Prevention of fat-induced insulin resistance by salicylate

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and may involve fat-induced activation of a serine kinase cascade involving IKK-β. To test this hypothesis, we first examined insulin action and signaling in awake rats during hyperinsulinemic-euglycemic clamps after a lipid infusion with or without pretreatment with salicylate, a known inhibitor of IKK-β. Whole-body glucose uptake and metabolism were estimated using [3-³H]glucose infusion, and glucose uptake in individual tissues was estimated using [1-¹⁴C]2-deoxyglucose injection during the clamp. Here we show that lipid infusion decreased insulin-stimulated glucose uptake and activation of IRS-1–associated PI 3-kinase in skeletal muscle but that salicylate pretreatment prevented these lipid-induced effects. To examine the mechanism of salicylate action, we studied the effects of lipid infusion on insulin action and signaling during the clamp in awake mice lacking IKK-β. Unlike the response in wild-type mice, IKK-β knockout mice did not exhibit altered skeletal muscle insulin signaling and action following lipid infusion. In summary, high-dose salicylate and inactivation of IKK-β prevent fat-induced insulin resistance in skeletal muscle by blocking fat-induced defects in insulin signaling and action and represent a potentially novel class of therapeutic agents for type 2 diabetes.

Insulin resistance plays a primary role in the development of type 2 diabetes (1) and is a characteristic feature of other health disorders including obesity, dyslipidemias, hypertension, and cardiovascular disease (2). The mechanism by which insulin resistance occurs is unknown but may be related to alterations in fat metabolism (3). More than 30 years ago, Randle and colleagues (4) introduced the concept of substrate competition between glucose and fatty acid (i.e., glucose-fatty acid cycle) and postulated that fatty acids cause insulin resistance through an increase in the NADH/NAD and acetyl CoA/CoA ratios and citrate, leading to the inhibition of pyruvate dehydrogenase (5) and phosphofructokinase activity (6). Subsequently, glucose 6-phosphate concentration increases, which leads to the inhibition of hexokinase activity and glucose uptake (7). Recent studies by our group have challenged this substrate competition mechanism and found that acute elevations in plasma fatty acids in humans result in decreased glucose transport activity, as reflected by decreased concentrations of intracellular glucose 6-phosphate (8) and glucose (9). Furthermore, we have demonstrated that an acute elevation of plasma fatty acids resulted in activation of protein kinase C-θ (PKC-θ) (10), a serine kinase, and postulated that this leads to serine phosphorylation of insulin receptor substrate (IRS)-1, which reduces the ability of IRS-1 to activate phosphatidylinositol 3-kinase (PI 3-kinase), an important mediator of insulin signaling and action (11).

Almost 100 years ago, Williamson and colleagues (12) showed that high-dose salicylate treatment reduced the severity of glycosuria in diabetic patients, and in 1957, Reid and colleagues (13) further demonstrated that 10–14 days of aspirin treatment improved the results of oral glucose tolerance tests in diabetic patients. The mechanism by which salicylate may affect whole-body glucose homeostasis remained unknown until a recent finding by Yin and colleagues (14) that salicylate inhibits the activity of IκB kinase-β (IKK-β), a known serine kinase. In light of these findings, we hypothesized that salicylate might prevent fat-induced insulin resistance in skeletal muscle by inhibiting the activity of IKK-β and subsequent serine phosphorylation of IRS-1 by IKK-β and decreased activation of IRS-1–associated PI 3-kinase. To test this hypothesis, we examined whether high-dose salicylate pretreatment would prevent fat-induced alterations in insulin activation of IRS-1–associated PI 3-kinase and skeletal muscle glucose uptake. In addition, we examined whether mice with inactivation of IKK-β are protected from fat-induced alterations in skeletal muscle insulin signaling and action.

