Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse
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Substantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance.
Mice deficient in TLR2 were used to investigate the in vivo roles of TLR2 in initiating and maintaining inflammation-associated insulin resistance and energy homeostasis.
We first recapitulated the observation with elevated expression of TLR2 and inflammatory cytokines in white adipose tissue and liver of ob/ob mice. Aged or high-fat-fed TLR2-deficient mice were protected from obesity and adipocyte hypertrophy compared with wild-type mice. Moreover, mice lacking TLR2 exhibited improved glucose tolerance and insulin sensitivity regardless of feeding them regular chow or a high-fat diet. This is accompanied by reductions in expression of inflammatory cytokines and activation of extracellular signal-regulated kinase (ERK) in a liver-specific manner. The attenuated hepatic inflammatory cytokine expression and related signalling are correlated with increased insulin action specifically in the liver in TLR2-deficient mice, reflected by increased insulin-stimulated protein kinase B (Akt) phosphorylation and IRS1 tyrosine phosphorylation and increased insulin-suppressed hepatocyte glucose production.
The absence of TLR2 attenuates local inflammatory cytokine expression and related signalling and increases insulin action specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.
KeywordsExtracellular signal-regulated kinase Inflammation Insulin resistance Toll-like receptor 2
Protein kinase B
Brown adipose tissue
Extracellular signal-regulated kinase
Insulin tolerance test
c-Jun N-terminal kinase
Monocyte chemotactic protein-1
Nuclear factor kappa B
White adipose tissue
Insulin resistance is the chief abnormality present in metabolic syndrome. Increasing evidence suggests causative links between inflammation and development of insulin resistance [1, 2]. Thus, the inflammatory nuclear factor kappa B (NF-κB) signalling pathway is activated and pro-inflammatory cytokines are produced in the white adipose tissue (WAT) and livers of insulin-resistant animals and humans [3, 4, 5]. Genetic or pharmacological inhibition of NF-κB pathways and cytokine neutralisation reverses insulin resistance [4, 6]. Previous studies demonstrated that inflammation-induced activation of protein kinases, such as IκB kinases (IKKs), c-jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs), inhibits tyrosine phosphorylation of IRS1 and suppresses downstream insulin signalling. This results in inefficient glucose uptake and use [7, 8, 9]. Thus, it is critical to identify the target(s) and mechanism(s) that initiate inflammatory responses leading to insulin resistance.
Insulin resistance is frequently associated with obesity  and dyslipidaemia . Obesity is thought to be the most important identified factor contributing to insulin resistance . Dyslipidaemia, characterised by abnormal amounts and composition of lipids and/or lipoproteins in blood, represents an additional risk factor for insulin resistance. NEFAs and triacylglycerols are elevated in obese and diabetic individuals and animals. Previous studies demonstrated that exposure to elevated NEFA causes activation of inflammatory signals and production of cytokines in multiple cell types, and these findings have been correlated with impairments of insulin actions [13, 14]. Thus, circulating lipid and associated inflammation appear to be a link between nutrient excess and insulin resistance. Recent studies have suggested that the induction of inflammatory pathways by dietary factors may mediate through the membrane receptors, including toll-like receptors (TLRs) .
TLRs play a central role in the innate immune response by recognising conserved patterns in diverse pathogenic molecules and activating pro-inflammatory signalling pathways [16, 17]. TLR2, a subclass of TLRs, is the main receptor recognising components of bacterial cell wall, lipoproteins and lipopeptides. Ligand-induced dimerisation of TLR2 with either TLR1 or TLR6 triggers recruitment of adaptor proteins, following a cascade of kinase activation, ultimately leading to activation of NF-κB and production of pro-inflammatory cytokines. The binding specificity of TLR2 with lipoprotein ligand is mediated through its dimerisation with either TLR1 or TLR6. The TLR2–TLR1 complex recognises triacylated lipoprotein, whereas the TLR2–TLR6 complex senses diacylated lipoprotein . Thus, the number of acyl chain of lipoproteins is finely differentiated by TLR1–TLR2 and TLR2–TLR6. In addition to the number of acyl chains, other characteristics, such as length and ester bond of acyl chains, are critical for the biological activity of TLR2-dependent signalling [19, 20]. These results suggest that the fatty acid composition plays a central role in ligand recognition and receptor activation for TLR2. Furthermore, other studies demonstrated that TLR2 is involved in NEFA-induced insulin resistance [21, 22, 23]. It is reasonable to postulate that the fatty acid moiety from nutrients could potentially activate TLR2 and transduce the inflammatory signals.
