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Tlr2 is critical for diet-induced metabolic syndrome in a murine model.

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  • 1. Texas Children's Hospital, Section of Pediatric Gastroenterology, 1102 Bates St., Texas Children's Feigin Center, Rm. 860B, Houston, TX, 77030, USA.
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    • Himes RW 1
    (1 author)

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FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 19 Oct 2009, 24(3):731-739
https://doi.org/10.1096/fj.09-141929 PMID: 19841034 PMCID: PMC2830137

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Abstract 

Obesity and its associated comorbidities, termed metabolic syndrome, are increasingly prevalent, and they pose a serious threat to the health of individuals and populations. Gene-environment interactions have been scrutinized since the kinetics of the increased prevalence of obesity would argue against a purely genetic etiology. Toll-like receptors (TLRs), widely expressed and highly conserved transmembrane receptors, are at the intersection of diet and metabolism, and may therefore be important determinants of weight gain and its sequellae. We sought specifically to determine the role of Tlr2 in the development of obesity and metabolic syndrome utilizing two dietary models that approximate contemporary diet compositions. Using C57BL/6 Hsd mice (wild type, WT) and mice with a targeted mutation in Tlr2 (Tlr2(-/-)), we showed that mice lacking TLR2 are substantially protected from diet-induced adiposity, insulin resistance, hypercholesterolemia, and hepatic steatosis. In adipose tissue, Tlr2 deletion was associated with attenuation of adipocyte hypertrophy, as well as diminished macrophage infiltration and inflammatory cytokine expression.-Himes, R. W., Smith, C. W. Tlr2 is critical for diet-induced metabolic syndrome in a murine model.

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FASEB J. 2010 Mar; 24(3): 731–739.
PMCID: PMC2830137
PMID: 19841034

Tlr2 is critical for diet-induced metabolic syndrome in a murine model

Ryan W. Himes*†,1 and C. Wayne Smith*‡
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Abstract

Obesity and its associated comorbidities, termed metabolic syndrome, are increasingly prevalent, and they pose a serious threat to the health of individuals and populations. Gene-environment interactions have been scrutinized since the kinetics of the increased prevalence of obesity would argue against a purely genetic etiology. Toll-like receptors (TLRs), widely expressed and highly conserved transmembrane receptors, are at the intersection of diet and metabolism, and may therefore be important determinants of weight gain and its sequellae. We sought specifically to determine the role of Tlr2 in the development of obesity and metabolic syndrome utilizing two dietary models that approximate contemporary diet compositions. Using C57BL/6 Hsd mice (wild type, WT) and mice with a targeted mutation in Tlr2 (Tlr2−/−), we showed that mice lacking TLR2 are substantially protected from diet-induced adiposity, insulin resistance, hypercholesterolemia, and hepatic steatosis. In adipose tissue, Tlr2 deletion was associated with attenuation of adipocyte hypertrophy, as well as diminished macrophage infiltration and inflammatory cytokine expression.—Himes, R. W., Smith, C. W. Tlr2 is critical for diet-induced metabolic syndrome in a murine model.

Keywords: adipose tissue, hepatic steatosis, insulin resistance, obesity

Obesity is a worldwide health threat. Metabolic syndrome, the name given the constellation of conditions consisting of abdominal obesity accompanied by insulin resistance or diabetes mellitus, atherogenic dyslipidemia, hepatic steatosis, hypertension, and low-grade systemic inflammation, is associated with coronary artery disease, stroke, and possibly excessive mortality (1, 2). The treatment of established obesity is exceedingly difficult; therefore, efforts aimed at understanding its pathogenesis are needed to inform preventive strategies and avert a burgeoning public health crisis.

Toll-like receptors (TLRs) are highly conserved transmembrane receptors with widespread expression (3), including expression on the cells most intimately involved in the pathogenesis of metabolic syndrome, namely adipocytes and macrophages. Each TLR recognizes a specific repertoire of ligands and is capable of initiating activation of NF-κB, a master regulator of the molecular inflammatory response. Though classically described as receptors for antigens associated with a variety of bacteria, viruses, and fungi, recent reports suggest that TLRs also sense and respond to nutritional fatty acids (4, 5). In this capacity, TLRs may be viewed as a possible bridge between nutrition and molecular inflammation, the harbinger of metabolic syndrome. With this as background, the role of TLR signaling in models of diet-induced obesity becomes an important question.

