OBJECTIVE-Tall-like receptor (TLR)4 has been implicated in the pathogenesis of free fatty acid (FFA)-induced insulin resistance by activating inflammatory pathways, including inhibitor of B (IB)/nuclear factor B (NFB). However, it is not known whether insulin-resistant subjects have abnormal TLR4 signaling. We examined whether insulin-resistant subjects have abnormal TLR4 expression and TLR4-driven (IB/NFB) signaling in skeletal muscle. RESEARCH DESIGN AND METHODS-TLR4gene expression and protein content were measured in muscle biopsies in 7 lean, 8 obese, and 14 type 2 diabetic subjects. A primary human myotube culture system was used to examine whether FFAs stimulate IB/NFB via TLR4 and whether FFAs increase TLR4 expression/content in muscle.RESULTS-Obese and type 2 diabetic subjects had significantly elevated TLR4 gene expression and protein content in muscle. TLR4 muscle protein content correlated with the severity of insulin resistance. Obese and type 2 diabetic subjects also had lower IB␣ content, an indication of elevated IB/NFB signaling. The increase in TLR4 and NFB signaling was accompanied by elevated expression of the NFB-regulated genes interleukin (IL)-6 and superoxide dismutase (SOD)2. In primary human myotubes, acute palmitate treatment stimulated IB/NFB, and blockade of TLR4 prevented the ability of palmitate to stimulate the IB/NFB pathway. Increased TLR4 content and gene expression observed in muscle from insulin-resistant subjects were reproduced by treating myotubes from lean, normal-glucose-tolerant subjects with palmitate. Palmitate also increased IL-6 and SOD2 gene expression, and this effect was prevented by inhibiting NFB.CONCLUSIONS-Abnormal TLR4 expression and signaling, possibly caused by elevated plasma FFA levels, may contribute to the pathogenesis of insulin resistance in humans. Diabetes 57: [2595][2596][2597][2598][2599][2600][2601][2602] 2008 T he mechanism(s) by which free fatty acids (FFAs) cause insulin resistance is not fully understood. Considerable evidence suggests that the deleterious effect of FFAs on insulin action is caused by intramyocellular FFA metabolites that stimulate inflammatory pathways leading to impaired insulin signaling/action (1). However, recent reports demonstrate that FFAs directly can stimulate plasma membrane receptors (2,3), suggesting an alternate model in which FFAs cause insulin resistance by stimulating inflammatory pathways through the direct activation of plasma membrane receptors. Consistent with this hypothesis, FFAs have been shown to bind to toll-like receptor (TLR)4 (4), a transmembrane receptor, and TLR4-driven inflammatory cascades, such as the inhibitor of B (IB)/nuclear factor B (NFB) pathway, are implicated in the pathogenesis of insulin resistance (5-7).TLRs play an important role in the innate immune system by activating inflammatory pathways in response to microbial agents (8). TLR4 functions as the receptor for lipopolysaccharide (LPS) of gram-negative bacterial cell walls (8). Saturated FFAs acylated in the lipid A moiety of...

Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = −0.46, P = 0.005) and LBP (r = −0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals.