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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Toll-like receptor-4 (Tlr-4), a key pattern recognition receptor involved in innate immune response, is activated by saturated fatty acids (SFAs). To investigate the involvement of this receptor in obesity caused by consumption of diets high in fat, we utilized male Tlr-4-deficient 10ScN mice and 10J controls. Mice were fed either low fat (low-fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or high saturated fat + palmitate (HFP) diets ad libitum for 16 weeks. Relative to the LFC diet, the HFC diet resulted in greater epididymal fat pad weights and adipocyte hypertrophy in both Tlr-4-deficient and normal mice. However, the 10ScN mice were completely protected against the obesigenic effects of the HFP diet. Moreover, macrophage infiltration and monocyte chemotactic protein-1 (MCP-1) transcript abundance were lower in adipose tissue of 10ScN mice fed the HFP diet, and the hyperinsulinemic response was negated. Tlr-4-deficient mice also had markedly lower circulating concentrations of MCP-1 and much less nuclear factor-κB (NFκB) protein in nuclear extracts prepared from adipose tissue, irrespective of diet. In contrast, Tlr-4 deficiency did not attenuate the induction of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) expression in adipose tissue. These data indicate that Tlr-4 deficiency selectively protects against the obesigenic effects of SFA and alters obesity-related inflammatory responses in adipose tissue.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Obesity is associated with chronic low-grade inflammation (1,2,3,4) that is characterized by increased circulating concentrations of proinflammatory cytokines (5,6,7) and acute phase proteins (8,9), and decreased concentrations of the anti-inflammatory protein, adiponectin (10). Although the mechanisms underlying the onset and progression of this inflammatory state remain somewhat ambiguous, two recent findings have strongly implicated the adipose tissue itself as a major contributor. First, genetic and diet-induced obesity (DIO) models have not only shown increased expression of proinflammatory cytokines in the adipose tissue (1,3,11,12) but also a marked adipose-specific infiltration of macrophages which show gene expression profiles consistent with inflammation (13,14,15). Secondly, the adipocyte itself is capable of mounting a classical innate immune response initiated by ligand activation of Toll-like receptor-4 (Tlr-4), followed by activation of nuclear factor-κB (NFκB), increases in proinflammatory gene expression and the release of these cytokines from cultured adipocytes (16,17,18). Furthermore, both Tlr-2 and Tlr-4 expression are increased in adipose tissue in association with obesity and noninsulin-dependent diabetes mellitus (19).

Whereas it is clear from multiple obesity models that adipocytes, and macrophages localized in adipose tissue, are producing proinflammatory mediators, the mechanistic links among adipocyte hypertrophy, dietary fat, inflammation, and macrophage recruitment and activation have not been delineated. However, direct regulation of Tlr-4 by certain saturated fatty acids (SFAs) is a likely component of this process because palmitate and other SFAs act directly to stimulate proinflammatory cytokine expression and NFκB activation in cultured adipocytes and macrophages (20,21). Furthermore, Suganami et al. (21) recently determined that free fatty acids released from hypertrophic adipocytes can signal macrophages through Tlr-4 and stimulate release of tumor necrosis factor-α (TNF-α). Additional studies by other groups (20,22,23,24) have shown that Tlr-4 deficient mice are less susceptible to fat-induced inflammation and insulin resistance and that C3H/HeJ mice are protected against hyperglycemia and inflammation in adipose tissue when Tlr-4 signaling is blunted (22).

Although the link between a functional Tlr-4 receptor and obesity-induced inflammation in adipose tissue seems quite clear, whether this receptor contributes to the onset and progression of obesity is controversial. Elegant in vivo studies with a Tlr-4 knockout mouse model have shown blunted inflammation, yet a greater adiposity in females (23). In contrast, Tsukumo et al. (25) recently reported evidence that a loss of function mutation in Tlr-4 protects against high fat, DIO. Albeit, there is currently a paucity of data which address the possibility that saturated vs. unsaturated dietary fats influence the ability of Tlr-4 to promote adipose accretion. Consequently, we used the 10ScN mouse strain, which has a 74-kilobase deletion from chromosome 4 that precludes expression and production of Tlr-4, to test the hypothesis that Tlr-4 specifically mediates obesity and inflammation associated with a diet high in saturated fat.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Animals and animal care

Male Tlr-4-deficient C57BL/10ScN mice (cat. no. 003752) derived from the C57BL/10 subline and corresponding control male C57BL/10J mice (cat. no. 000665), were obtained from Jackson Laboratories (Bar Harbor, ME) at 6–8 weeks of age. Subsequently, lack of Tlr-4 mRNA was confirmed with real-time PCR. Animals were housed individually in stainless steel wire-mesh cages at 21 °C in a room with an automatically controlled 12-h light:dark cycle. Mice were acclimated to the environment and provided unlimited access to food and water. Animals from each genotype were then randomly assigned to one of three experimental diets (n = 25/genotype/diet): (i) low-fat control (LFC), high-fat control (HFC), or high-fat palmitate (HFP) for 16 weeks. Both high-fat diets were semipurified, powdered diets based on American Institute of Nutrition recommendations (26) that were modified to induce obesity by providing 60% of calories from lipids (Table 1). The high-fat diets were identical, except that the source of fat used was either soybean oil in the HFC (Harlan Teklad, Madison, WI) or a mixture of lard (Harlan Teklad) and purified palmitate (Nu-Check Prep, Elysian, MN) in the HFP. The LFC diet contained 12% of calories from lipid. Food intake was measured daily and used to calculate total energy intake. Mice were fasted overnight (6–8 h) and killed by CO2 asphyxiation for all blood and tissue collections. Blood was collected by heart puncture, placed on ice, and serum was collected by centrifugation and frozen at −80 °C until analyzed. Animals were weighed and abdominal fat pads and liver were removed and weighed. All extracted tissues were immediately frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction and immunohistochemical analysis. All experimental protocols for animal care and use were approved by the Institutional Animal Care and Use Committee at Iowa State University, Ames, Iowa.

Table 1.  Diet composition
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Analysis of metabolic parameters

Serum concentrations of glucose (Biovision, Mountain View, CA) and insulin (Linco, St Charles, MO) were measured using commercial assay kits. Leptin, monocyte chemotactic protein-1 (MCP-1), adiponectin, and interleukin-6 (IL-6) serum concentrations were measured using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Serum free fatty acid was measured with nonesterified fatty acids assay-C (Wako Pure Chemical Industries, Richmond, VA) kit and protein carbonyl levels were determined with Protein Carbonyl Assay Kit (Cayman Chemical, Ann Arbor, MI).

Quantitative real-time PCR

Total RNA was extracted from frozen adipose tissue (500 mg) using a commercially available acid-phenol reagent (TRIzol; Invitrogen, Carlsbad, CA). Potential DNase contamination was removed with DNase-free (Ambion, Austin, TX). First-strand cDNA was synthesized using SuperScript III First-Strand Synthesis System for reverse transcriptase-PCR (Invitrogen). Primer sequences for mouse for sense and antisense primers are listed in Table 2. Thermal cycling conditions for PCR reactions were 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Polymerase reaction products amplified by these primers were cloned into pGEMT vector (Promega, Madison, WI) and sequenced for verification. Real-time reactions were carried out on an iCycler real-time machine (BioRad, Hercules, CA) using the IQ SYBR Green Supermix kit (BioRad). The abundance of each gene product was calculated by regressing against the standard curve generated in the same reaction with their respective plasmid. All genes of interest were normalized to β-actin and expressed as log starting quantity.

Table 2.  Primer sequences for quantitative real-time PCR
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Immunohistochemistry

Frozen adipose tissue was fixed overnight at room temperature in 10% zinc-formalin solution and embedded in paraffin. Five micron sections were cut at 50 μm intervals and mounted on glass slides, deparaffinized in xylene, and stained for expression of F4/80 with monoclonal antibody (Serotec, Raleigh, NC) as previously described (14). For each mouse, four different fields were selected, and adipocyte area was determined using AxioVision v4.5.0.0 (Carl Zeiss, Germany). F4/80-positive macrophages were then counted at ×40 original magnification and divided by total cell number to obtain percent F4/80-positive cells.

