TLR4 links innate immunity and fatty acid–induced insulin resistance (original) (raw)

This article addresses the hypothesis that TLR4, the obligatory receptor for bacterial LPS and a key molecular component of the innate immune system, can play an additional role as well, as a “sensor” for endogenous lipids that may contribute to the pathogenesis of lipid-induced insulin resistance. The plausibility of this hypothesis derives from several prior observations. First, obesity and diabetes have been shown, through a variety of approaches, to be associated with activation of inflammatory pathways in key metabolic tissues as well as macrophages (1, 2, 7, 12, 14, 20–23), and these inflammatory signals and their downstream effectors have been implicated as mediators of resistance to insulin action that characterize these states (1, 2, 7). FFAs, whose levels are commonly increased in obesity, have been implicated as proximate causes of insulin resistance in several models (8–14, 24, 25) and have also been shown to be capable of inducing inflammatory signaling in several tissues (9, 26–32). FFAs have been shown to cause insulin resistance in vivo — as evidenced by their impairment of insulin’s ability to activate signal transduction, inhibit hepatic glucose production, and stimulate glucose uptake in skeletal muscle and adipose tissue (8, 9, 13, 25–27, 33). The molecular pathways by which FFAs induce insulin resistance have been investigated in insulin-sensitive tissues such as fat and skeletal muscle. FFAs have been shown to activate JNK and stimulate TNF-α expression in 3T3-L1 adipocytes, while blockage of JNK or TNF-α can prevent FFA-induced insulin resistance, suggesting that fatty acid–induced insulin resistance may be mediated in part by these proinflammatory signaling pathways (26). Moreover, FFAs also activate the proinflammatory signal IKK/NF-κB in 3T3-L1 adipocytes, leading to expression of the cytokines TNF-α and IL-6 (27, 28). However, the critical question of how FFAs are sensed by adipocytes or other tissues in order to activate proinflammatory signaling networks remains uncertain. Whether such sensing involves an intracellular target of FFA, is a consequence of FFA metabolism to a subsequent mediator, or requires an action of FFA at the plasma membrane has not been determined.

Several plausible theories have been advanced to explain the ability of FFAs to activate intracellular inflammatory signals. These include uptake and intracellular actions of FFAs, either through their subsequent metabolism to species such as long-chain CoA derivatives or via their action as ligands to modify nuclear receptors. Proof that such mechanisms underlie fatty acid–induced inflammatory signaling has yet to be provided. An additional or alternative mechanism might involve actions of FFAs to modify plasma membrane pathways that are capable of initiating inflammatory signals. Several G protein–coupled receptors have recently been shown to respond to FFAs as a ligand (34, 35), but these have not been implicated in FFA-induced inflammation or insulin resistance. TLRs, including TLR4, are potential candidates for such a role, as these receptors are known to initiate signals that activate NF-κB, JNK, and SOCS pathways (15, 16, 19), all of which participate in regulation of inflammation and have been proven as well to be capable of inducing insulin resistance (12, 14, 20, 21, 36). In this regard, it has been shown that the C12 fatty acid lauric acid, a major component of lipid A of LPS, is capable of activating TLR4 signaling (17, 18). Nutritional fatty acids have been shown, using in vitro systems, to activate TLR4 signaling as well (17, 18). Thus, multiple lines of evidence converge on the possibility that FFAs might initiate inflammatory signaling and insulin resistance, at least in part, by activating TLR4 signaling in one or more tissues and that such signaling might contribute to insulin resistance, or at least that component related to FFAs. In this study, we have combined a number of in vitro and in vivo approaches to assess this hypothesis.

The function of TLR4 has been most extensively studied in cells of the immune system, including macrophages. The relevance of macrophages to the pathogenesis of type 2 diabetes was not previously viewed as likely. However, over the past several years, a potential role for macrophages as mediators of insulin resistance has emerged from elegant genetic studies (21–23), so potential activation of macrophage TLR4 by FFAs can now be readily linked to insulin resistance. In addition, to the extent that TLR4 is expressed in tissues directly involved in insulin’s metabolic actions, such as adipose tissue and muscle, an ability of FFAs to activate TLR4 in these tissues could be important as well. Our findings in this study are relevant to both of these possibilities.

