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
), 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
), proteasome-mediated degradation of IRS-1 (30
), or phosphatase-mediated dephosphorylation of IRS-1 (32
). 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
) and activation of IRS-1–associated PI 3-kinase (27
). 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
), 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.