Tissue and plasma fatty acid profile

Lipids from adipose and serum samples were extracted by the method of Lepage and Roy (27) with minor modifications. In brief, 0.5 g of tissue was homogenized in 2.5 ml 4:1 methanol:hexane, and 200 μl of 3.7 mmol heptadecanoic acid/L-methanol were added to each sample as an internal standard. Fatty acid methyl esters were analyzed by gas chromatography on a Hewlett-Packard model 6890 (Hewlett-Packard, Palo Alto, CA) fitted with an Omegawax 320 (30 m × 0.32 mm internal diameter, 0.25 μm) capillary column (Sigma-Aldrich, St Louis, MO). Hydrogen was the carrier gas. The temperature program ranged from 80 to 250 °C with a temperature rise of 5 °C/min. The injector and detector temperatures were 250 °C, and 1 μl of sample was injected and run splitless. Fatty acids were identified by their retention times on the column with respect to appropriate standards.

Electrophoretic mobility shift assay and activated NFκB p65 enzyme-linked immunosorbent assay

Adipose tissue nuclear extracts and mobility shift assay were prepared and validated as previously described (16). The consensus NF-B oligonucleotides (Santa Cruz, Santa Cruz, CA) were end-labeled with [32P]ATP (PerkinElmer, Waltham, MA) using T4 polynucleotide kinase (Santa Cruz). Binding of nuclear proteins to the labeled probe was done by incubating 50 μg nuclear proteins with 50,000 counts/min of labeled probe for 30 min at room temperature in a binding buffer (2 mmol/l 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, 50 mmol/l KCl, 2 mmol/l EDTA, 10% glycerol, and 1% bovine serum albumin (wt/vol)) in the presence of 2 μg poly(dI-dC) (Sigma-Aldrich, St Louis, MO) in a final reaction volume of 40 μl. For quantitative NFκB p65 DNA binding, 10 μg of nuclear protein was assayed for the presence of activated p65 by enzyme-linked immunosorbent assay using antibodies specific for activated p65 following binding to nuclear factor-κB consensus sequence (TransAM Active Motif, Carlsbad, CA).

Statistical analyses

Data were tested for normality and analyzed using the mixed-model analysis with diet and genotype considered fixed effects, and the time dietary treatments were implemented for subgroups as a random effect. Significant interactions of main effects are indicated as diet × genotype effects. All indicated P values were two tailed and Bonferroni corrected. Differences were considered significant at P < 0.05 and a tendency at P < 0.10. Values are presented as least squares means ± s.e.m.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Tlr-4 deficiency protects mice from increased adiposity caused by consumption of a diet high in saturated fat, without reducing caloric intake

Previous studies have indicated that high-fat diets induce greater adiposity in Tlr-4-deficient mice (22–24). Consequently, we examined whether the fatty acid composition of the diet influences the obesigenic effect of high-fat diets in male Tlr-4 mutant (10ScN) and normal (10J) mice. Regardless of diet, 10ScN mice weighed on average 3.8 g less than 10J mice after 16 weeks on the experimental diets (genotype, P < 0.0001, Figure 1a). Furthermore, the HFC diet increased body weight in both genotypes (diet, P < 0.0001), but average weekly caloric intake (Figure 1b) was reduced by the HFC diet (diet, P < 0.0001). Additionally, epididymal fat pad weight was increased by the HFC diet in both genotypes. However, only the mice lacking Tlr-4 were protected against the obesigenic effect of the HFP diet (Figure 1c, diet × genotype, P = 0.05). Similar results were obtained for epididymal adipocyte size (Figure 1d) in that all mice fed the HFC diet had larger cells, but only the normal strain fed the HFP diet had enlarged cells vs. the LFC (diet × genotype, P = 0.05).

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Figure 1. Toll-like receptor-4 deficiency protects mice from increased adiposity due to consumption of a diet high in saturated fat. All measures were taken after 16 weeks (wk) on experimental diets (n = 25 unless noted otherwise). (a) Body weight of 10ScN (white bars) and 10J (black bars) mice (diet effect P < 0.0001, genotype effect, P < 0.0001, diet × genotype interaction P = 0.8436). (b) Average weekly energy intake (kcal/wk) of 10ScN and 10J mice (diet effect P < 0.0001, genotype effect P = 0.3381, diet × genotype interaction P = 0.9364). (c) Epididymal fat pad weight of 10ScN and 10J mice (diet effect P < 0.0001, genotype effect P < 0.0001, diet × genotype interaction P = 0.058). (d) Adipocyte size (μm2) of 10ScN and 10J mice (n = 6, diet effect P = 0.0079, genotype effect P < 0.0001, diet × genotype interaction P = 0.052). All displayed values represent least squares (LSs) means ± s.e.m. for diet × genotype interaction. When interaction term was P ≤ 0.15, Bonferroni correction was used to determine significant difference between means as indicated by different letters. Significant main effects (genotype and diet) were also indicated by “*” (genotype effect) and “#” (diet effect) over LS means. HFC, high-fat control; HFP, high-fat palmitate; LFC, low-fat control.

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To confirm diet-induced changes in fatty acid profiles of epididymal adipose tissue, we measured SFA, n-3, and n-6 fatty acid contents (Table 3). As expected, relative to the LFC and HFP diets, both palmitate and total SFA were reduced in mice fed the HFC diet (diet, P < 0.002), and n-3 fatty acids (docosahexenoic acid and eicosapentenoic acid) were lower in mice fed the HFP diet (diet, P < 0.002). Furthermore, the n-6 fatty acid content was higher in mutant mice (genotype, P < 0.005) and was also reduced by the HFP diet (diet, P < 0.03). However, the effects of diet and genotype on n-3 and n-6 fatty acid contents were not sufficient to alter the n-6 to n-3 ratio in this particular adipose depot.

Table 3.  Adipose tissue fatty acid profiles in normal and Tlr-4 mutant mice fed control, high fat, and high-fat palmitate diets for 16 weeks
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Tlr-4 deficiency improves markers of insulin sensitivity and inflammation

To gain insight into the effect of high fat intake on insulin sensitivity in Tlr-4-deficient mice, we measured fasting serum glucose (Figure 2a) and insulin (Figure 2b) following 16 weeks on experimental diets. Glucose concentrations were lower in the mutant strain (genotype, P = 0.0068) and were reduced in both genotypes fed either high-fat diet (diet, P = 0.0014). Insulin concentrations were also lower in Tlr-4 mutant mice (genotype, P < 0.0001). Alternatively, the HFC diet nearly doubled serum insulin in these mice, but this response only approached significance (diet, P = 0.111), and there was no effect on the normal strain (diet × genotype, P = 0.1529). However, these differences resulted in lower glucose to insulin ratios in mutant mice fed the HFC diet compared to the LFC or HFP diets, and the normal 10J mice had similar glucose to insulin ratio, regardless of diet (diet × genotype, P = 0.0275).

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Figure 2. Toll-like receptor-4 deficiency improves markers of insulin sensitivity and inflammation in mice fed a diet high in saturated fat. (a) Serum glucose (n = 10, diet effect P = 0.0014, genotype effect P = 0.0068, diet × genotype interaction P = 0.5685), (b) serum insulin (n = 10, diet effect P = 0.111, genotype effect P < 0.0001, diet × genotype interaction P = 0.1529), (c) glucose to insulin ratio (n = 10, diet effect P = 0.0377, genotype effect P < 0.0001, diet × genotype interaction P = 0.0275), (d) serum leptin (n = 10, diet effect P = 0.0004, genotype effect P = 0.4993, diet × genotype interaction P = 0.1465), (e) serum adiponectin (n = 10, diet effect P = 0.2512, genotype effect P = 0.043, diet × genotype interaction P = 0.1082), and (f) serum monocyte chemotactic protein-1 (MCP-1) (n = 10, diet effect P = 0.956, genotype effect P < 0.0001, diet genotype interaction P = 0.7812) were measured in 10ScN (white bars) and 10J (black bars) mice following 16 weeks on diet. All displayed values represent least square (LS) means ± s.e.m. for diet × genotype interaction. When interaction term was P ≤ 0.15, Bonferroni correction was used to determine significant difference between means as indicated by different letters. Significant main effects (genotype and diet) were also indicated by “*” (genotype effect) and “#” (diet effect) over LS means. ADN, adiponectin; LFC, low-fat control; HFC, high-fat control; HFP, high-fat palmitate.

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We then measured several markers of inflammation that have been associated with obesity. The serum concentration of leptin was greater in all mice fed the HFC diet (diet, P = 0.0004, Figure 2d), and serum adiponectin was lower in the mutant strain (genotype, P = 0.043, Figure 2e). In addition, the HFC diet increased serum adiponectin relative to the control and HFP diets but only in the mutant strain (genotype × diet, P = 0.0308). Serum MCP-1 was sevenfold higher in 10J vs. mutant mice (genotype, P < 0.0001, Figure 2f) but was not influenced by diet in either genotype.

