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...

Activation of AMP-activated protein kinase (AMPK) by exercise induces several cellular processes in muscle. Exercise activation of AMPK is unaffected in lean (BMI ϳ25 kg/m 2 ) subjects with type 2 diabetes. However, most type 2 diabetic subjects are obese (BMI >30 kg/m 2 ), and exercise stimulation of AMPK is blunted in obese rodents. We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator-activated receptor coactivator (PGC)-1␣, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% VO 2max ) and moderate (70% VO 2max ) intensities, with a 4 -6 week interval. Exercise had no effect on LKB1 activity. Exercise had a time-and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise-induced increases in PGC-1 expression. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects. Diabetes 56:836 -848, 2007

Skeletal muscle insulin resistance plays a key role in the pathogenesis of type 2 diabetes. It recently has been hypothesized that excessive activity of the inhibitor of B (IB)/nuclear factor B (NFB) inflammatory pathway is a mechanism underlying skeletal muscle insulin resistance. However, it is not known whether IB/NFB signaling in muscle from subjects with type 2 diabetes is abnormal. We studied IB/NFB signaling in vastus lateralis muscle from six subjects with type 2 diabetes and eight matched control subjects. Muscle from type 2 diabetic subjects was characterized by a 60% decrease in IB␤ protein abundance, an indicator of increased activation of the IB/NFB pathway. IB␤ abundance directly correlated with insulin-mediated glucose disposal (R d ) during a hyperinsulinemic (40 mU ⅐ m ؊2 ⅐ min ؊1 )-euglycemic clamp (r ‫؍‬ 0.63, P ‫؍‬ 0.01), indicating that increased IB/NFB pathway activity is associated with muscle insulin resistance. We also investigated whether reversal of this abnormality could be a mechanism by which training improves insulin sensitivity. In control subjects, 8 weeks of aerobic exercise training caused a 50% increase in both IB␣ and IB␤ protein. In subjects with type 2 diabetes, training increased IB␣ and IB␤ protein to levels comparable with that of control subjects, and these increments were accompanied by a 40% decrease in tumor necrosis factor ␣ muscle content and a 37% increase in insulin-stimulated glucose disposal. In summary, subjects with type 2 diabetes have reduced IB protein abundance in muscle, suggesting excessive activity of the IB/NFB pathway. Moreover, this abnormality is reversed by exercise training. Diabetes 55:760 -767, 2006