The effect of high-dose salicylate pretreatment on fat-induced insulin resistance was examined in awake rats. Plasma salicylate concentrations were raised to approximately 2 mM after a 19-hour salicylate pretreatment and remained elevated during clamps in the salicylate and lipid-salicylate groups (Figure 1a). Plasma fatty acid concentrations were increased to approximately 2.5 mM after a 5-hour lipid infusion and remained elevated during clamps in the lipid and lipid-salicylate groups (Figure 1b). In contrast, plasma fatty acid concentrations were suppressed by approximately 80% during clamps in the control and salicylate groups. A 19-hour salicylate pretreatment or 5-hour lipid infusion did not alter plasma glucose and insulin concentrations (Table 1). During the clamps, plasma insulin concentrations were raised to approximately 3,000 pM, whereas plasma glucose was clamped at approximately 5.9 mM in all groups (Table 1). The rate of glucose infusion needed to maintain euglycemia increased rapidly in the control and salicylate groups and reached a steady state within 30 minutes. In contrast, the glucose infusion rate required to maintain euglycemia in the lipid group was markedly reduced (Table 1), indicating that lipid infusion caused insulin resistance in these animals. Salicylate pretreatment prevented the lipid-induced decrease in steady-state glucose infusion rate.

Figure 1

Metabolic parameters and insulin-stimulated whole-body and skeletal muscle (soleus) glucose uptake in awake control (black bars), salicylate (light gray bars), lipid (open bars), and lipid-salicylate (dark gray bars) rats. (a) Plasma salicylate concentrations during basal and hyperinsulinemic-euglycemic clamp. (b) Plasma FFA concentrations during basal and hyperinsulinemic-euglycemic clamp. (c) Insulin-stimulated whole-body glucose uptake in vivo. (d) Insulin-stimulated skeletal muscle glucose uptake in vivo. Values are means ± SE for six to seven experiments. *P < 0.05 versus control group.

Table 1

Metabolic parameters during basal and hyperinsulinemic-euglycemic clamp periods and steady-state glucose infusion rate (GINF)^A

Insulin-stimulated whole-body glucose uptake was decreased by 37% with the lipid infusion (Figure 1c). Salicylate pretreatment prevented decreases in insulin-stimulated whole-body glucose uptake caused by lipid infusion. Decreases in insulin-stimulated whole-body glucose uptake were due to a 41% decrease in insulin-stimulated glucose uptake in skeletal muscle (soleus) with lipid infusion (Figure 1d). Salicylate pretreatment also prevented the lipid-induced decrease in insulin-stimulated skeletal muscle glucose uptake. Furthermore, insulin-stimulated whole-body glycolysis and glycogen/lipid synthesis were significantly decreased by 27% and 43%, respectively, with the lipid infusion (Figure 2, a and b). Salicylate pretreatment prevented these decreases in insulin-stimulated whole-body glucose metabolic flux caused by lipid infusion. Similar to the decreases in insulin-stimulated whole-body glucose metabolic flux with lipid infusion, insulin-stimulated skeletal muscle glycolysis was decreased by 36% with lipid infusion (Figure 2c), but the most profound change was a 66% decrease in insulin-stimulated skeletal muscle glycogen synthesis with lipid infusion (Figure 2d). Salicylate pretreatment also prevented the lipid-induced decreases in insulin-stimulated skeletal muscle glucose metabolism.

Figure 2

Insulin-stimulated whole-body and skeletal muscle (soleus) glucose metabolic flux in awake control (black bars), salicylate (light gray bars), lipid (open bars), and lipid-salicylate (dark gray bars) rats. (a) Insulin-stimulated whole-body glycolysis in vivo. (b) Insulin-stimulated whole-body glycogen/lipid synthesis in vivo. (c) Insulin-stimulated skeletal muscle glycolysis in vivo. (d) Insulin-stimulated skeletal muscle glycogen synthesis in vivo. Values are means ± SE for six to seven experiments. *P < 0.05 versus control group.

Decreases in insulin-stimulated glucose uptake and metabolism in skeletal muscle were associated with a 59% decrease in insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle (gastrocnemius) with lipid infusion (Figure 3b). Also, insulin-stimulated IRS-1–associated PI 3-kinase activity in skeletal muscle was decreased by 38% with lipid infusion (Figure 3c). These findings suggest that skeletal muscle insulin resistance with lipid infusion may be secondary to the observed defects in skeletal muscle insulin signaling. Lipid-induced decreases in both insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1–associated PI 3-kinase activity in skeletal muscle were prevented with salicylate pretreatment. In contrast, insulin-stimulated tyrosine phosphorylation of insulin receptor was not altered with lipid infusion or salicylate pretreatment (Figure 3a). These findings further suggest that lipid-induced defect and the effect of salicylate on skeletal muscle insulin signaling lie downstream to the insulin receptor and likely reside at the level of insulin receptor substrate.

Figure 3

Insulin signaling in skeletal muscle (gastrocnemius) of the control (black bars), salicylate (light gray bars), lipid (open bars), and lipid-salicylate (dark gray bars) rats. (a) Insulin-stimulated tyrosine phosphorylation of insulin receptor. (b) Insulin-stimulated tyrosine phosphorylation of IRS-1. (c) Insulin-stimulated IRS-1–associated PI 3-kinase activity. Values are means ± SE for six to seven experiments. *P < 0.05 versus control group.

In contrast to the defects in skeletal muscle insulin signaling and action with lipid infusion, basal EGP and insulin’s ability to suppress basal EGP were not altered with lipid infusion or salicylate pretreatment (Figure 4, a and b). Moreover, insulin-stimulated glucose uptake in epididymal white adipose tissue was not altered with lipid infusion or salicylate pretreatment (Figure 4c). These findings indicate that increased plasma fatty acid concentrations caused a selective decrease in insulin responsiveness in skeletal muscle, which may be attributed to decreases in skeletal muscle insulin signaling and action, and that these defects were prevented with salicylate pretreatment.

Figure 4

Insulin action in liver and epididymal white adipose tissue in the control (black bars), salicylate (light gray bars), lipid (open bars), and lipid-salicylate rats (dark gray bars). (a) Basal rates of endogenous glucose production. (b) Percent suppression of basal endogenous glucose production during insulin-stimulated state. (c) Insulin-stimulated glucose uptake in epididymal white adipose tissue. Values are means ± SE for six to seven experiments.

To further determine the mechanism of salicylate action, we examined whether mice with inactivation of IKK-β are protected from fat-induced insulin resistance in skeletal muscle. A 5-hour lipid infusion increased plasma fatty acid concentrations threefold during the basal period and more than 15-fold during the clamps compared with the control group (Table 2 and Figure 5a). During the clamps, plasma insulin concentrations were raised to approximately 730 pM, whereas plasma glucose was clamped at approximately 6.5 mM in all groups (Table 2). Lipid infusion markedly decreased the glucose infusion rate required to maintain euglycemia in the control mice, indicating that lipid infusion caused insulin resistance in these mice. In contrast, lipid infusion did not alter steady-state glucose infusion rate in the IKK-β KO mice compared with the control and IKK-β KO mice without lipid infusion (Table 2). Basal EGP and insulin’s ability to suppress basal EGP were unaltered with lipid infusion in the control and IKK-β KO mice (Figure 5b).

Figure 5

Metabolic parameters and insulin-stimulated whole-body and skeletal muscle (gastrocnemius) glucose uptake in awake control (black bars), IKK-β KO (light gray bars), control-lipid (open bars), and IKK-β KO-lipid (dark gray bars) mice. (a) Plasma FFA concentrations during basal and hyperinsulinemic-euglycemic clamp. (b) Insulin-stimulated percent suppression of basal EGP. (c) Insulin-stimulated whole-body glucose uptake in vivo. (d) Insulin-stimulated skeletal muscle glucose uptake in vivo. Values are means ± SE for three to five experiments. *P < 0.05 versus control group; ^†P < 0.05 versus IKK-β KO-lipid group.