Obesity-induced inflammation in adipose tissue is differentially regulated by saturated vs. unsaturated dietary fats and is partly ameliorated by Tlr-4 deficiency

To determine whether inflammatory markers were related to changes in adipose tissue, we quantified macrophage infiltration and measured several inflammatory markers in adipose tissue. The percentage of F4/80-positive cells (i.e., macrophages) in adipose tissue of 10J control mice was greater in mice fed either HF diet (diet, P < 0.0001, Figure 3a). Alternatively, Tlr-4 deficiency attenuated the SFA-induced, but not HFC-mediated, macrophage accumulation in 10ScN mice (genotype × diet interaction, P = 0.0002). Furthermore, the greater percentage of F4/80-positive cells was associated with the formation of distinct crown-like structures (Figure 3b).

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Figure 3. Accumulation of F4/80-positive macrophages is attenuated in adipose tissue of high Toll-like receptor-4-deficient (10ScN) mice fed the HFP diet. Five micron sections were cut at 50 μm intervals and mounted on glass slides, deparaffinized in xylene, and stained for expression of F4/80 with monoclonal antibody. Adipose sections were visualized under ×40 original magnification. (a) Percent F4/80-positive cells (n = 5, genotype effect P = 0.6456, diet effect P = 0 < 0.0001, genotype × diet interaction P = 0.0002) were measured in 10ScN (white bars) and 10J (black bars) mice following 16 weeks on diet. All displayed values represent least square (LS) means ± s.e.m. for diet × genotype interaction. When interaction term was P ≤ 0.15, Bonferroni correction was used to determine significant difference between means as indicated by different letters. Significant main effects (genotype and diet) were also indicated by “*” (genotype effect) and “#” (diet effect) over LS means. (b) Representative images from 10ScN and 10J mice fed low-fat (LFC), high-fat (HFC), or HFP diet. LFC, low-fat control; HFC, high-fat control; HFP, high-fat palmitate.

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We then examined the mRNA abundance of selected proinflammatory chemokines and cytokines. MCP-1 expression was increased in the epididymal adipose tissue of 10J mice fed the high-fat diets (genotype × diet, P = 0.0208, Figure 4a). There was also a trend for increased CCR2 transcript abundance in mice fed either HF diet (diet, P = 0.0894, Figure 4b). Expression of the proinflammatory cytokines, TNF-α (Figure 4c), and IL-6 (Figure 4d) were increased by over twofold and threefold in adipose tissue of mice fed the HFP diet (diet, P = 0.0132 and P < 0.0001, respectively). Furthermore, the magnitude of the increase in IL-6 was greater in mutant vs. normal mice fed the HFP diet (genotype × diet, P = 0.0736). Tlr-2 expression was increased over twofold with DIO, irrespective of genotype (diet effect, P = 0.035, Figure 4e).

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Figure 4. Adipose tissue inflammatory gene expression in Toll-like receptor-4-deficient (10ScN) mice is differentially regulated in response to the HFP diet. (a) Monocyte chemotactic protein-1 (MCP-1) (n = 6, diet effect P = 0.6965, genotype effect P = 0.3072, diet × genotype interaction P = 0.0208), (b) CCR2 (n = 6, diet effect P = 0.0894, genotype effect P = 0.3303, diet × genotype interaction P = 0.3515), (c) tumor necrosis factor-α (TNF-α) (n = 6, diet effect P = 0.0132, genotype effect P = 0.5592, diet × genotype interaction P = 0.9318), (d) IL-6 (n = 6, diet effect P < 0.0001, genotype effect P = 0.9782, diet × genotype interaction P = 0.0736), and (e) Tlr-2 (n = 6, diet effect P = 0.0350, genotype effect P = 0.4492, diet × genotype interaction P = 0.6464) were measured in 10ScN (white bars) and 10J (black bars) mice following 16 weeks on diet. All displayed values represent least square (LS) means ± s.e.m. for diet × genotype interaction. When interaction term was P ≤ 0.15, Bonferroni correction was used to determine significant difference between means as indicated by different letters. Significant main effects (genotype and diet) were also indicated by “*” (genotype effect) and “#” (diet effect) over LS means. LFC, low-fat control; HFC, high-fat control; HFP, high-fat palmitate.

