The main finding of this study is that obese and type 2 diabetic subjects have increased TLR4 gene expression/content and TLR4-driven (IκB/NFκB) signaling in skeletal muscle. To examine whether FFAs directly activate TLR4 signaling in human muscle, myotubes were treated with palmitate, which rapidly stimulated the IκB/NFκB cascade. Furthermore, blocking TLR4 completely inhibited the ability of palmitate to stimulate this pathway. Because most obese nondiabetic and type 2 diabetic subjects have elevated plasma FFA concentrations (33
), our results suggest that TLR4 may play an important pathogenic role in the mechanism by which FFAs induce an inflammatory response in insulin-resistant subjects.
To elucidate the cause for the elevation in TLR4 content present in the muscle from the obese and type 2 diabetic subjects, human myotubes were treated with palmitate for 3 days. Chronic palmitate treatment caused a significant increase in myotube TLR4 content, whereas prolonged exposure to insulin, TNF-α, or IL-6, whose plasma concentrations are typically elevated in insulin-resistant (obese and type 2 diabetic) subjects, had no effect. In line with this finding, palmitate increased TLR4 gene expression, an effect that was mediated via NFκB. Consistent with investigations performed in human endothelial cells that showed that LPS increases TLR4 expression (34
), we found that treatment with the specific TLR4 agonist monophosphoryl lipid A also induces TLR4 gene expression in human myotubes. This suggests that palmitate-induced TLR4 gene expression is dependent on TLR4 activation. Nonetheless, this effect was not inhibited by TLR4-neutralizing antibodies, suggesting that, in addition to TLR4 stimulation, other mechanisms, such as accumulation of intracellular lipids (ceramides and diacylglycerol), may be involved in FFA-induced TLR4 expression. Because the elevations in TLR4 expression/content in the muscle from insulin-resistant subjects were reproduced in myotubes by prolonged exposure to palmitate, one could speculate that these increases in TLR4 expression/content are acquired defects, secondary to excess FFA supply. Nonetheless, future studies will be needed to establish whether there also is a genetic (inherited) basis underlying the elevation in TLR4 expression in insulin-resistant muscle.
) reported that TLR2, another member of the TLR family, is involved in palmitate-induced insulin resistance in mouse-derived C2C12 myotubes. To examine this possibility in humans, we measured TLR2 gene expression in muscle from insulin-resistant subjects. Both obese and type 2 diabetic individuals displayed elevated TLR2 gene expression in muscle. However, TLR2 protein was not detected in muscle by direct Western blotting, although a signal (too weak for quantitation) corresponding to TLR2 was detected after immunoprecipitation. Taking into account that absolute mRNA levels of TLR4 versus TLR2 in human muscle were roughly similar (not shown), the low TLR2 protein content observed in human muscle is likely due to post-transcriptional regulation and/or protein instability (i.e., degradation). Poor antibody immunoefficiency could be another reason for the low TLR2 protein content observed in human muscle, although this seems unlikely considering that TLR2 protein was readily detectable in human mononuclear cells. Importantly, TLR2 neutralizing antibodies did not affect the ability of palmitate to activate IκB/NFκB signaling. Based on these results, it is unlikely that TLR2 plays a major role in lipid-induced insulin resistance in human muscle.
Another relevant finding in this study is the increased gene expression of the inflammatory proteins IL-6 and SOD2 in the muscle from obese and type 2 diabetic subjects. Experiments performed in monocyte-like (U937) cells (12
) and fibroblasts (13
) have demonstrated that the expression of these proteins is regulated by NFκB. Human myotubes were treated with palmitate to explore whether elevated circulating FFA levels in obese and type 2 diabetic subjects might be responsible for the increased gene expression of IL-6 and SOD2 in muscle. Palmitate robustly increased mRNA expression of IL-6 and SOD2 in the myotubes. Moreover, blockade of NFκB completely blocked the ability of palmitate to induce the gene expression of these inflammatory proteins. Therefore, elevation in plasma FFA concentration may contribute to increased gene expression of IL-6 and SOD2 in muscle from insulin-resistant subjects, and this effect is likely mediated by NFκB.
Accumulating evidence suggests that monocytes/macrophages play an important role in the pathogenesis of insulin resistance by infiltrating insulin-sensitive tissues, such as muscle and fat (36
). Thus, it is possible that the increases in TLR4 expression and signaling observed in the muscle tissue from insulin-resistant subjects are due to inflammatory cell infiltration of the muscle, rather than from intrinsic upregulation of TLR4 expression/signaling within the myofibers. For this reason, we performed Western analysis and real-time PCR of EMR1, a marker for monocyte/macrophage infiltration, in muscle tissue of insulin-resistant subjects and in cultured myotubes. Unlike CD14+
monocytes (AllCells, Emeryville, CA), which expressed EMR1, neither EMR1 protein nor mRNA was detected in the muscle tissue or myotubes (not shown). It is therefore unlikely that the differences in TLR4 expression/signaling observed between groups are secondary to monocyte/macrophage infiltration of muscle.
In conclusion, insulin-resistant subjects have increased TLR4-driven signaling in muscle. We propose a model in which elevated plasma FFA levels in obese and type 2 diabetic subjects activates this signaling pathway by 1) directly interacting (binding) with TLR4 and 2) increasing TLR4 gene expression and protein content, leading to a net increase in the number of TLR4 receptors available for stimulation by elevated plasma FFA levels. Strategies aimed at reducing TLR4 expression, or at blocking TLR4 signaling, may prove useful in enhancing insulin sensitivity in insulin-resistant individuals.