Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet
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Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice.
Male and female littermate Tlr2 +/+ and Tlr2 −/− mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production.
While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2 −/− mice were protected from the adverse effects of HFD compared with Tlr2 +/+ littermate controls. Female Tlr2 −/− mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2 −/− mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2 −/− mice did not increase IL-1β expression in response to a NEFA mixture, whereas Tlr2 +/+ control tissues did.
These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.
KeywordsDiabetes Insulin resistance IL-1 Pancreatic islet Toll like receptor 2
Bone-marrow-derived dendritic cell
Glucose-stimulated insulin secretion
IL-1 receptor antagonist
I.p. glucose tolerance test
Insulin tolerance test
Nuclear factor κB
Type 2 diabetes develops as a result of a combination of both insulin resistance and pancreatic beta cell failure, resulting in hyperglycaemia. Chronic activation of the innate immune system is associated with type 2 diabetes, and evidence now suggests that both insulin resistance and beta cell failure are regulated by this inflammatory state in humans [1, 2, 3]. Indeed, clinical data have shown improved insulin resistance after treatment of type 2 diabetes patients with salicylate (via inhibition of nuclear factor κB [NF-κB]) [4, 5], and improved beta cell insulin secretion in patients after treatment with the IL-1 receptor antagonist (IL-1Ra) .
Local tissue inflammation is a characteristic of the human pathology of insulin resistance in obesity and beta cell failure in type 2 diabetes [1, 2, 7]. Animal models of type 2 diabetes also show increased expression of inflammatory markers specific for cytokines, chemokines and immune cells in both insulin-responsive tissues (liver, muscle, adipose tissue) and pancreatic islets [3, 8, 9, 10, 11]. This local tissue inflammation has now been causally linked to insulin resistance and beta cell function in a number of genetic knockout models and therapeutic intervention studies [3, 11]. Genetic ablation studies in the high-fat diet (HFD) model have recently shown that pro-inflammatory CD11c+ cells are responsible for this inflammatory response in insulin sensitive tissues, impacting on insulin sensitivity . However, the upstream/cell surface molecular mechanisms responsible for triggering increased tissue inflammation in obesity and type 2 diabetes are still not known.
Toll-like receptors (TLRs) are expressed in multiple tissues and are integral to mounting an immediate defence against the presence of pathogens [13, 14]. Upon binding to their cognate ligands, TLRs recruit intracellular signalling molecules (e.g. MYD88), leading to the activation of NF-κB and the secretion of proinflammatory cytokines and chemokines. Intriguingly, both TLR2 (which heterodimers with TLR1, or TLR6) and TLR4 recognise lipid-based structures; classically bacterial lipopeptides and lipopolysaccharide, respectively. Recent studies have shown that NEFAs, including palmitate and oleate, can activate both TLR2 and TLR4 signalling to induce proinflammatory cytokine production in various tissues, leading to an impairment of tissue-specific effects [15, 16, 17, 18, 19]. In addition, both Tlr2 −/− and Tlr4 −/− mice show improvements in the metabolic syndrome associated with HFD-induced obesity [17, 20, 21, 22]. Despite this, the mechanism underlying resistance to HFD-induced metabolic dysregulation in Tlr2 −/− mice is unclear.
Here we show that TLR2 signalling contributes to the adverse effects of HFD feeding in mice, impacting on insulin resistance, beta cell insulin secretion, energy substrate utilisation and tissue inflammation. These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis.
Please see the Electronic supplementary material (ESM) for further detailed methods.
Materials and animals
Tlr2 −/− mice backcrossed on C57BL/6J for four to five generations were from the Jackson Laboratory (Strain B6.129-Tlr2 tm1Kir /J) (Bar Harbor, ME, USA) and bred in house with C57BL/6J mice to generate all Tlr2 +/+ and Tlr2 −/− sex- and age-matched littermate mice used for in vitro and in vivo studies.
Animals were housed under specific-pathogen-free conditions at the Institute of Labortierkunde, Vetsuisse faculty of the University of Zurich (Zurich, Switzerland). Experiments were performed according to Swiss veterinary law and institutional guidelines. The committee for animal welfare at the Katholieke Universiteit Leuven approved all tissue isolation protocols from mice used for Tlr2 tissue expression profiling.