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To determine whether the proinflammatory transcription factor NFκB may be involved in adipose-specific inflammation, we utilized electrophoretic mobility shift assays as a qualitative assessment of NFκB translocation. There was a marked reduction in the nuclear localization of NFκB in Tlr-4-deficient mice regardless of diet (Figure 5a). Similar quantitative results were obtained using a NFκB p65 DNA binding enzyme-linked immunosorbent assay, which indicated that adipose nuclear NFκB concentrations in mutant mice were 50% less than that in 10J mice (genotype, P = 0.0015, Figure 5b).

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Figure 5. Toll-like receptor-4 (Tlr-4) deficiency attenuates nuclear factor-κB (NFκB) activation in adipose tissue of Tlr-4 deficient (10ScN) mice. Animals were fasted 6–8 h and epididymal fat pad was collected. Nuclear extracts were obtained from adipose tissue and subsequently used for (a) NFκB electrophoretic mobility shift assay, each lane representing an individual sample (n = 2); (b) NFκB p65 DNA binding enzyme-linked immunosorbent assay. Nuclear extracts were used to quantify NFκB DNA binding; 10ScN (white bars) and 10J (black bars) (n = 4, diet effect P = 0.3913, genotype effect P = 0.0015, diet × genotype interaction P = 0.4301). All displayed values represent least square (LS) means ± s.e.m. for diet × genotype interaction. When interaction term was P ≤ 0.15, Bonferroni correction was used to determine significant difference between means as indicated by different letters. Significant main effects (genotype and diet) were also indicated by “*” (genotype effect) and “#” (diet effect) over LS means. LFC, low-fat control; HFC, high-fat control; HFP, high-fat palmitate.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

The data presented herein establish a novel relationship between obesity and Tlr-4. The absence of a functional Tlr-4 due to the 74-kilobase deletion on chromosome 4 in the 10ScN strain selectively protects against the obesigenic effect of a diet high in saturated fat and palmitate but not that of an isocaloric diet high in unsaturated fatty acids in male 10ScN mice. Although we did not quantify total body fat mass in this study, adipocyte size mirrored epididymal fat pad weights and thus reinforce our conclusion that the absence of Tlr-4 protected against DIO but only when the diet is based on saturated fats. It is also important to note that this resistance to adipocyte hypertrophy occurred without a corresponding reduction in energy consumption, and is thus of metabolic origin, rather than a simple limitation in caloric intake. These findings may implicate energy expenditure, and this possibility and potential underlying mechanisms are currently under investigation. Additionally, it is intriguing that recent findings indicate that the gut microbial population influences adipose accretion through effects on energy availability and regulation of the fasting-induced adipocyte factor. The fasting-induced adipocyte factor protein is a circulating lipoprotein lipase inhibitor expressed by the intestinal epithelium and adipocyte, and knockout models have shown that the absence of fasting-induced adipocyte factor protects against overaccumulation of adipose tissue caused by introduction of a normal microbiota in germ-free mice (28). Consequently, it seems possible that the absence of Tlr-4 precludes changes in the gut microbiota in response to saturated fats that would alter fasting-induced adipocyte factor expression and thereby influence triglyceride storage in adipocytes.

Recent reports (22,23,24,25) have indicated mixed results with regards to DIO in Tlr-4 mutant or knockout mice. Our results are consistent with those of Tsukumo et al. (25), who also used males but from the C3H/HeJ model. In contrast, Shi et al. (23) used females from a Tlr-4 knockout model backcrossed onto the C57BL6J background for six generations and determined that these mice succumbed to DIO when fed a high-fat diet containing lard. Furthermore, Poggi et al. (24) reported heavier epididymal adipose weights and adipocyte hypertrophy in male C3H/HeJ mice consuming a high-fat diet based on anhydrous milk fat, which likely varies considerably in fatty acid composition. Although the mechanisms underlying these disparate results are not yet apparent, Tsukumo et al. (25) suggested that metabolic and physiologic differences relating to strain and background are likely to be important factors, and our data clearly indicate a strong linkage between Tlr-4 function, dietary fat source, and DIO. Equally important, Cani et al. (29) provided convincing evidence that mice consuming a high-fat diet containing both corn oil and lard had increased circulating concentrations of bacterial lipopolysaccharide that triggered obesity and inflammation. Because mice lacking the CD14 coreceptor for Tlr-4, which precludes formation of a functional Tlr-4 complex, were resistant to this phenomenon, these researchers suggested that endotoxemia facilitated by dietary fat intake largely mediates the effects of a high-fat diet on obesity and inflammation via a Tlr-4-mediated signaling pathway. Collectively, the data substantiate the possibility that saturated fats promote obesity postabsorptively by direct activation of Tlr-4, and (or) by promoting the uptake of gut-derived endotoxin, which also activates Tlr-4 signaling. Consequently, it will be of utmost importance in the future to establish the effects of specific Tlr-4 mutations and knockout models on obesity with respect to specific dietary fat sources and fatty acid profiles.