Previous work has implicated Tlr4 in the pathogenesis of metabolic syndrome; however, different experimental approaches have led to varying conclusions. In work from the Flier laboratory (5), it was shown that Tlr4-knockout mice consuming a high-saturated-fat diet were more obese after 26 wk of feeding than were wild-type (WT) mice. That difference in body composition was associated with increased food consumption in the knockout animals, suggesting centrally mediated changes in satiety control mechanisms. On the contrary, Tsukumo et al. (6) observed that mice with a loss-of-function mutation in TLR4 exposed to high-fat diet for 8 wk actually weighed less than mice with functional TLR4. In comparison, their data showed similar food consumption between both groups but increased oxygen consumption in the TLR4 mutants fed a high-fat diet, invoking expenditure rather than intake as the etiology for the observed differences in body composition. Strikingly, however, both papers showed improved insulin sensitivity in the mice with impaired/absent TLR4, reinforcing a fundamental, though complex, role for TLRs in propagating metabolic syndrome.

The role of Tlr2 in metabolic syndrome has not yet been systematically examined, though several lines of evidence converge on the view that it may be important. In addition to its well-known role as a receptor for components of gram-positive bacteria (3), it can bind dietary fatty acids (4) and induce insulin resistance (7). Moreover, the seminal work of Turnbaugh et al. (8), demonstrating a shift in the microbial flora of obese rodents and humans to one enriched for gram-positive organisms, raises the interesting question as to whether these bacteria may also drive metabolic inflammation through TLR2. A single publication reported that transient siRNA inhibition of TLR2 resulted in improved insulin sensitivity in mice fed a high-fat diet for 2 mo (9), similar to the findings of Tsukumo et al. (6) and Shi et al. (5) in TLR4 models. They did not, however, report the effects of TLR2 inhibition on other elements of metabolic syndrome, nor did the transient nature of their experimental approach allow for the study of body weight or composition. Herein, we describe the phenotype and molecular consequences of Tlr2 deletion in two models of diet-induced metabolic syndrome and find support for our hypothesis that Tlr2 critically modulates this process.

MATERIALS AND METHODS

Animals and diets

All studies utilized male C57BL/6 Hsd mice (WT) and mice with a targeted deletion of Tlr2 (Tlr2−/−; a kind gift from Dr. Jesus Vallejo, Baylor College of Medicine, Houston, TX, USA), which were backcrossed 9 generations onto the C57BL/6 Hsd background. Experimental feeding commenced at 4 to 5 wk of age and was of variable duration, as described for each experiment. Animals were randomized to receive ad libitum conventional rodent chow (CRC; 2020X; kcal %: fat 16%, carbohydrate 62%, protein 22%; Harlan Teklad, Indianapolis, IN, USA), a custom diet designed to mirror an American-style diet (AD; 101588; kcal %: fat 32%, carbohydrate 51%, protein 17%; Dyets, Bethlehem, PA, USA) or a commercially available Western diet (WD; 112734; kcal %: fat 42%, carbohydrate 42%, protein 16%; Dyets). We have previously shown that the WD induces significant inflammatory effects in abdominal adipose tissue (10,11,12).

Animals were housed in a conventional barrier vivarium at the Children’s Nutrition Research Center, and experiments were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.

Body composition analysis and calorimetry

A Lunar PIXImus dual-energy X-ray absorptiometer (DEXA) was used to obtain body composition in anesthetized animals.

Indirect calorimetry and food consumption were assessed at the Children’s Nutrition Research Center Mouse Metabolic Research Unit using the comprehensive laboratory animal monitoring system (Columbus Instruments, Columbus, OH, USA) for 36 h in a cohort of mice (n=3/group) after acclimatization to single housing and a new cage environment.

Serum measurements

Fasting serum glucose, cholesterol, low-density lipoprotein (LDL), and triglycerides were determined using an automated clinical chemistry analyzer at the Center for Comparative Medicine at Baylor College of Medicine. Serum alanine aminotransferase (ALT) was determined by colorimetric means using Infinity ALT reagent (Thermo Scientific, Louisville, CO, USA). Serum leptin and insulin were determined using commercial mouse ELISA kits (Crystal Chem, Downers Grove, IL, USA) according to the manufacturer’s protocol. Serum free fatty acids were measured with a commercially available kit (Wako Pure Chemical Industries, Osaka, Japan) by the Diabetes and Endocrinology Research Center core lab at Baylor College of Medicine.

Histology

Portions of liver and epididymal adipose tissue were preserved in zinc fixative (BD Biosciences, San Jose, CA, USA) and subsequently processed and paraffin embedded. Five-micrometer sections were mounted for hematoxylin and eosin (H&E) staining. A separate portion of each liver was frozen in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura, Torrance, CA, USA). Five-micrometer sections were mounted for oil red O staining. Adipocyte size was quantitatively assessed using H&E-stained sections, and an unbiased stereological counting method is described elsewhere (13).

Quantitative RT-PCR

Total RNA was isolated from tissues using RNeasy columns (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using AMV enzyme (Roche Applied Science, Mannheim, Germany). Custom-designed sequence-specific primers were used with SYBR Green chemistry (Roche Applied Science, Mannheim, Germany) on an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). Gene expression was normalized to the housekeeping gene Gapdh. See Supplemental Data for primer sequences.

Data analysis

Values are presented as means ± se. Two-factor ANOVA followed by Fisher’s least-significant-difference procedure was used to determine statistical significance. For food consumption and energy expenditure data derived from calorimetry experiments, P values reflect animal body weight as a covariate (14).

RESULTS

Body composition and calorimetry

A defining component of metabolic syndrome is adiposity, and indeed, WT mice exposed to AD or WD for 5 wk gained more body fat than those fed CRC (27.62±1.5, 30.47±1.5 vs. 17.35±1.5%, respectively; Fig. 1A). Tlr2−/− mice were completely protected from both AD- and WD-induced adiposity, having comparable body fat composition to CRC-fed mice of either genotype (Tlr2−/− CRC 19.07±1.5%, AD 17.3±1.5%, WD 19.05±1.5% vs. WT CRC 17.35±1.5%; P=ns; Fig. 1A). The lean body mass of both genotypes was similar (20.46±0.56 vs. 20.13±0.62 g for WT and Tlr2−/−; P=0.6951) underscoring the adipose compartment as the primary determinant of differences in body composition. Serum levels of the adipokine leptin confirmed adiposity as assessed by DEXA (Fig. 1B). Growth curves from 5-wk (Fig. 1C) and 14-wk (Fig. 1D) feeding experiments demonstrate similar growth velocities for both genotypes.

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American diet or Western diet feeding induces adiposity. A) Exposure to either diet model for 5 wk resulted in increased body fat, as assessed by DEXA scan, which was completely prevented by Tlr2 deletion (n=4/group). B) Changes in adiposity were reflected in circulating leptin levels (n=6–7/group). C, D) Growth curves from 5 wk (C) and 14 wk (D) feeding experiments demonstrate similar growth velocity in all groups. *P < 0.001 vs. WT CRC; +P < 0.001, ϕP < 0.05 vs. corresponding WT.

Over 36 h of observation in the Mouse Metabolic Research Unit, there were no differences in caloric intake between mice in any group (WT CRC 13.8±0.8 kcal, Tlr2−/− CRC 12.9±0.8 kcal, WT AD 13.5±0.8 kcal, Tlr2−/− AD 11.8±0.8 kcal; P=ns; Fig. 2A). Energy expenditure was modestly increased in WT AD mice compared to the WT CRC group (0.45±0.01 vs. 0.39±0.01 kcal/h; P=0.001; Fig. 2B). Within diets, Tlr2 deletion only affected CRC-fed mice, with the Tlr2−/− group having slightly higher energy expenditure than the WT CRC group (0.43±0.01 vs. 0.39±0.01 kcal/h; P=0.008; Fig. 2B).

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Food consumption and energy expenditure in the American diet model. A) Over 36 h of observation, no differences were detected in caloric intake among any group (n=3/group). B) Energy expenditure was significantly increased in all groups relative to the WT CRC group (n=3/group). *P = 0.001 vs. WT CRC; +P = 0.008 vs. corresponding WT; body weight used as a covariate for both analyses.

Glucose homeostasis and insulin sensitivity

Analysis of fasting serum glucose and insulin after 5 wk of AD feeding failed to demonstrate significant differences compared to the CRC group. This finding may indicate that derangements in carbohydrate metabolism are not yet present at this early time point, so we subsequently measured these parameters after 14 wk of AD feeding. Fasting serum glucose was found to be increased in WT mice fed AD compared to CRC (366.7±25.5 vs. 279.4±19.7 mg/dl; P=0.007; Fig. 3A). Tlr2 deletion prevented this increase among AD-fed mice (275.7±25.5 vs. 366.7±25.5 mg/dl; P=0.012; Fig. 3A) but had no effect on serum glucose among the CRC fed group. Similarly, fasting serum insulin was increased after 14 wk of AD feeding (1.29±0.14 vs. 0.54±0.13 ng/ml; P<0.001; Fig. 3B), and Tlr2 deletion was associated with protection against that increase for AD (0.29±0.14 vs. 1.29±0.14 ng/ml; P<0.001; Fig. 3B) but not CRC-fed mice. As a measure of insulin resistance, the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated. As would be predicted, there was an increase in HOMA-IR between WT mice fed CRC and AD (2.74±1.3 vs. 12.63±1.7 AU; P<0.001; Fig. 3C), implying relative insulin resistance. On the other hand, Tlr2 deletion was associated with normalization of HOMA-IR for mice in the AD group (1.4±1.7 vs. 12.6±1.7 AU; P<0.001; Fig. 3C), suggesting maintenance of insulin sensitivity. WD-fed mice had serum glucose and insulin analyzed after 5 wk of feeding. Like their AD-fed counterparts, Tlr2−/− WD mice had serum glucose values similar to either CRC-fed group (258.6±22.5 vs. 288.1±34.1 or 250.4±19.2 mg/dl; P=ns; Fig. 3D). There was a WD-dependent increase in serum insulin that did not vary by genotype (Fig. 3E). However, congruent with findings in the AD model, HOMA-IR was increased in WT WD mice (9±1.2 vs. 1.16±1.1 AU; P<0.001; Fig. 3F) and this difference was abolished in the Tlr2−/− mice (2.1±1.2 vs. 9±1.2 AU; P<0.001; Fig. 3F), resulting in comparable HOMA-IR scores among WT CRC, Tlr2−/− CRC, and Tlr2−/− WD groups.

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Diet-induced insulin resistance is abolished in Tlr2−/− mice. A–C) After 14 wk of feeding, fasting serum glucose (n=3–5/group; A) and insulin (n=4–5/group; B) were elevated among WT AD mice but not the Tlr2−/− AD group, leading to normalization of the HOMA-IR (n=3–5/group; C). D–F) In mice fed WD for 5 wk, serum glucose was similarly reduced in the Tlr2−/− group (n=5–7/group; D); however, insulin (n=6–7/group; E) was not different. HOMA-IR, on the other hand, was reduced to basal levels in Tlr2−/− WD mice (n=4–5/group; F). *P < 0.008 vs. WT CRC; +P < 0.0013 vs. corresponding WT.

Serum lipids

Fasting measurement of serum total cholesterol demonstrated the increase anticipated in WT mice exposed to AD or WD at 5 wk compared to CRC (173±10.7, 230±10.7 vs. 127±10.7 mg/dl; P<0.003; Fig. 4A). Tlr2−/− mice fed either experimental diet had total cholesterol levels similar to the CRC group. Confirming the development of an atherogenic lipid profile, WT AD- and WD-fed mice had elevated LDL levels relative to WT CRC (26.4±3, 51.5±3 vs. 15.2±3 mg/dl; P<0.01; Fig. 4B). As with total cholesterol, serum LDL increases were largely prevented in Tlr2−/− mice in both experimental diet groups. Serum triglycerides were elevated only in the WT WD group compared to WT CRC (132.2±6.8 vs. 99.4±6.8 mg/dl; P=0.01; Fig. 4C). Nonetheless, there were statistically important reductions in fasting serum triglycerides in the Tlr2−/− groups fed either AD or WD (72.2±6.8 vs. 93.4±6.8 mg/dl, P=0.027, and 95.8±6.8 vs. 132.2±6.8 mg/dl, P<0.001, respectively, Fig. 4C).

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Fasting serum lipids after 5 wk of diet exposure. A, B) Diet-induced increases in serum cholesterol (A) and LDL (B) were attenuated in Tlr2−/− mice in both models (n=5/group). C) Serum triglycerides increased only in the WT WD group; however, Tlr2 deletion led to reduced triglycerides in both experimental models (n=5/group). D) Serum free fatty acids were increased in all Tlr2−/− groups irrespective of diet (n=6/group). *P < 0.025 vs. WT CRC; +P < 0.028 vs. corresponding WT.

On the other hand, serum free fatty acids were significantly increased among Tlr2-deleted mice irrespective of diet (Fig. 4D). WT AD mice had slightly lower serum free fatty acids than did the WT CRC group (0.91±0.02 vs. 0.83±0.02 mEq/L; P<0.025; Fig. 4D).

Liver histology

Oil red O-stained sections of liver were examined after 5 wk of feeding to qualitatively assess the degree of hepatosteatosis. Neither genotype fed CRC demonstrated appreciable staining, indicating a relative paucity of intracellular lipid (Fig. 5A). In contrast, the WT AD group showed diffuse, primarily microvesicular staining consistent with the presence of hepatosteatosis that was almost completely absent in Tlr2−/− mice fed AD (Fig. 5A). WT WD mice exhibited more florid macrovesicular steatosis, which was also prevented for the most part in the Tlr2−/− WD group (Fig. 5A).

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Hepatic lipid content and serum ALT. A) Among WT mice, feeding AD or WD for 5 wk resulted in the intrahepatic deposition of lipid, which was substantially prevented in Tlr2 deleted animals; oil red O stain, original view ×10. B) Serum ALT was increased significantly only among WT WD mice compared to WT CRC (n=7–8/group). *P = 0.005 vs. WT CRC.

Having established an apparent association between hepatic steatosis and Tlr2, we next sought to determine whether there was a correlation between Tlr2 and hepatic inflammation and necrosis, characteristic of nonalcoholic steatohepatitis (NASH). Serum ALT levels were different among only WT WD and CRC mice (67.7±7.8 vs. 35.5±8.4 U/L; P=0.005; Fig. 5B). Examination of H&E-stained sections of liver revealed occasional foci of mixed inflammatory cells variably associated with necrosis and most consistent with focal granulomas, a normal finding in rodent liver tissue (15). Because their occurrence is not pathological and did not vary by group, these models recapitulate steatosis but not NASH after 5 wk of exposure.

Adipose tissue

H&E-stained sections of epididymal adipose tissue from CRC- and AD-fed mice were prepared to assess both adipocyte morphology and the degree of leukocytic infiltration since changes in both have been observed in growing adipose tissue depots. Compared to all other groups, mice in the WT AD group had the expected increase in the size of adipocytes (Fig. 6A), which we confirmed by quantitative stereology (data not shown). Concomitantly, the frequency of infiltrating leukocytes forming circumferential aggregates around adipocytes (so-called crown-like structures; ref. 16) increased in this group. Notably, both the increase in adipocyte size and the greater number of crown-like structures in WT AD mice were substantially attenuated in the Tlr2−/− AD group (Fig. 6A). Quantitative PCR (QPCR) for the expression of the Emr1 murine macrophage antigen (17, 18) confirmed a 5- and 7-fold induction in WT AD and WD mice, respectively, compared to WT CRC mice (Fig. 6B), that was significantly abrogated in Tlr2−/− mice, corroborating our histological observations.