Table 2

Metabolic parameters during basal and hyperinsulinemic-euglycemic clamp periods in the control, IKK-β KO, lipid-infused control, and lipid-infused IKK-β KO mice at ∼20 weeks of age

Insulin-stimulated whole-body glucose uptake was decreased by 28% with lipid infusion, and this change was due to a 56% decrease in insulin-stimulated skeletal muscle glucose uptake with lipid infusion in the control mice (Figure 5, c and d). In contrast, lipid infusion did not alter insulin-stimulated whole-body and skeletal muscle glucose uptake in the IKK-β KO mice compared with the control mice (Figure 5, c and d). Furthermore, insulin-stimulated whole-body glycolysis was unaltered, whereas whole-body glycogen/lipid synthesis was significantly decreased by 62% with lipid infusion in the control mice (Figure 6, a and b). Insulin-stimulated skeletal muscle glycolysis and glycogen synthesis were significantly decreased by 56% and 58%, respectively, with lipid infusion in the control mice (Figure 6, c and d). These defects in insulin-stimulated whole-body and skeletal muscle glucose metabolism with lipid infusion were prevented in the IKK-β KO mice (Figure 6).

Figure 6

Insulin-stimulated whole-body and skeletal muscle (gastrocnemius) glucose metabolic flux in awake control (black bars), IKK-β KO (light gray bars), control-lipid (open bars), and IKK-β KO-lipid (dark gray bars) mice. (a) Insulin-stimulated whole-body glycolysis in vivo. (b) Insulin-stimulated whole-body glycogen/lipid synthesis in vivo. (c) Insulin-stimulated skeletal muscle glycolysis in vivo. (d) Insulin-stimulated skeletal muscle glycogen synthesis in vivo. Values are means ± SE for three to five experiments. *P < 0.05 versus control group; ^†P < 0.05 versus IKK-β KO-lipid group.

Decreases in insulin-stimulated glucose uptake and metabolism in skeletal muscle of control mice were associated with a 43% decrease in insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle (gastrocnemius) with lipid infusion (Figure 7a). Also, insulin-stimulated IRS-1–associated PI 3-kinase activity in skeletal muscle was decreased by 37% with lipid infusion in the control mice (Figure 7b). Lipid-induced decreases in both insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1–associated PI 3-kinase activity in skeletal muscle were prevented in the IKK-β KO mice (Figure 7, a and b). These findings suggest that the lipid-induced decreases in insulin-stimulated skeletal muscle glucose uptake in the control mice were secondary to the defects in muscle insulin signaling and that the IKK-β inactivation prevented these lipid-induced defects in muscle insulin signaling and action.

Figure 7

Insulin signaling in skeletal muscle (gastrocnemius) and insulin action in fat of the control (black bars), IKK-β KO (light gray bars), control-lipid (open bars), and IKK-β KO-lipid (dark gray bars) mice. (a) Insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle. (b) Insulin-stimulated IRS-1–associated PI 3-kinase activity in skeletal muscle. (c) Insulin-stimulated glucose uptake in intrascapular brown adipose tissue. (d) Insulin-stimulated glucose uptake in epididymal white adipose tissue. Values are means ± SE for three to five experiments. *P < 0.05 versus control group; ^†P < 0.05 versus IKK-β KO-lipid group.

In addition to lipid-induced defects in skeletal muscle insulin signaling and action, insulin-stimulated glucose uptake in brown adipose tissue was decreased by 54% with lipid infusion in the control mice, and this lipid-induced defect was also prevented in the IKK-β KO mice (Figure 7c). Interestingly, insulin-stimulated glucose uptake in epididymal white adipose tissue showed a tendency to increase with lipid infusion in the IKK-β KO mice but did not reach statistical significance (Figure 7d).

In this study, we found that a 5-hour lipid infusion decreased insulin-stimulated glucose uptake and metabolism in rat skeletal muscle. Given that glucose transport is rate-controlling for glucose utilization in skeletal muscle (20, 21), decreases in glucose transport may account for the parallel decreases in glycolysis and glycogen synthesis with lipid infusion. These abnormalities were associated with defects in insulin activation of tyrosine phosphorylation of IRS-1 and IRS-1–associated PI 3-kinase in skeletal muscle. Recent studies have shown that IRS-1–associated PI 3-kinase is an important intracellular mediator of insulin signaling in skeletal muscle (22), and insulin stimulation of both glucose transport and glycogen synthase activity has been associated with activation of IRS-1–associated PI 3-kinase in skeletal muscle (23). These findings suggest that defects in skeletal muscle insulin action may be secondary to the observed defects in skeletal muscle insulin signaling with lipid infusion. Interestingly, the decrease in insulin-stimulated muscle glycogen synthesis was more profound than the decrease in muscle glucose uptake. This may be due to additional downstream defects in insulin activation of PI 3-kinase that in turn affect insulin-stimulated glycogen synthase activity in skeletal muscle with lipid infusion. In contrast to the defects in insulin activation of tyrosine phosphorylation of IRS-1 and IRS-1–associated PI 3-kinase in skeletal muscle, insulin-stimulated tyrosine phosphorylation of the insulin receptor in the muscle was unaltered with lipid infusion. This finding suggests that the defect in insulin signaling with lipid infusion was not due to an alteration in plasma membrane or membrane-associated insulin receptors (24). It further suggests that the mechanism of blunted insulin signaling with lipid infusion occurs downstream of the insulin receptor and at the level of the IRS-1. Furthermore, a 5-hour lipid infusion did not alter insulin’s ability to suppress EGP or stimulate glucose uptake in white adipose tissue.

The mechanism by which lipid infusion may affect muscle insulin signaling may involve increases in intracellular fatty acid derived metabolites (i.e., fatty acyl CoA, diacylglycerol, ceramides) and subsequent activation of a serine/threonine kinase (e.g., PKC-θ, IKK-β). Chalkley et al. (25) have reported that a 5-hour lipid infusion increased muscle triglyceride and long-chain fatty acyl CoA contents, and this increase in fatty acyl CoA might lead to an increase in diacylglycerol, a known potent activator of PKC-θ (26). Moreover, recent studies by our group have shown that an acute elevation of plasma fatty acids for 5 hours resulted in activation of PKC-θ, which was associated with decreased tyrosine phosphorylation of IRS-1 (10). Mechanisms responsible for decreased insulin-stimulated tyrosine phosphorylation of IRS-1 may involve serine phosphorylation of IRS-1 (27–29), proteasome-mediated degradation of IRS-1 (30, 31), or phosphatase-mediated dephosphorylation of IRS-1 (32, 33). In this regard, serine phosphorylation of IRS-1 impairs the ability of IRS-1 to associate with the insulin receptor, which inhibits subsequent insulin-stimulated tyrosine phosphorylation of IRS-1 (10, 24, 34) and activation of IRS-1–associated PI 3-kinase (27, 28). Moreover, 5-hour lipid infusion did not alter IRS-1 protein content in rat skeletal muscle (222 ± 21 vs. 246 ± 26 arbitrary units in controls). Thus, accumulation of diacylglycerol or fatty acyl CoA’s due to lipid infusion (i.e., increased delivery of fatty acid into skeletal muscle) may be responsible for defects in insulin’s ability to activate IRS-1–associated PI 3-kinase in skeletal muscle and subsequent insulin action. Furthermore, it is also possible that increased concentrations of other intracellular fatty acid derived metabolites, such as ceramide, may play a role in the lipid-induced insulin resistance in skeletal muscle. Summers and colleagues have shown that ceramide suppressed insulin-stimulated glucose transport in 3T3-L1 adipocytes by inhibiting phosphorylation and activation of Akt/protein kinase B, a serine/threonine protein kinase that is activated by insulin in a PI 3-kinase-dependent manner (35) and involved in the translocation of GLUT4 to the cell membrane (36).