Although the predominant site of TLR2 production is on cells of the innate immune system , TLR2 is found on a number of insulin-responsive cells, including adipose, skeletal muscle and liver cells [24, 25]. However, the roles of TLR2 in initiating and maintaining inflammatory-associated insulin resistance and energy homeostasis in vivo have not been established. Here, we show that mice with TLR2 deficiency are protected from developing insulin resistance and adiposity. The increased insulin sensitivity is associated with increased insulin-signal transduction, improved glucose metabolism, and reduced inflammatory cytokine expression and related signalling specifically in the liver.
Mice deficient in TLR2 (Tlr2−/−) were kindly provided by S. Akira  and maintained on a C57BL/6 genetic background. Studies were carried out using both male and female Tlr2−/− mice and age-matched wild-type (WT) C57BL/6 mice, obtained from National Cheng Kung University Laboratory Animal Center. Leptin-deficient (ob/ob) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were fed ad libitum either regular chow (RC) (Purina Laboratory Rodent Diet 5001, PMI Nutrition International, Richmond, IN, USA) or a high-fat (HF) diet (58Y1; TestDiet, Richmond, IN, USA).
Tissue collection and RNA analysis
Tissues were collected and stored in RNAlater (Ambion, Austin, TX, USA), and RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Samples of mRNA were analysed with SYBR Green-based real-time quantitative RT-PCR (Applied Biosystems, Foster City, CA, USA), with β-actin or cyclophilin A as the reference gene in each reaction. Sequences of the primers used for RT-PCR assays are shown in Electronic supplementary material (ESM) Table 1.
For the insulin signalling, 62.5 mU/kg insulin was administered through the portal vein, and muscle tissues were collected 5 min after injection as described by Hirosumi et al. . Liver and WAT tissue samples were collected 2 min after injection with 200 and 500 mU/kg insulin through the portal vein, respectively. Further methods can be found in the ESM.
Upregulation of TLR2 and inflammatory cytokines in WAT and liver of obese mice
Increased insulin sensitivity in Tlr2−/− mice
Decreased body weight and fat mass in Tlr2−/− mice
Microscopically, adipocytes in gonadal WAT of RC-fed aged Tlr2−/− mice were smaller than WT cells (mean area 821 μm2 in Tlr2−/− vs 1503 μm2 in WT) (Fig. 3f,g). The numbers of adipocytes were similar in the Tlr2−/− and WT mice (1.89 × 107 in TLR2−/− vs 1.73 × 107 in WT). HF feeding led to fat deposition in both Tlr2−/− and WT mice and the difference in adipocyte size between genotypes was preserved (mean area 2242 μm2 in Tlr2−/− vs 3276 μm2 in WT) (Fig. 3h,i). The decrease in adipocyte size of Tlr2−/− mice under HF feeding was associated with a decrease in the number of adipocytes (1.21 × 107 in Tlr2−/− vs 2.62 × 107 in WT). Thus, the deficiency of TLR2 brought a shift in the distribution of adipocyte sizes towards smaller adipocytes in WAT (Fig. 3g,i). The reduction in fat mass of Tlr2−/− mice appears to result primarily from the reduction in adipocyte size with a modest decrease in adipocyte number. In vitro differentiation of embryonic fibroblasts showed that adipogenic ability judged by the formation of cells staining positive with Oil red O after hormone stimulation was preserved in Tlr2−/− mice (Fig. 3j), ruling out the possibility of defective adipogenic programming with TLR2 deficiency.
Lipid profile and inflammatory mediators in Tlr2−/− mice
Attenuated local inflammatory cytokine expression and signalling in Tlr2−/− mice
Inflammation-induced activation of IKK, JNK and ERK has been shown to phosphorylate IRS1 at its serine residue(s), which further interrupts tyrosine phosphorylation on IRS1 and suppresses insulin signalling [7, 8, 9]. To further evaluate whether the attenuated inflammatory cytokine expression and signalling in the liver of Tlr2−/− mice correlates with serine phosphorylation of IRS1, we detected the phosphorylation at Ser307 and Ser612 of IRS1. No difference in phosphorylation at Ser307 and Ser612 of IRS1 between WT and Tlr2−/− livers was detected, regardless of diet (Fig. 6e,f). These results suggest that TLR2 deficiency did not affect phosphorylation at Ser307 or Ser612 of IRS1 in the liver.
Improved insulin signalling specifically in the liver of Tlr2−/− mice
Improved glucose metabolism in the liver of Tlr2−/− mice
To determine the insulin sensitivity in the skeletal muscle, we performed the in vitro glucose uptake assay. Basal glucose uptake in muscle in vitro was similar between Tlr2−/− and WT mice (Fig. 8d). Insulin-stimulated glucose uptake in muscle of Tlr2−/− mice was 1.6 times those of WT, although the difference did not reach the statistical significance. Consistent with this, triacylglycerol content in liver, which is associated with hepatic insulin resistance, was significantly reduced in Tlr2−/− mice, while muscle triacylglycerol content was not altered in Tlr2−/− mice (Fig. 8e).
Substantial evidence indicates inflammation as a key mechanism linking metabolic disturbance to nutrient excess. To establish this link, we hypothesised that TLR2 transduces the inflammatory signals and further contributes to insulin resistance. Our findings recapitulated the observation that obesity is associated with elevated expression of inflammatory cytokines in WAT and liver. Moreover, mice lacking TLR2 exhibited enhanced insulin sensitivity, which is accompanied by increased transduction of insulin signal, improved glucose metabolism, and attenuated inflammatory cytokine expression and related signalling specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.
Body weight represents the homeostasis between energy intake and expenditure. The deficiency of TLR2 caused a reduction in body weight and fat mass without alteration in food intake. Increased energy expenditure and locomotor activity, as well as body temperature, point to increased energy dissipation in Tlr2−/− mice. It is known that increased physical activity can influence the insulin sensitivity of skeletal muscle, which is consistent with our findings of a modest increase of insulin-stimulated glucose uptake in Tlr2−/− muscle. However, the differences in body weight and fat mass were not noticeable unless mice were aged up to 11 months or stimulated with a high-energy diet. The reductions in WAT mass and adipocyte size of Tlr2−/− mice could result from the defect of pre-adipocytes differentiating into adipocytes and/or maturation of adipocytes. To address the bona fide role of TLR2 in adipocyte differentiation and maturation, we subjected embryonic fibroblasts to adipogenesis in vitro. TLR2 deficiency did not influence the abilities of adipocyte differentiation and triacylglycerol accumulation, suggesting that these properties are not affected by TLR2 deficiency. Although the mechanism involved in increased energy expenditure in Tlr2−/− mice is not clear, the reduced deposition of excess energy in the body potentially contributes to better metabolic profiles and glucose metabolism.
Improvement of insulin sensitivity with TLR2 deficiency is present in mice fed RC, suggesting TLR2-induced insulin resistance takes place in the basal physiological condition without the need of further HF stimulation. This raises the speculation that TLR2 ligand and the signalling it mediates are present in the basal state. Although the actual endogenous TLR2 ligands have not been identified conclusively, it has been demonstrated that TLR2 recognises a large number of lipid-containing molecules , as well as endogenous proteins released from damaged tissues or injured cells . It is interesting to note that the critical portion of many TLR2 ligands contains a similar fatty acid component. Removal or change of these fatty acids results in loss of TLR2 activation capability , implicating an important role of these fatty acids in ligand recognition and receptor activation. Particularly, palmitic and stearic acids are known to be major fatty acids acylated in the lipoprotein or lipopeptide to activate TLR2 [31, 32]. NEFA possess the capacity to induce stress/inflammatory signals not only in immune cells but also other cell types and tissues [13, 14, 33]. This phenomenon is of particular importance in conditions of nutrient excess such as obesity and diabetes or after ingestion of a fatty meal.
Inflammation-induced activation of protein kinases, such as IKK, JNK and ERK, has been shown to inhibit tyrosine phosphorylation of IRS1 [7, 8, 9] and suppress insulin action and its downstream signalling. Serine phosphorylation of IRS1 is a general mechanism to interrupt IRS1 function and insulin-signal transduction [34, 35]. For example, phosphorylation of IRS1 at Ser307 reduces IRS1 coupling to activated insulin receptors  and enhances IRS1 degradation . Phosphorylation of IRS1 at Ser612 decreased PI3K docking to IRS1 . IRS1 Ser307 can be phosphorylated by IKK and JNK [7, 8], whereas IRS1 Ser612 can be phosphorylated by ERK . Our results showing enhanced insulin-stimulated Akt Ser473 phosphorylation and IRS1 tyrosine phosphorylation specifically in the liver of Tlr2−/− mice provide a biochemical correlate for increased hepatic insulin sensitivity. In the search for protein kinases involved in insulin signalling that are induced by inflammatory mediators, we did not detect alterations in the activation of JNK and IKK in the livers of Tlr2−/− mice. ERK activation was dramatically reduced in the livers of Tlr2−/− mice, providing a possible link between TLR2 and hepatic inflammation. However, further investigation of the hypothesised phosphorylation target on IRS1 did not reveal any change in phosphorylation of Ser307 and Ser612. These results suggest that the hypothesised phosphorylation target on IRS1 mediated through TLR2 is not Ser307 or Ser612. Nevertheless, our results implicate ERK as a downstream intracellular mediator of the TLR2-induced inflammatory response influencing the insulin signal pathway through IRS1 modification.
Our observation of improved insulin sensitivity in the absence of TLR2 supports and elaborates previous findings. For example, palmitate induced IL-6 production and inflammatory signalling, leading to inhibition of insulin-activated signal transduction through TLR2 in myotubes . In addition, inhibition of TLR2 expression by TLR2 antisense oligonucleotide improves insulin sensitivity and signalling in muscle and WAT from mice fed the HF diet . However, tissue specificity and transient treatment of antisense oligonucleotide limit the interpretation of long-term effect of TLR2 on complex physiological homeostasis. Although mice lacking TLR2 have recently been found to be protected from diet-induced adiposity and systemic insulin resistance , mechanistic insight and tissue specificity for the improvement of insulin sensitivity with TLR2 deficiency have been lacking. Collectively, these findings implicate TLR2 as a key mediator of inflammation, causing insulin resistance under conditions of nutrient excess in many key insulin-responsive tissues.
Despite the apparent upregulation of inflammatory cytokines in WAT of ob/ob mice, the induction of inflammatory cytokines and TLR2 was also present in the livers of ob/ob mice in our study. These findings suggest that the nutrient-induced inflammatory response and insulin resistance takes place in both WAT and liver. In support of this hypothesis, TLR2-deficiency-associated decreases in inflammatory cytokine expression and signalling and enhancement of insulin signalling were conspicuous in the liver. In contrast, the induction of inflammatory cytokines was not evident in the muscle of ob/ob mice. Consistent with this, the gene expression and signalling molecules related to inflammation and insulin resistance in muscle appeared unchanged in Tlr2−/− mice. Thus, the modification of peripheral insulin sensitivity through TLR2-induced inflammatory cytokine expression and signalling is likely to be mediated primarily in the liver.
Recently, the role of TLR4 in the cross-talk between nutrient-induced inflammation and insulin resistance has been investigated [40, 41, 42, 43], and the hypothesised location for TLR4 action mediating the impairment of insulin sensitivity is predominantly in WAT. Different TLR production profiles affect the responsiveness of particular cell types and tissues to the ligand stimulation. In addition, different TLR ligands may induce activation of different downstream signalling pathways, leading to a diverse array of target gene expression and cellular responses through distinct TLRs. Given the differences in ligand preference, signalling pathways and tissue distribution between TLR2 and TLR4, it is reasonable to speculate that TLR2 and TLR4 may receive different ligand stimulation from nutrient factors and transduce inflammatory signals. Furthermore, there was no compensatory alteration in the gene expression of Tlr4 in WAT, liver and muscle (data not shown). Our results unequivocally provide TLR2 as another TLR family receptor involved in the interface of inflammatory response and metabolic disturbance.
In conclusion, our results demonstrated that TLR2 contributes to upregulation of inflammatory cytokine expression and signalling and interference of insulin action specifically in the liver. Although the detailed mechanism by which factors activate TLR2 or interact with its associated factors is not known, the results presented here represent a new paradigm that endogenous molecule(s) can utilise the innate immune receptor TLR2 to trigger the local inflammation-related signalling in the liver and subsequently affect systemic metabolism. Our findings provide a rationale for the development of the hepatic inflammatory response and insulin resistance in response to nutrient factors.
We thank A. Pendse, N. Takahashi and C.-H. Lee for discussions; Z.-H. Lin, H.-F. Jheng, the National Laboratory Animal Centre Pathology Core (C.-T. Liang), H.-T. Wu, Y.-H. Lee and the Taiwan Mouse Clinic for technical assistance; and S. Akira for kindly providing Tlr2−/− mice. This work was supported by the grant from the National Health Research Institute (NHRI-EX98-9823SC).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.