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Adipose tissue morphology and gene expression. A) Sections of epididymal adipose tissue demonstrate AD-induced hypertrophy of adipocytes and more numerous eosinophilic crown-like structures among WT mice. Tlr2−/− AD mice showed the expected lack of adipocyte hypertrophy but also fewer crown-like structures; H&E stain. Scale bars = 100 μm. B–E) Retroperitoneal adipose tissue QPCR (n=5/group). WT AD and WD mice had increased expression of murine macrophage antigen Emr1 (B), which may have been mediated, in part, by higher expression of the chemotactic factor Ccl2 (C). Emr1 and Ccl2 expression was significantly lower in Tlr2−/− mice. Though TNF-α expression did not differ among any group (D), Il6 expression increased in both diet models but was significantly diminished by Tlr2 deletion (E). *P < 0.019 vs. WT CRC; +P < 0.029 vs. corresponding WT.

To investigate factors that could modulate the differential infiltration of macrophages into adipose tissue during experimental feeding, we then examined the expression of Ccl2, a potentially important chemokine for recruitment of macrophages in adipose tissue. Its expression was up-regulated >3-fold in WT AD mice and >6-fold in WT WD mice compared to the WT CRC group (Fig. 6C) but reduced significantly in Tlr2−/− mice fed AD or WD (Fig. 6C).

Because both experimental feeding groups were found to be more insulin resistant relative to WT CRC by HOMA-IR score (Fig. 3C, F), we examined the adipose tissue expression of canonical inflammatory cytokines Tnfa and Il6, since they have been implicated in the pathogenesis of insulin resistance (19). Although Tnfa was not differentially expressed among any group after 5 wk of feeding (Fig. 6D), we found an induction of Il6 in both the WT AD and WD groups relative to WT CRC (Fig. 6E). On the other hand, Tlr2−/− mice were substantially protected from this increase, having Il6 expression similar to that of CRC-fed groups.

Mechanisms of hepatosteatosis

Human nonalcoholic fatty liver disease (NAFLD) is thought to result from one or more of the following mechanisms: increased delivery of lipid to the liver, increased de novo lipogenesis, reduced lipid oxidation, and reduced lipid export from the liver (20). Because at least in the AD model, which we tested with calorimetry, there were no differences in caloric intake, we performed QPCR for key genes in the aforementioned pathways to elucidate potential mechanisms underlying our observations.

Srebp1c, which encodes a transcription factor regulating a battery of lipogenic genes, demonstrated diet-dependent suppression irrespective of genotype in both models (Fig. 7A). However, because Srebp1c is subject to post-transcriptional regulation, the expression of a downstream gene, fatty acid synthase (Fas) was also assayed. Like the expression of Srebp1c, however, Fas expression did not differ among either genotype fed AD or WD (Fig. 7B), providing further support that de novo lipogenesis does not explain the disparate degree of hepatosteatosis between these groups.

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Hepatic gene expression and serum VLDL. A–C) Hepatic QPCR (n=4–5/group). A) AD or WD feeding was associated with suppression of hepatic Srebp1c expression. B) Analysis of its downstream gene, Fas, confirmed AD and WD-dependent transcriptional suppression of the lipogenic pathway. C) Ppara, a key transcription factor in lipid oxidation, showed significant reduction in Tlr2−/− mice relative to WT AD mice. D) Serum VLDL was measured as a gauge of hepatic lipid export. Among AD- or WD-fed mice only, Tlr2 deletion was associated with decreased serum VLDL (n=5/group). *P < 0.04 vs. WT CRC; +P < 0.027 vs. corresponding WT.

To assess hepatic lipid oxidative activity, the expression of hepatic transcription factor Ppara was determined. Although there were no differences in expression between WT CRC and AD or WD groups, there was a statistically significant reduction in its expression in Tlr2−/− AD mice compared to WT AD mice (Fig. 7C). Such a change in gene expression would be expected to lead to an increased degree of hepatosteatosis in the Tlr2−/− AD group rather than decreased amount that we observed.