We have demonstrated that a mixture of palmitate and oleic acid acts through TLR4 to induce NF-κB signaling in 293 cells, confirming earlier observations (17, 18). In addition, we have shown that palmitate acts through TLR4 on mouse peritoneal macrophages to induce IκBα degradation and activate JNK. We have also addressed the possibility that TLR4 is expressed on adipocytes and can mediate inflammatory signaling in this cell type. Our findings indicate that TLR4 is expressed on the 3T3-L1 adipocytes in a differentiation-dependent manner and is expressed in normal adipose tissue. The level of expression is similar to that of unstimulated RAW cells. The expression of TLR4 mRNA in total adipose tissue extracts increased in 2 models of obesity, and this could be due in part to increased numbers of macrophages known to reside in fat tissue of obese animals. In 3T3-L1 adipocytes, FFAs induced increased expression of SOCS3 and IL-6 mRNA. In isolated mouse adipocytes, FFAs induced release of TNF-α and IL-6, and, importantly, this action of FFA was not observed in adipocytes from TLR4–/– mice.

To further examine the role of TLR4 in lipid-mediated inflammatory signaling in an in vivo setting, we utilized the model of i.v. infusion of lipid plus heparin. This model has been used repeatedly to demonstrate the ability of acute elevation of lipids in vivo to suppress insulin-stimulated glucose metabolism and insulin signaling (10, 11, 13). In this system, a 5-hour lipid infusion in mice reduces the action of insulin to stimulate glucose disposal into muscle and fat and suppresses insulin-mediated signal transduction (10, 11, 13). In agreement with previous results, lipid infusion suppressed insulin-mediated glucose disposal, as measured by euglycemic clamp, and glucose uptake into muscle and fat, as assessed by 2-deoxyglucose uptake in WT mice. In contrast, in mice lacking TLR4, this adverse effect of lipid infusion was markedly suppressed.

To study the mechanism for the observed reduction in lipid-induced insulin resistance in TLR4–/– mice, we assessed the ability of lipid infusion to induce inflammatory signaling in adipose tissue. We found 2 indications that lipid infusion activated NF-κB signaling in adipose tissue, and this did not occur in TLR4–/– mice. First, we determined by EMSA analysis that lipid infusion activated NF-κB in adipose tissue. Second, we used ChIP assays to show that lipid infusion induced binding of NF-κB to sequences in the promoter of IL-6 and MCP-1 within adipose tissue. Strikingly, this did not occur in mice lacking TLR4 in both cases. Given the time course, it is most likely that this was due to activation of NF-κB in adipocytes, but these findings are also consistent with activation of macrophages either resident in adipose tissue or recruited to adipose tissue during the course of the infusion. The potential biologic significance of the findings for insulin resistance in adipose tissue could be similar in either case. The finding that lipid infusion reduced the ability of insulin to acutely activate signaling through IRS-1 likely underlies the reduced glucose uptake into muscle after lipid infusion. As TLR4 expression on muscle cells has been described (37, 38), this is likely a direct effect of lipid infusion on muscle cells, though indirect effects may also be present.

Having demonstrated both in vitro and in vivo that FFAs can act through TLR4 on macrophages and adipocytes to induce inflammatory signaling and suppress insulin signaling and insulin-mediated regulation of glucose metabolism, we next sought to determine the impact of TLR4 on high-fat diet–induced obesity and metabolic sequelae. In female mice, TLR4 status had a clear effect on energy homeostasis, with increased weight gain and fat mass, exaggerated by high-fat diet. This was attributed to increased food intake rather than changes in energy expenditure. Despite the increased obesity linked to TLR4 deficiency, insulin tolerance tests in female TLR4–/– mice showed improved insulin sensitivity. Consistent with the initial hypothesis of our studies, this protection from high-fat diet–induced insulin resistance was associated with failure of high-fat diet to induce expression of inflammatory mediators or indicators in liver and fat. These data suggest that, in female C57BL/6J mice, TLR4 is in part required for the ability of high-fat diet to induce inflammatory mediators in peripheral tissues and may thereby contribute as well to the insulin resistance that develops. The results do not support the conclusion that TLR4 is the exclusive mechanism, however, as insulin resistance of lesser degree was seen after high-fat diet in TLR4–/– mice. The fact that these changes in energy balance and insulin sensitivity were not evident in male mice despite suppression of inflammatory markers indicates that TLR4 and these inflammatory mediators are among a larger set of factors involved in linking diet, obesity, and insulin resistance, and these factors are subject to further modification by sex-specific factors. In addition, our data strongly suggest that while lipid infusion protocols can provide valuable insights into potential mechanisms for lipid-induced changes in insulin signaling and insulin resistance, this model is not necessarily predictive of the findings in mice fed high-fat diets. Whether the differences between these models are the result of different circulating FFA levels, the nature of specific fatty acids involved, variable duration of exposure, or other factors remains to be determined. With respect to FFA levels, our data closely resemble those of Kim et al. (39), who found that mean FFA levels were elevated 4-fold by high-fat diet but 5- to 6-fold after lipid infusion.

Additional study is needed to determine the cells/tissues in which TLR4 signaling is required for regulation of systemic insulin sensitivity. FFAs cause inflammatory responses in insulin-sensitive tissues such as skeletal muscle, resulting in both local and systemic insulin resistance. Two inflammatory kinases, PKC-θ and IKKα, appear to play roles in fatty acid–induced insulin resistance in muscle, since PKC-θ or IKKα knockout or inactivation of IKKα by high-dose salicylate can prevent FFA-induced defects in insulin signaling and insulin resistance in skeletal muscle (10, 11). In a human study, lipid-induced insulin resistance was associated with activation of IKK/NF-κB signaling in skeletal muscle (29). Further studies confirmed that FFAs activate IKK/NF-κB signaling in myotubes, leading to cytokine expression (30, 31). Since skeletal muscle cells express functional TLR4 that activates proinflammatory signaling in response to LPS (37, 38), it is possible that FFA-induced insulin resistance in skeletal muscle is mediated by FFA activation of TLR4 expressed on muscle cells. Indeed, our data show that TLR4 deficiency substantially limits impaired insulin signaling and insulin resistance caused by lipid infusion in muscle. However, additional studies, involving, for example, specific deletion of TLR4 in individual tissues, will be required to determine to what extent the improved insulin sensitivity in response to FFAs in a given tissue such as muscle is due to the direct effect of TLR4 deficiency in that tissue, as opposed to systemic effects secondary to changes exerted in adipose tissue or macrophages.

The obesity observed in female TLR4–/– mice is worthy of discussion. The mechanism involves changes in food intake and presumably indicates some changes in the CNS circuitry involved in appetite regulation. At this time, it is unclear by what mechanism this arises secondary to TLR4 deficiency. It is possible that TLR4 deficiency in a peripheral tissue results in altered levels of a systemic regulator of appetite or that TLR4 deficiency in CNS has a more direct role. TLR4 expression has been identified in several brain regions (40), but whether such receptors play a role energy balance will require additional studies.

In summary, although FFAs are known to play a key role in the etiology of insulin resistance, the molecular sensing mechanisms through which high levels of FFAs are transduced to produce intracellular inflammatory signaling, which then induces insulin resistance, have remained obscure. We have shown here that FFAs are capable of utilizing the innate immune receptor TLR4 to induce proinflammatory cytokine expression in macrophages, adipocytes, and liver. Moreover, TLR4 signaling appears to be required for a component of insulin resistance induced by FFAs in adipocytes and in vivo after lipid infusion and high-fat diets. These data therefore suggest a previously unappreciated link between the innate immune system and metabolism and suggest that TLR4 may be involved in other adverse effects of FFAs on tissues and processes that constitute the metabolic syndrome.