Tissue expression of Tlr2
Tissue expression analysis using Affymetrix gene arrays was performed as previously described .
Animals were fed a high-fat diet (HFD; Diet D12331) manufactured by Research Diets (New Brunswick, NJ, USA) as previously described [10, 23]. For glucose-tolerance testing, mice were injected i.p. with 2 mg/g body weight glucose (IPGTT) and insulin was measured by ELISA (Mercodia, Uppsala, Sweden). Insulin-tolerance testing (ITT) was performed by injecting female mice i.p. with 0.85 U/kg human insulin (Novo Nordisk, Denmark), and male mice i.p. with 1.0 U/kg insulin, as previously described .
Isolated adipocyte experiments
Adipocyte isolation and glucose incorporation were performed as described previously . Aliquots of all adipocyte fractions were used to determine mean cell diameters, as previously described . At least 100 adipocytes per fraction of four independent experiments were analysed.
Indirect calorimetry and physical activity
Energy expenditure and respiratory quotient (RQ) were measured for 48 h (two dark and light phases) by using two open-circuit calorimetry systems (Integra system, AccuScan Instruments, Columbus OH, USA). Physical activity was measured by telemetry every 5 min using Dataquest A.R.T. 3.1 software.
Cytokines and chemokines
Plasma samples were assayed using mouse Luminex kits (Millipore, Billerica, MA, USA) as previously described .
Glucose clamp studies
After 5 h fast, hyperinsulinaemic–euglycaemic clamps were performed in freely moving mice as previously described .
Total liver lipid extraction
Total liver lipids were determined as recently described .
RNA extraction and real-time PCR
Total tissue RNA was prepared as described  from adipose tissue, liver, muscle, isolated islets, bone-marrow-derived macrophages (BMDMs), and bone-marrow-derived dendritic cells (BMDCs) according to the manufacturer’s instructions (Qiagen, Hombrechtikon, Switzerland). Commercially available mouse primers (Applied Biosystems, CA, USA) were used. For ex vivo detection of islet inflammatory genes after HFD feeding, islets were initially allowed to recover for 4 h in suspension before extraction of total RNA.
NEFAs were prepared using endotoxin-free BSA as recently described .
Mouse liver and pancreatic cryosections were incubated with an anti-F4/80 primary Ab (clone CI:A3-1, BMA Biomedicals, Switzerland), or isotype control rat IgG2b (AbD Serotec) and counterstained with haematoxylin and eosin. F4/80 was visualised using goat anti-rat Ab (112-005-167) (Jackson ImmunoResearch, Newmarket, UK) and a donkey anti-goat Ab conjugated to HRP (Jackson 705-035-147). Images were captured on an Axioplan 2 imaging system (Zeiss, Feldbach, Switzerland) and F4/80 positive area/islet positive area was quantified using Image J software (NIH). For liver sections, three random fields of view were analysed in two sections per animal separated by 200 μm (an average of 3,600 ± 100 cells per animal). For the pancreas, all islets in four sections separated by 200 μm were analysed per animal (an average of 47 ± 7 islets per animal).
Pancreatic islet isolation and BMDM/BMDC preparation
Mouse islets were isolated by collagenase digestion, followed by Histopaque gradient centrifugation and cultured as previously described . For in vitro islet experiments, islets were plated on extracellular matrix (Novamed, Jerusalem, Israel). Islets were allowed to adhere and spread on the extracellular matrix dishes for 48 h before initiation of experiments.
BMDMs and BMDCs were prepared as previously described [27, 28]. Briefly, bone marrow was isolated from femurs and tibias of 8- to 10-week-old female mice and cultured in macrophage medium (50% DMEM) supplemented with 20% horse serum and 30% L929 conditioned medium (a source of macrophage colony-stimulating factor). After 7 days cells were plated into 24-well plates (5 × 106 cells/well) for experiments and extraction of RNA. To generate BMDCs, bone marrow prepared from femurs and tibias was cultured in non-tissue-culture-treated Petri dishes with RPMI medium supplemented with 2-mercaptoethanol (50 μM), 10% FCS and 200 U/ml granulocyte macrophage colony-stimulating factor, as previously described . After 7 days, non-adherent cells were plated into 24-well plates (5 × 106 cells/well) for experiments and extraction of RNA. The specificity of the two differentiation protocols to generate BMDMs (CD45+F4/80+CD11b+Cd11c−) and BMDCs (CD45+CD11b+CD11c+) was verified by FACS analysis. BMDM and BMDC were resuspended in FACS buffer (2% FCS, 10 mmol/l EDTA in PBS) and incubated with anti-mouse CD16/32 (Fc-block, BD Biosciences, Heidelberg, Germany) for 5 min, then stained with anti-mouse CD45-biotin/SAV-APC/Cy7 (BD Biosciences), F4/80-FITC (BD Biosciences), CD11c-PE (Invitrogen) and CD11b-APC (BD Biosciences) for 20 min. The cells were analysed with a Partec FloMax flow cytometer (Munster, Germany). 7-AAD (7-amino-actinomycin D, Invitrogen) was used to exclude nonviable cells in flow cytometric analysis. Cells were treated for 6 h with BSA or a 0.5 mmol/l palmitate:oleate (2:1 molar ratio) preparation before RNA extraction.
Data are expressed as means ± S.E. with the number of individual experiments presented in the figure legends. All data were analysed using the nonlinear regression analysis programme PRISM (GraphPad, CA, USA), and significance was tested using Student’s t test and analysis of variance (ANOVA) with Dunnett’s or Bonferroni’s post-hoc test for multiple comparison analysis. Significance was set at p < 0.05.
Tlr2 tissue expression and regulation by HFD feeding
Tlr2 −/− mice are resistant to the adverse effects of HFD feeding
In agreement with reduced adiposity, circulating leptin, MCP-1 and TNF-α levels were significantly reduced in female Tlr2 −/− animals after 20 weeks of HFD feeding (ESM Table 1). However, we did not detect differences in circulating resistin, IL-6, cholesterol, NEFAs or ketones. In support of improved insulin sensitivity, circulating triacylglycerol levels were significantly reduced in Tlr2 −/− mice on HFD (ESM Table 1).
Tlr2 −/− mice are protected from liver insulin resistance, hepatosteatosis and liver inflammation on HFD
Tlr2 −/− mice are protected from impaired beta cell insulin secretion and islet inflammation on HFD
Next, we analysed islet inflammation due to HFD feeding in female Tlr2 +/+ and Tlr2 −/− mice. Increases in islet Il-1β mRNA owing to HFD positively correlated with increased macrophage/dendritic cell marker expression: Cd68, F4/80 (also known as Emr1) and Cd11c (also known as Itgax) (Fig. 6b) (for Cd68 r 2 = 0.91, p = 0.0003; for F4/80 r 2 = 0.92, p = 0.0006; for Cd11c r 2 = 0.60, p = 0.02; n = 8). By contrast, islets from Tlr2 −/− mice on HFD were protected from increased Il-1β, Cd68 and F4/80 mRNA (Fig. 6c–e). Furthermore, Il-6, Tnf-α, Mcp-1 and Kc mRNA was also significantly reduced in Tlr2 −/− vs Tlr2 +/+ islets on HFD, with no effect on islet Cd36 expression (Fig. 6c–f). No reductions in these inflammatory markers were observed in islets isolated from sex- and age-matched chow-fed Tlr2 −/− compared with Tlr2 +/+ mice (not shown). In contrast to F4/80 mRNA data, and similar to the liver, the amount of islet F4/80+ cells were unchanged in HFD Tlr2 −/− mice (percentage area of F4/80+ cells of total islet area: 1.9 ± 0.4% in Tlr2 +/+ [n = 4] vs 1.8 ± 0.4% in Tlr2 −/− [n = 4]), possibly suggesting an altered activation status of liver and islet macrophages/dendritic cells in Tlr2 −/− vs Tlr2 +/+ mice on HFD.
Finally, analysis of total peripheral leucocytes did not show differences in cytokine mRNA (Il-1β, Tnf-α) or macrophage marker expression (Cd68, F4/80) between Tlr2 genotypes on HFD (data not shown), supporting the tissue specificity of the inflammatory response.
Impaired inflammatory response of BMDCs and islets to NEFAs in Tlr2 −/− mice
The impact of inflammation on insulin resistance and beta cell dysfunction has been confirmed clinically in patients with type 2 diabetes [3, 6]. Nevertheless, the triggering mechanism responsible for induction of the inflammatory response in obesity and type 2 diabetes is not known. Recent studies have implicated TLR4 and its associated co-receptor, CD14, in HFD- and NEFA-stimulated tissue inflammation and insulin resistance [17, 21, 22, 31]. Here, we show that TLR2 signalling is also detrimental for proper glucose homeostasis under HFD conditions. Overall, TLR2 deficiency protected from HFD-induced insulin resistance and beta cell dysfunction via regulation of energy substrate utilisation and tissue inflammation.
During the preparation of this manuscript, another study on the role of genetic deletion of TLR2 during HFD feeding was published . Overall, their data on improved whole body glucose homeostasis and liver lipid content are consistent with our data. Our data extends on these data, however, indicating that lack of TLR2 positively regulates energy-substrate utilisation, hepatic and adipose tissue insulin sensitivity, beta cell insulin secretion and liver and islet tissue inflammation. Furthermore, our in vitro data suggest a role for TLR2 in tissue immune cells (BMDCs) or parenchymal tissue itself (islets) in regulating the inflammatory response to elevated fatty acids during HFD feeding.
Our male and female Tlr2 −/− mice were more prone to obesity on HFD than the male Tlr2 −/− mice used in the study of Himes and Smith . This may be explained by differences in diets used, or the use of littermate controls in our study, which was not explicitly stated in the other study. Indeed, we also performed our experiments on non-littermate-controlled Tlr2 +/+ and Tlr2 −/− mice initially, with data matching those of Himes and Smith in male mice (not shown). Repeating the study on littermate mice resulted in the sexually dimorphic phenotype shown here in response to HFD, and a milder resistance to obesity phenotype than published . Interestingly, our data are similar to a published report on Tlr4 −/− mice , which also showed sexual dimorphism in their resistance to the adverse effects of HFD feeding, with males showing only a partial resistance compared with females. We cannot explain these differences, but it will be interesting to investigate the effects of sex on tissue inflammation with respect to obesity and diabetes.
A reduced RQ, together with a reduction in adiposity and liver lipid content in Tlr2 −/− animals, suggests that fatty acids are being shunted away from the liver to be increasingly oxidised in other tissues (such as skeletal muscle) in HFD Tlr2 −/− mice. This would be consistent with a diminished capacity of the liver to take up lipids due to suppressed Cd36 mRNA expression. TLR2 has been shown to associate with CD36 at the plasma membrane following receptor ligand activation, suggesting that the absence of TLR2 activation in the liver may protect from fatty acid uptake .
Reduced adiposity and reduced hepatosteatosis were also consistent with overall improvements in whole body and hepatic insulin sensitivity as determined by the hyperinsulinaemic–euglycaemic clamp. Thus, our data show for the first time that TLR2 regulates hepatic insulin sensitivity during HFD, extending findings that reduced TLR2 expression by antisense oligonucleotides improved insulin signalling in muscle and white adipose tissue of HFD-fed mice .
Beyond improved insulin sensitivity, we also observed improvements in beta cell insulin secretion in response to glucose load, both in vivo and in vitro. Thus, the overall improved glucose tolerance of HFD Tlr2 −/− mice was due to improved insulin sensitivity and beta cell insulin secretion, which correlated well with a partially attenuated tissue inflammatory response in the liver and islet.
We went on to investigate why tissue inflammation might be suppressed in Tlr2 −/− mice on HFD. Reductions in tissue inflammation may be due to reduced immune cell content of tissues, a reduced activation status of tissue immune cells or reduced inflammation of the parenchymal tissue. Despite reductions in Cd68, and/or F4/80 mRNA in adipose tissue, liver and in islets, we did not observe differences between genotypes in the number of F4/80+ cells by immunohistochemistry in the liver or islet. Thus, we hypothesised that the activation status of proinflammatory immune cells in Tlr2 −/− mice or the parenchymal tissue itself had a reduced capacity to mount an inflammatory response under HFD conditions. Pro-inflammatory CD11c+ cells have been causally linked to tissue inflammation and the induction of insulin resistance in response to HFD in the B6 mouse . Our data showing increased Cd11c mRNA expression during HFD feeding in adipose tissue, liver tissue and islets are consistent with the notion that CD11c+ cells are recruited to these tissues during obesity [19, 33]. Indeed, Tlr2 −/− BMDCs (CD45+CD11b+Cd11c+) showed a refractory response to NEFA-induced Il-1β mRNA in vitro, whereas BMDMs (CD45+F4/80+CD11b+Cd11c−) did not. The pancreatic islet was also tested as a representative tissue with respect to NEFA induction of Il-1β mRNA. Consistent with the effects seen in BMDCs and our in vivo effects, Tlr2 −/− islets were also refractory to palmitate-induced IL-1β mRNA. We previously found no effect of oleate on mouse islet Il-1β mRNA (not shown) . These data suggest that Tlr2 −/− CD11c+ cells are resistant to the effects of NEFAs present in HFD, possibly resulting in the reduced tissue inflammation seen in the liver and islets of HFD Tlr2 −/− mice. However, given the broad tissue expression of Tlr2 mRNA, we cannot conclude whether the observed reductions in tissue inflammation in vivo are due to an immune cell, or parenchymal cell, origin.
Recent data also suggest a role for TLR2 in human type 2 diabetes. One study has shown increased production of TLR2 protein on circulating monocytes following feeding of a high-fat high-carbohydrate meal in healthy lean human participants . Evidence for increased TLR2 protein levels on monocytes and increased circulating TLR2 ligands in recently diagnosed type 2 diabetes patients has also been reported . Monocytes from these type 2 diabetes individuals showed increased proinflammatory cytokine secretion following TLR2 stimulation . Further, adipose tissue from participants with obesity and type 2 diabetes compared with controls also showed increased protein levels of TLR2 . Whether TLR2 activation also contributes to insulin resistance and beta cell dysfunction in humans with type 2 diabetes awaits further investigation.
In summary, these data show that deficiency in TLR2-mediated signalling has a positive impact on glucose homeostasis, insulin sensitivity, insulin secretion and energy-substrate utilisation during HFD feeding. The overall improved metabolic phenotype of Tlr2 −/− mice on HFD is likely to be due to both reduced fat accumulation and reduced tissue inflammation, impacting on tissue-specific functions to improve both insulin sensitivity and secretion.
We thank R.M. Zinkernagel and A.A. Navarini (University Hospital of Zurich) for the Tlr2 −/− mice. We thank M. Borsig, A. Fitsche and G. Kristiansen for technical assistance. This work was supported by grants from the Swiss National Science Foundation (M. Y. Donath, D. Konrad), the European Foundation for the Study of Diabetes (EASD/MSD to M. Y. Donath), the Juvenile Diabetes Research Foundation (M. Y. Donath), the Katholieke Universiteit Leuven (GOA 2004/11 and GOA 2008/16, to F.S.), the Gebert Rüf Stiftung (GRS-027/06 to O. Tschopp), the Swiss SystemsX.ch Initiative to the project ‘LiverX’ of the Competence Center for Systems Physiology and Metabolic Diseases (O. Tschopp and S. M. Schultze) and the University Research Priority Program ‘Integrative Human Physiology’ at the University of Zürich (J.A. Ehses and M. Y. Donath).
Duality of interest
M. Y. Donath is a consultant for Amgen, XOMA, Novartis, Merck, Solianis, and Nycomed. M. Y. Donath is listed as the inventor on a patent (WO6709) filed in 2003 for the use of an IL-1 receptor antagonist for the treatment of or prophylaxis against type 2 diabetes. The patent is owned by the University of Zurich, and M. Y. Donath has no financial interest in the patent. All other co-authors have no duality of interest to report.
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