Serum very low density lipoprotein (VLDL) concentrations were measured as an index of hepatic lipid export. Mice from WT CRC, Tlr2−/− CRC, and WT AD groups all had similar levels of serum VLDL (Fig. 7D). Tlr2−/− mice fed AD or WD, however, had a statistically significant decline in circulating VLDL (18.6±1.3 vs. 14.4±1.3 mg/dl for WT AD and Tlr2−/− AD, P=0.027; WT WD 26.4±1.3 vs. 19.1±1.3 mg/dl for WT WD and Tlr2−/− WD, P<0.001; Fig. 7D). In keeping with the Ppara expression data, however, this finding would likely lead to increased rather than decreased steatosis in the Tlr2−/− group, contradictory to histological observations.

DISCUSSION

We have utilized two different dietary approaches, each based on contemporary consumption patterns in the developed world, to establish short-term models of obesity with certain features of metabolic syndrome. The AD is enriched in ω-6 polyunsaturated fats and sucrose, whereas the WD is composed of saturated fats and sucrose and is supplemented with cholesterol, although it should be emphasized that several differences between the diets preclude direct comparison with one another. By standard clinical criteria, both models were efficient inducers of obesity, insulin resistance, hypercholesterolemia, and hepatic steatosis. Increased expression of inflammatory cytokines and chemokines in adipose tissue of both models underscores the assertion that this important tissue is involved in propagating molecular inflammation. Unlike the WD model, in which hyperlipidemia and insulin resistance were both detected early (5 wk), in the AD model, derangements in lipid homeostasis preceded abnormal carbohydrate metabolism, which was not present at the 5-wk time point but was apparent at 14 wk. Differences in diet composition may account for this discrepancy. On the basis of data derived from calorimetry experiments, we have also shown that in the AD model, mice consumed isocaloric amounts of food irrespective of genotype or diet, at least over a limited period of observation. We cannot, however, exclude the small cumulative differences in consumption over the entire 5-wk feeding period. Energy expenditure was increased to a similar degree in all groups relative to WT CRC, so this too is unlikely to be responsible for the disparate phenotypes.

We have demonstrated that cardinal features of metabolic syndrome are almost completely prevented by Tlr2 deletion, indicating a central role for this pathogen-recognition receptor in modulating an important metabolic disease independent of energy intake or expenditure. Although Tlr2−/− mice exposed to either experimental model were fully protected from insulin resistance as assessed by HOMA-IR, whether this association holds true independent of adiposity is unknown, since it was not possible to tease them apart in our model systems. This is a relevant distinction to make, however, since evidence from the TLR literature is that body weight is not always directly related to insulin sensitivity (5). Our results also indicate that Tlr2-knockout mice were spared from increases in total serum cholesterol and LDL cholesterol, which may indicate a role for Tlr2 in regulating serum lipids as well. This would be consistent with the observations of Liu et al. (21), who showed that Tlr2−/− Apoe−/− double-knockout mice had reduced lipid accumulation and macrophage recruitment to the aortic sinus in a model of atherosclerosis compared to Apoe−/− mice. Fasting serum triglycerides were also reduced in Tlr2−/− mice under both experimental models. The absence of hypertriglyceridemia in the WT AD group may reflect the known triglyceride reducing effects of polyunsaturated fatty acids (22), the primary fat source in that model.