Chronic inflammation in adipose tissue is a comorbidity of obesity that is strongly associated with the onset and progression of insulin resistance, and ultimately the transition to frank diabetes. We have shown previously that treating cultured adipocytes with palmitate results in activation of NFκB and proinflammatory cytokine expression and release into the culture media (30). Furthermore, using in vivo and in vitro experiments, Shi et al. (23) determined that a functional Tlr-4 is a requisite component of the proinflammatory effects of palmitate or saturated fat on adipocytes. With respect to the present data, several points are notable. First of all, the only inflammatory marker attenuated by the absence of Tlr-4 was MCP-1. Whereas the high-fat diets, irrespective of whether saturated or unsaturated in composition, resulted in increased MCP-1 expression in epididymal adipose tissue of normal mice, neither diet altered expression in mutant mice. Furthermore, the expression of both IL-6 and TNF-α were markedly increased by the HFP diet, irrespective of genotype, and CCR2 expression was increased by the high-fat diets in both genotypes. These results indicate not only that some inflammatory responses to high-fat diets may be mediated independently of Tlr-4, at least at the mRNA level, but also that some responses to high fat intake occur whether the fat comprises predominantly saturated or unsaturated fatty acids. It should also be noted that even in the absence of epididymal adipose expansion and adipocyte hypertrophy in the mutant mice fed the HFP diet, both IL-6 and TNF-α expression were increased. This possibly indicates a metabolic component to the proinflammatory effects of SFAs in adipocytes, as suggested previously, because inhibition of fatty acyl Co-A synthase attenuates the activation of nuclear factor-κB by palmitate but exacerbates IL-6 release into the culture media (30). Finally, the absence of a functional Tlr-4 not only precluded the adipocyte hypertrophy in mice fed the HFP diet but also blocked infiltration of adipose tissue with macrophages. This is consistent with the hypothesis that it is adipocyte hypertrophy that ultimately signals for macrophage recruitment, and it is clear that this response does not depend on a functional Tlr-4.

The serum MCP-1 and adipose NFκB data show a striking effect of genotype in that both were markedly depressed in the mutant mice and were largely unresponsive to diet. Although serum MCP-1 concentrations were barely measurable in mutant mice, MCP-1 adipose expression differed little from that in normal mice. This perhaps indicates that adipose tissues contribute little MCP-1 to the circulating concentration in these mice, but we cannot discount the possibility that adipose concentrations were sufficient to be of physiological significance. As regards nuclear localization of adipose NFκB, both qualitative and quantitative assessments indicated that the nuclear content of this important transcription factor is significantly lower in mutant vs. normal mice with little effect of diet. We have shown previously that the transcriptional activity of NFκB in adipocytes can be uncoupled from proinflammatory cytokine responses (30), but it is perhaps important that Shi et al. (23) noted a marked effect of dietary fat on NFκB activity in a knockout model.

In summary, our study indicates that the Tlr-4 mutation in male 10ScN mice selectively blocks saturated fat-induced obesity and that this response is associated with absence of adipose infiltration with macrophages and improved markers of insulin sensitivity, as reflected in the lower ratios of glucose to insulin. These results point to a unique relationship between Tlr-4 and SFAs, and underscore the potential for interactions between specific dietary fatty acids and Tlr-4 mutations to impact the development of obesity and the associated inflammation.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

This work was supported by a grant from the Center for Designing Foods to Improve Nutrition at Iowa State University.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES