of IRS-1-associated PI 3-kinase. Initially, we pursued this aim in animal models using two approaches: first, by examining whether high-dose salicylate pretreatment would prevent the above-described fatty acid-induced alterations in insulin activation of IRS-1-associated PI 3- kinase and skeletal muscle glucose metabolism; and second, by examining whether mice with inactivation of IKK-b were protected from fat-induced alteration in skeletal muscle insulin signaling and action. 23 In awake rats, we estimated whole-body glucose turnover using [3-3 H]glucose, glucose uptake in individual tissues using [1-14 C]deoxy-glucose injection during hyperinsulinemic euglycemic clamps associated with lipid infusion in animals with or without pretreatment with salicylate. This study demonstrated that the deleterious effects of lipid infusion on whole body and tissue glucose metabolism (muscle glycolysis and glycogen synthesis) and on insulin action (tyrosine phosphorylation of IRS-1 and IRS-1-associated PI 3-kinase activity) observed in the animals without salicylate pretreatment were prevented in the animal pretreated with salicylate. Moreover, the IKK-b knockout mice did not exhibit the altered skeletal muscle insulin action and glucose metabolism during the lipid infusion, further confirming that both high-dose salicylate and inhibition of IKK-b equally prevented fat-induced skeletal muscle insulin resistance. However, the effects of aspirin in patients with type 2 diabetes remained less clear. For this reason, we decided to examine this hypothesis in patients with type 2 diabetes. We measured basal and insulin-stimulated rates of whole-body glucose metabolism before and after high-dose aspirin therapy (7 g/day), using hyperinsulinemic euglycemic clamps in combination with indirect calorimetry and [6,6-2 H 2 ]glucose turnover measurements. In addition, mixed meal tolerance testing was carried out before, during and following the aspirin treatment. 24 We found that a 2-week trial of high-dose aspirin treatment was accompanied by significant decreases in hepatic glucose production (22%), fasting plasma glucose (24%), fatty acids (50%) and triglycerides (48%), and a 19% increase in peripheral glucose disposal. Aspirin therapy also resulted in significant reductions in postprandial hyperglycemia during the mixed meal tolerance test, which can likely be attributed to the combined effects of increased peripheral insulin sensitivity and higher plasma insulin concentrations (due to decreased insulin clearance). Aspirin therapy also resulted in significant reductions in both fasting and postprandial plasma fatty acid concentration, suggesting that high-dose aspirin may also have an insulin-sensitizing effect or direct antilipolytic effect on adipocytes leading to reduced rates of lipolysis and lower plasma fatty acid levels, which in turn could lead to enhanced insulin sensitivity in the liver and muscle. Of course, in view of the potential toxicities associated with chronic high-dose aspirin, we would strongly advocate against its use for the treatment of type 2 diabetes; however, these data do support the potential role of IKK-b in mediating insulin resistance in patients with type 2 diabetes. A unifying hypothesis for common forms of human insulin resistance An attractive hypothesis to account for the effects of fatty acids in muscle cells is shown in Figure 1. This model holds that increasing intracellular fatty acid metabolites (diacylglycerol, fatty acyl CoAs, etc) activates a serine/threonine kinase cascade (possibly initiated by protein kinase Cy, 18,19 protein kinase C, 25 IKK-b, 22,23 c-jun amino-terminal kinases (JNKs)), 26 leading to phosphorylation of serine sites (eg Ser 307) on insulin receptor substrates. 27 Serine-phosphorylated forms of these proteins fail to associate with and activate PI 3-kinase, resulting in decreased activation of glucose transport and other downstream events. If this hypothesis is correct, any perturbation that results in accumulation of intracellular fatty acyl CoAa or other fatty acid metabolites in the muscle and liver, either through increased delivery and/or decreased mitochondrial metabolism, might be expected to induce insulin resistance. Evidence supporting this hypothesis comes from recent studies in transgenic mice that are almost totally devoid of fat because their adipocytes express the A-ZIP/F-1 protein, which blocks the function of several classes of transcription factors. 28 These mice are severely insulin resistant, due to defects in insulin action, particularly IRS-dependent activation of PI 3-kinase, in the muscle and liver. 29 Interestingly, these abnormalities were associated with a two-fold increase in muscle and liver triglyceride content, and upon transplantation of fat tissue into these mice, triglyceride content in the muscle and liver returned to normal, as did insulin signaling and action. These findings are consistent with the hypothesis that insulin resistance develops in obesity, type 2 diabetes and lipodystrophy because of alterations in the partitioning of fat between the adipocyte and muscle or liver. This change leads Plasma Glucose Fatty Acids GLUT 4 + PI 3 kinase Glucose IRS-1/IRS-2 tyrosine phosphorylation - Fatty acyl CoA diacylglycerol IRS-1/IRS-2 serine/threonine phosphorylation + JNK-1 insulin + PKC θ PKC ßII PKC δ IKK ß Figure 1 Proposed mechanism for fatty acid-induced insulin resistance in human muscle. An increase in the delivery of fatty acids to muscle or a decrease in the intracellular metabolism of fatty acids leads to an increase in intracellular fatty acid metabolites, such as diacylglycerol, ceramides and fatty acyl CoA. These metabolites activate a serine/threonine kinase cascade possibly initiated by PK Cy, PKCbII, PKC d or by IKK-b or JNK. leading to phosphorylation of serine/threonine sites of the insulin receptor substrates (IRS-1 and IRS-2), which in turn reduces the ability of IRSs to activate PI 3- kinase. As a consequence, glucose transport activity and other events downstream of insulin receptor signaling are diminished. S9

5 S10 to the intracellular accumulation of triglycerides and, probably more importantly, of certain intracellular fatty acid metabolites (fatty acyl CoAs, diacylglycerol, among others) in these insulin-responsive tissues, which leads to acquired insulin signaling defects and insulin resistance. This hypothesis might also explain how thiazolidinediones improve insulin sensitivity in muscle and liver tissue: by activating PPARg receptors in the adipocyte and promoting adipocyte dedifferentiation, these agents might promote a redistribution of fat from the liver and muscle into the adipocyte, much as fat transplantation does in fat-deficient mice. 30 This hypothesis is supported by some recent thiazolidinedione studies in high-fat-fed rats. 31 It might also be expected that any alteration in the ability of muscle and liver to metabolize fatty acids, such as inherited or acquired defects in mitochondrial function, would also lead to intracellular accumulation of fatty acid metabolites and subsequent defects in insulin signaling and action. Indeed, a recent study has demonstrated that insulin resistance in the elderly can be attributed to increased intramyocellular lipid content due to an age-associated reduction in mitochondrial oxidative-phosphorylation acivity. 32 Given the polygenic nature of type 2 diabetes, it is likely that examples of both of these possibilities will be identified in patients with type 2 diabetes. This mechanism, if it proves to be correct, offers many new therapeutic targets for novel insulin-sensitizing agents. Conclusion In conclusion, we present data in this review to support the following hypotheses: (1) that insulin resistance in skeletal muscle can mostly be attributed to defects in insulinstimulated glucose transport; (2) in contrast to the original mechanism proposed by Randle et al, where fatty acids cause insulin resistance by inhibiting pyruvate dehydrogenase activity, we found that fatty acids cause insulin resistance in human skeletal muscle by interfering with insulinstimulated glucose transport activity; (3) reduced insulin activation of glucose transport activity can be attributed to a defect in insulin-stimulated IRS-1-associated PI 3-kinase activity at the level of IRS-1 tyrosine phosphorylation. This in turn can be ascribed to fatty acid activation of a serine kinase cascade possibly involving PKCy (rodents) or PKC (fill in isoform) (humans), IKK-b and JNKs that lead to increased serine phosphorylation of IRS-1 at critical sites, which interfere and block tyrosine phosphorylation of IRS-1 tyrosine sites, which are required to bind and activate PI 3- kinase; (4) any perturbation that leads to an increase in intramyocellular lipid (fatty acid metabolites) content, such as acquired or inherited defects in mitochondrial fatty acid oxidation, defects in adipocyte fat metabolism or simply increased fat delivery due to increased energy intake, will lead to insulin resistance through this common pathway. Understanding these key cellular mechanisms of insulin resistance should help elucidate new targets for treating type 2 diabetes. References 1 De Fronzo RA. The triumvirate beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 1988; 37: Warram JH, Martin BC, Krolewski AS, Soeldner JS, Kahn CR. Slow glucose removal rate and hyperinsulinemia precede the development of Type 2 diabetes in the offspring of diabetic parents. Ann Int Med 1990; 113: Lillioja S, Mott DM, Spraul M, Ferraro R, Foley JE, Ravussin E, Knowler WC, Bennett PH, Bogardus C. Insulin resistance and insulin secretory dysfunction as precursors of non-insulindependent diabetes mellitus. Prospective studies of Pima Indians. N Engl J Med 1993; 329: O Rahilly S. Science, medicine, and the future. Non-insulin dependent diabetes mellitus: the gathering storm. BMJ 1997; 314: Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13 C nuclear magnetic resonance spectroscopy. N Engl J Med 1990; 322: Rothman DL, Shulman RG, Shulman GI. 31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate: evidence for reduced insulin-dependent muscle glucose transport or phosphorylation activity in non-insulin dependent diabetes mellitus. J Clin Invest 1992; 89: Rothman DL, Magnusson I, Cline G, Gerard D, Kahn CR, Shulman RG, Shulman GI. Decreased muscle glucose transport/ phosphorylation is an early defect in the pathogenesis of noninsulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 1995; 92: Perseghin G, Price TB, Petersen KF, Roden M, Cline GW, Gerow K, Rothman DL, Shulman GI. Increased glucose transport/phosphorylation and muscle glycogen synthesis after exercise training in insulin resistant subjects. N Engl J Med 1996; 335: Cline GW, Petersen KF, Krssak M, Shen J, Hundal RS, Trajanoski Z, Inzucchi S, Dresner A, Rothman DL, Shulman GI. Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N Engl J Med 1999; 341: McGarry JD. What if Minkowski had been ageusic? An alternative angle on diabetes. Science 1992; 258: Perseghin G, Ghosh S, Gerow K, Shulman GI. Metabolic defects in lean nondiabetic offspring of NIDDM parents. A cross-sectional study. Diabetes 1997; 46: Krssak M, Falk Petersen K, Dresner A, DiPietro L, Vogel SM, Rothman DL, Roden M, Shulman GI. Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1 H NMR spectroscopy study. Diabetologia 1999; 42: Perseghin G, Scifo P, De Cobelli F, Pagliato E, Battezzati A, Arcelloni C, Vanzulli A, Testolin G, Pozza G, Del Maschio A, Luzi L. Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1 H- 13 C NMR spectroscopy assessment in offspring of type 2 diabetic parents. Diabetes 1999; 48: Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucosefatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963; i: Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, Shulman GI. Mechanism of free fatty acid induced insulin resistance in humans. J Clin Invest 1996; 97: Petersen KF, Hendler R, Price TB, Perseghin G, Rothman DL, Held N, Amatruda J, Shulman GI. 13 C/ 31 P NMR studies on the mechanism of insulin resistance in obesity. Diabetes 1998; 47:

6 17 Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI. Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin Invest 1999; 103: Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI. Free fatty acidinduced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade. Diabetes 1999; 48: Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)- associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002; 277: Ebstein W. Zur therapie des diabetes mellitus, insbesondere uber die anwendung des salicylsauren natron bei demselben. Berl Klin Wochenschr 1877; 24: Williamson RT, Lond MD. On the treatment of glycosuria and diabetes mellitus with sodium salicylate. BMJ 1901; 1: Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IkB kinase-b. Nature 1998; 396: Kim J, Kim Y, Fillmore J, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI. Prevention of fatinduced insulin resistance by salicylate. J Clin Invest 2001; 108: Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 2002; 109: Itani SI, Ruderman NB, Schmieder F, Boden G. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha. Diabetes 2002; 51: Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS. A central role for JNK in obesity and insulin resistance. Nature 2002; 420: Lee YH, Giraud J, Davis RJ, White MF. c-jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem 2003; 278: Moitra J, Mason MM, Olive M, Krylov D, Gavrilova O, Marcus- Samuels B, Feigenbaum L, Lee E, Aoyama T, Eckhaus M, Reitman ML, Vinson C. Life without white fat: a transgenic mouse. Genes Dev 1998; 12: Kim JK, Gavrilova O, Chen Y, Reitman ML, Shulman GI. Mechanism of insulin resistance in A-ZIP/F-1 fatless mice. J Biol Chem 2000; 275: Gavrilova O, Marcus-Samuels B, Graham D, Kim JK, Shulman GI, Castle AL, Vinson C, Eckhaus M, Reitman ML. Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice. J Clin Invest 2000; 105: Ye JM, Frangioudakis G, Iglesias MA, Furler SM, Ellis B, Dzamko N, Cooney GJ, Kraegen EW. Prior thiazolidinedione treatment preserves insulin sensitivity in normal rats during acute fatty acid elevation: role of the liver. Endocrinology 2002; 143: Petersen KF, Befroy D, Dufour S, Dziura J, Ariyan C, Rothman DL, DiPietro L, Cline GW, Shulman GI. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science 2003; 300: S11