On the basis of histological examination of the liver, both of our models recapitulate NAFLD (cf. NASH), insofar as steatosis is present without pathological inflammation, necrosis, or fibrosis. That both experimental diets resulted in hepatosteatosis is not surprising given the nature of their composition; however, the lack of steatosis observed in Tlr2-deleted mice was not anticipated, particularly in the AD model, in which we showed isocaloric food consumption. We examined hepatic gene expression in an attempt to elucidate mechanisms that could explain this finding but did not discover a plausible mechanism in our limited evaluation. Although it plays a central role in lipogenesis, transcriptional regulation of hepatic Srebp1c does not appear to account for the contrasting degree of steatosis observed, as it and its downstream gene Fas, were not differentially expressed among experimentally fed mice. Moreover, expression of hepatic Ppara, a transcription factor in the β-oxidation pathway, was decreased in Tlr2−/− mice on AD relative to WT, which would be expected to enhance hepatosteatosis rather than to diminish it. Likewise, serum VLDL was also reduced in Tlr2−/− mice in both experimental diet groups, which again would be predicted to portend increased steatosis. However, the observation that serum free fatty acids were elevated in Tlr2-knockout mice from each diet group was an unexpected but potentially significant finding. We believe this may indicate defective uptake or utilization of fatty acids in liver and other peripheral tissues, resulting in excess spillover into the vascular compartment. Intriguingly, the scavenger receptor CD36 (fatty acid translocase) imports fatty acids into cells (23,24,25) and has been shown to be an accessory receptor for TLR2 (26,27,28). It is conceivable that absent TLR2, CD36-mediated fatty acid import is impaired in liver and other tissues, explaining how mice with equivalent caloric intake and energy expenditure could have a dissimilar degree of adiposity and steatosis along with an increase in serum free fatty acids. In support of that hypothesis, a Cd36-knockout mouse has been generated (29), and there are compelling phenotypic similarities between it and the Tlr2−/− mice in our study. Compared to their sufficient counterparts, both knockout strains have lower body weight (30), lower serum glucose (29, 30) and insulin (31), and less hepatic triglyceride content (32), as well as higher serum free fatty acids (29, 30).

We have also shown that molecular events in adipose tissue fundamental to the development of obesity are present in our models and attenuated to a great degree in Tlr2-knockout mice. Histologically, Tlr2−/− mice fed AD had smaller adipocytes than WT AD mice, and there were fewer crown-like structures present in this group compared to controls. These aggregates have been characterized by others as infiltrating macrophages surrounding distressed adipocytes (16, 33,34,35), and congruent with our histological observation, the increased expression of the murine macrophage marker Emr1 was completely prevented in Tlr2−/− mice fed AD and reduced in Tlr2−/− WD mice. One possible explanation for this finding is that Ccl2 expression was significantly decreased in Tlr2−/− AD and WD mice, which would be predicted to lead to fewer macrophages being recruited to adipose tissue. Functional consequences of diminished macrophage infiltration into adipose tissue include qualitative and quantitative changes in the cytokine milieu. We showed that Tlr2-deleted animals fail to up-regulate Il6 in adipose tissue in response to AD or WD, which may explain the improved insulin sensitivity in these groups.

We have shown that obesity and other cardinal features of metabolic syndrome can be prevented in Tlr2-deleted mice using two physiologically relevant diet models. These experiments add to the literature by confirming the work of Caricilli et al. (9) and documenting for the first time the additional observation that Tlr2 plays an integral role in the obesity phenotype as well, independent of caloric intake or energy expenditure. We further demonstrated histologically and by QPCR that Tlr2 deletion alters the well-described events in adipose tissue that are believed to be the molecular basis for metabolic syndrome. Some limitations of our data also merit discussion. First, we relied on the HOMA-IR score as an index of insulin resistance. Although commonly utilized in research, its use in nonhuman research has been criticized (36), and it may lack the sensitivity of other options, such as insulin tolerance tests and euglycemic clamps. Also, although our dietary models are designed to mirror contemporary diets, they are not specifically matched so as to allow head-to-head comparison between them; thus, this interesting question remains open. Lastly, our model systems do not allow us to tease apart the effect of Tlr2 deletion from other elements of metabolic syndrome, independent of changes in whole-body adiposity, although our current research efforts seek to address this knowledge gap.

Acknowledgments

This work was supported by National Institutes of Health (NIH) institutional research training grant T32 DK 07644, U.S. Department of Agriculture grant ARS 6250-51000-046-01A, and NIH grant R01 DK078847. We are grateful for the expert assistance provided by the Mouse Metabolic Research Unit at the Children’s Nutrition Research Center, the Texas Medical Center Digestive Disease Center (NIH/National Institute of Diabetes and Digestive and Kidney Diseases Center grant P30 DK56338) and the Baylor College of Medicine Diabetes and Endocrinology Research Center (DK-079638).

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