Although recent evidence shows that chronic inflammation is a central contributing factor in the development of insulin resistance in obesity, the pathway(s) that transduce the inflammatory signal in obesity are unclear. Here we show that knock out of Tlr4, which is a key receptor involved in activation of the innate immune/inflammatory response, in hematopoietic cells, prevents HFD/obesity-induced hyperinsulinemia, hyperglycemia, and abrogates insulin resistance in liver and adipose tissue. Importantly, the improved insulin action in adipose tissue and liver of these mice occurred in conjunction with reduced macrophage infiltration of adipose tissue, as well as reduced expression of proinflammatory cytokines, such as TNF-α, both in adipose tissue and liver. We further verified the importance of hematopoietic cell Tlr4 in the induction of insulin resistance by using a gene therapy approach to knockdown Tlr4 in autologous hematopoietic stem cells. Considering that Tlr4 is a receptor for fatty acids, our results suggest that Tlr4 acts as an important transducer of the extracellular signal from fatty acids to activation of intracellular inflammatory pathways in hematopoietic cells (most likely macrophages), with subsequent release of proinflammatory cytokines that cause insulin resistance.
Chronic low-grade tissue inflammation has recently garnered considerable attention as a necessary contributor to insulin resistance in obesity. Tlr's play a critical role in activating the innate immune response, and consequently, have been implicated in the induction of insulin resistance in obesity. Tlr4 is an attractive candidate for linking innate immunity to insulin resistance, because it is expressed in most cell types, and Tlr4 is also a receptor for fatty acids, which are increased in obesity (Lee et al., 2001
; Lee et al., 2003
). Indeed, several recent studies have demonstrated that mice with knockout of Tlr4 (Shi et al., 2006
) or a loss-of-function mutation in Tlr4 (Tsukumo et al., 2007
) are protected against fatty acid- and obesity-induced insulin resistance. However, since Tlr4 is expressed in many important insulin-responsive cell types (e.g. muscle, adipocytes, hepatocytes), a limitation of these studies is that they do not specifically isolate the contribution of the innate immune system (e.g. macrophage, neutrophils etc.) to changes in insulin sensitivity. To address this and other questions, we used BMT to generate chimeric mice with knockout of Tlr4 specifically in hematopoietic cells. Because innate immune cells are derived from hematopoietic stem cells, our model results in the knockout of Tlr4 in macrophages as well as in other hematopoietic cells. Interestingly, our results demonstrate that BMT-Tlr4-/-
mice are protected against HFD/obesity-induced hyperinsulinemia, insulin intolerance, and insulin resistance in adipose tissue and the liver. These results are in line with recent studies in which we found that myeloid-specific knockdown of JNK1 (which is a downstream target of Tlr4 signaling) and IKK improve insulin sensitivity in HFD fed mice.
Two previous studies have examined the issue of insulin resistance in mice in which Tlr4 is either knocked out or disabled (Shi et al., 2006
; Tsukumo et al., 2007
). In the paper by Shi et al. (Shi et al., 2006
), the authors show that the Tlr4 knockout protects animals from the effects of acute lipid infusions to cause insulin resistance, however, the tissues responsible for this systemic effect could not be specified. This finding during acute lipid infusions did not translate that well into the setting of chronic HFD. Thus, they found that the Tlr4 deletion had no effect on body weight or insulin sensitivity in HFD fed male mice, but did lead to increased obesity with insulin sensitivity in females. In contrast, Tsukomo et al. (Tsukumo et al., 2007
) studied mice with a loss of function Tlr4-mutation and found that male animals gained less body weight than controls on HFD, and became less insulin resistant. However, in the setting of a lean and insulin sensitive phenotype, it is not clear whether it is the leanness of the mouse, or the knockout of Tlr4, per se, which causes the insulin sensitivity, and the tissue type responsible for the phenotype could not be determined. Since our chimeric mice express Tlr4 deficiency only in bone marrow derived hematopoietic cells, both control and knockout mouse models gained an equal amount of weight on HFD and had equal expansion of both subcutaneous and visceral adipose depots. Thus, differences in adiposity between our two groups (BMT-wt and BMT-Tlr4-/-) is not a confounding factor, making it possible to ascertain the contribution of hematopoietic cell Tlr4 signaling to insulin sensitivity. It is not clear why the Shi et al., and Tsukumo et al. studies are so different from each other, but perhaps it is due to the fact that one group studied Tlr4 null animals (Shi et al., 2006
), whereas, the other studied a mouse strain carrying a loss of function mutation in the Tlr4 receptor (Tsukumo et al., 2007
). Of course, other strain differences may also be contributing factors. Clearly, the animal model we used is much different, since we employed adoptive transfer to generate chimeric animals all on the same background in which the Tlr4 depletion is only carried in hematopoietic-derived cells with normal Tlr4 in all other tissues.