The defects in skeletal muscle insulin signaling and action with lipid infusion were completely prevented with salicylate pretreatment, and the mechanism by which salicylate prevents insulin resistance may involve inhibition of IKK-β. Recently, Yin et al. (14) have shown that salicylate may prevent activation by NF-κB of genes involved in the pathogenesis of the inflammatory response by inhibiting the activity of IKK-β and phosphorylation of IκB (at serines S32 and S36 in IκBα and S19 and S23 in IκBβ). DiDonato et al. (37) and Zandi et al. (38) have recently shown that serine phosphorylation of IκB triggers ubiquitination and degradation of the IκB and eventually activation of NF-κB, a transcription factor involved in immune and inflammatory response (39). Furthermore, TNF-α, a well-known proinflammatory cytokine, activates NF-κB by stimulating IKK activity (38), and salicylate has been demonstrated to inhibit TNF-α–induced stimulation of IKK activity (14). Interestingly, TNF-α has also been shown to promote insulin resistance in adipocytes (40) and skeletal muscle (41) by decreasing insulin activation of IRS-1 tyrosine phosphorylation (42) and IRS-1–associated PI 3-kinase (43). From these findings, we hypothesized that lipid infusion may cause skeletal muscle insulin resistance by activating IKK-β, which subsequently leads to increased serine phosphorylation of IRS-1. Consistent with this hypothesis, we found that salicylate pretreatment prevented lipid-induced insulin resistance in skeletal muscle by possibly inhibiting the IKK-β activity and preventing the lipid-induced decreases in tyrosine phosphorylation of IRS-1 and IRS-1–associated PI 3-kinase activity.

To further examine the hypothesis that salicylate prevents lipid-induced insulin resistance in skeletal muscle by inhibiting IKK-β, we determined the effects of 5-hour lipid infusion on skeletal muscle insulin signaling and action in mice with IKK-β inactivation (16). A 5-hour lipid infusion significantly decreased insulin-stimulated glucose uptake and metabolic flux (i.e., glycogen synthesis, glycolysis) in skeletal muscle of the control mice. These lipid-induced defects in skeletal muscle insulin action were secondary to defects in muscle insulin signaling, as observed in rats. In contrast, lipid infusion failed to alter insulin-stimulated glucose uptake, tyrosine phosphorylation of IRS-1, and IRS-1–associated PI 3-kinase activity in skeletal muscle of IKK-β knockout mice. These findings implicate the protective role of IKK-β inactivation on fat-induced development of skeletal muscle insulin resistance and further support the effects of salicylate on insulin action via inhibition of IKK-β activity in skeletal muscle. However, we cannot rule out that salicylate may have additional roles on ribosomal S6 kinase 2 activity (44), kinase Erk activity (45), NF-κB activity (37, 44, 45), or other potential targets of anti-inflammatory action (46) that in turn affect insulin action in skeletal muscle.

In summary, salicylate pretreatment prevents lipid-induced skeletal muscle insulin resistance by inhibiting lipid-induced decreases in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1–associated PI 3-kinase activation. The effect of salicylate, a known inhibitor of IKK-β, on insulin action is further supported by our findings that mice with inactivation of IKK-β are protected from lipid-induced skeletal muscle insulin resistance. Overall, these results provide important new insights into the mechanism of fat-induced insulin resistance in skeletal muscle and suggest a potentially novel class of therapeutic agents for type 2 diabetes.

This study was supported by grants from the United States Public Health Service: R01 DK-40936, P30 DK-45735, and U24 DK-59635 (G.I. Schulman); and R01 DK-51729 and R01 DK-45493 (S.E. Shoelson). Jason K. Kim is a Research Associate, and Gerald I. Shulman is an Investigator of the Howard Hughes Medical Institute. We are grateful to Yan-Lin Wang, Jianying Dong, and Aida Groszmann for technical assistance.

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