It is of interest, that while we found substantial effects of the hematopoietic Tlr4 knockout to cause systemic insulin sensitivity, these effects were primarily manifested in liver and adipose tissue. With respect to skeletal muscle, we did not observe changes in the insulin stimulated in vivo glucose disposal rate, and since 70-80% of in vivo insulin stimulated glucose disposal is into skeletal muscle, this implies no major changes in skeletal muscle insulin sensitivity. It is well known that on HFD, large increases in macrophage numbers occur in adipose tissue and that, in the liver, Kupffer cell inflammatory activation state is enhanced, and the number of Kupffer cells may also be increased. On the other hand, there are relatively few macrophages that appear in skeletal muscle on HFD, and these cells are mostly present in inter-muscular adipose deposits. Since inflammatory markers were markedly decreased in liver and adipose tissue of the BMT-Tlr4-/- mice, the data indicate that Tlr4 expression in hematopoietic derived cells is an important control point for HFD-induced inflammation in adipose tissue in liver. In skeletal muscle, it is possible that the Tlr4 on the muscle cell itself plays the major role in detecting lipid signals in the setting of HFD-induced skeletal muscle insulin resistance. Indeed, Tsukomo et al. provide evidence for this hypothesis, since they have directly shown that, when studied ex vivo, Tlr4 knockout muscle is protected from fatty acid induced insulin resistance. This is consistent with our own results in which we show that deletion of hematopoietic cell Tlr4 is sufficient to cause a systemic insulin sensitive phenotype, but that this was primarily manifested in liver and adipose tissue and not muscle.
Tlr4 in macrophages is necessary for activation of inflammatory pathways by fatty acids or lipopolysaccharides (LPS). Knockdown of Tlr2 and/or Tlr4 in macrophage cells attenuates fatty acid-induced activation of Jnk1 and abrogates TNF-α secretion into the media. In fact, JNK1 is an obligatory component for the ability of fatty acids and Tlr4 to activate inflammatory pathways, and to increase TNF-α secretion (Nguyen et al., 2007
). In support of these findings, we found that the expression of several proinflammatory cytokines, namely, IL-6, TNF-α and IL-12 p70 was markedly reduced in adipose tissue from BMT-Tlr4-/-
mice on HFD. It is likely that the reduced expression of TNF-α is directly related to the reduced macrophage infiltration, as macrophages are the primary source of TNF-α in obese adipose tissue (Weisberg et al., 2003
; Xu et al., 2003
). It is notable that IL-12 p70 was reduced in our mice, since IL-12 is important for the transition of naïve T-cells into Th1 cells (Hsieh et al., 1993
). This is relevant to our studies since the main targets of Th1 cells are macrophages, whereby Th1 cells act to induce a macrophage proinflammatory state. Thus, we hypothesize that Tlr4 is an obligate receptor for the transduction of an obesity-derived signal (i.e. increased fatty acids) to macrophages. In turn, activation of inflammation via macrophage Tlr4 potentiates recruitment of additional cells to adipose tissue, with subsequent polarization to a proinflammatory state. This feed-forward process is likely exacerbated by the fact that Tlr4 expression is increased in macrophages in obese adipose tissue (Nguyen et al., 2007
). Consistent with this, insulin's effect to suppress circulating FFA levels was much greater in the BMT-Tlr4-/- mice, indicative of improved adipose insulin action.
In the liver, macrophages are present in the form of Kupffer cells. Because Kupffer cells are bone marrow derived, our model allows us to determine the effect of Tlr4 in Kupffer cells on induction of inflammation (and insulin resistance) in the liver of obese animals. Similar to results in adipose tissue, we found that the expression of various proinflammatory markers was markedly reduced in the liver of BMT-Tlr4-/- mice. In fact, the HFD-induced increase in TNF-α and RANTES in liver was completely reversed in BMT-Tlr4-/- mice. In parallel with this decrease in inflammation, the ability of insulin to suppress HGP in BMT-Tlr4-/- mice was normalized to values seen in chow fed mice. These results suggest that activation of inflammatory pathways in immune cells in the liver is necessary for induction of hepatic insulin resistance on HFD.
We extended these findings by demonstrating that transplantation of mice with bone marrow containing lentiviral-driven siRNA knockdown of Tlr4 leads to improved insulin sensitivity on HFD. Interestingly, this occurred despite the fact that we did not achieve as high a level of knockdown as seen in the BMT-Tlr4-/-mice (>95% versus 80% knockdown for BMT-Tlr4-/- and LV-siTlr4, respectively), suggesting that complete knockdown of Tlr4 is not necessary in order for beneficial metabolic effects to occur.
During the review of this manuscript, Coenen et al. (Coenen et al., 2009
) published a paper using the BMT Tlr4-/-
approach and reported a decrease in ATM content with reduced markers of adipose tissue inflammation on HFD in the KO mice. They also found a decrease in atherosclerosis in the KOs, but no changes in glucose homeostasis. However, this study consisted of only female mice of the LDL KO background, and were non-obese Agouti animals and so were markedly different than those in the current study.
In summary, we have demonstrated that knockout of Tlr4 signaling in macrophages reverses insulin resistance in adipose tissue and liver in HFD fed, obese mice. This protection occurs in parallel with a marked reduction in macrophage infiltration in adipose tissue, and reduced inflammatory markers in adipose and liver. Altogether, these data indicate the importance of innate immunity and hematopoietic derived cells, particularly macrophages and Kupffer cells, in the induction of insulin resistance in obesity. Our data also identify Tlr4 in hematopoietic derived cells as a potential target for the therapeutic treatment of insulin resistance.