C3H/HeJ mice carrying a toll-like receptor 4 mutation are protected against the development of insulin resistance in white adipose tissue in response to a high-fat diet
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Inflammation is associated with obesity and has been implicated in the development of diabetes and atherosclerosis. During gram-negative bacterial infection, lipopolysaccharide causes an inflammatory reaction via toll-like receptor 4 (TLR4), which has an essential function in the induction of innate and adaptative immunity. Our aim was to determine what role TLR4 plays in the development of metabolic phenotypes during high-fat feeding.
Materials and methods
We evaluated metabolic consequences of a high-fat diet in TLR4 mutant mice (C3H/HeJ) and their respective controls.
TLR4 inactivation reduced food intake without significant modification of body weight, but with higher epididymal adipose tissue mass and adipocyte hypertrophy. It also attenuated the inflammatory response and increased glucose transport and the expression levels of adiponectin and lipogenic markers in white adipose tissue. In addition, TLR4 inactivation blunted insulin resistance induced by lipopolysaccharide in differentiated adipocytes. Increased feeding efficiency in TLR4 mutant mice was associated with lower mass and lower expression of uncoupling protein 1 gene in brown adipose tissue. Finally, TLR4 inactivation slowed the development of hepatic steatosis, reducing the liver triacylglycerol content and also expression levels of lipogenic and fibrosis markers.
TLR4 influences white adipose tissue inflammation and insulin sensitivity, as well as liver fat storage, and is important in the regulation of metabolic phenotype during a fat-enriched diet.
KeywordsAdipose tissue High-fat diet Insulin resistance Lipid metabolism Liver steatosis Obesity TLR4 Toll-like receptor 4
IL1 receptor antagonist
Krebs–Ringer phosphate buffer
plasminogen activator inhibitor-1
peroxisome proliferator-activated receptor γ
toll-like receptor 4
TNF receptor superfamily, member 1b
white adipose tissue
Evidence from human populations  and animal research  has established correlative as well as causative  links between chronic inflammation and insulin resistance during obesity. However, the fundamental mechanisms for activating inflammatory pathways in obesity are poorly understood. One of the commonest causes of inflammation is microbial infection. The host’s acute-phase response to pathogens involves physiological changes including hypermetabolism, anorexia and body weight loss , but it is not known how this pathway intervenes in the chronic inflammatory state in obesity and in the control of insulin sensitivity.
Lipopolysaccharide (LPS) is continuously produced in the gut by the death of gram-negative bacteria and translocated into intestinal capillaries and the general circulation , where it can reach peripheral tissue such as adipose tissue and cause a local inflammatory reaction. Interestingly, LPS is found in the circulation of apparently healthy subjects [6, 7] and its activity may be associated with high-fat diet and dyslipidaemia. Infusion of triacylglycerol enhances LPS responses in humans, including the production of cytokines, suggesting that LPS can be locally released from lipoproteins in peripheral tissues through lipoprotein lipase action .
Portal endotoxin is involved in the progression of non-alcoholic steatohepatitis [9, 10], which mainly accounts for hepatic insulin resistance . Genetically obese rodents quickly develop steatohepatitis after exposure to low doses of LPS , while polymyxin B diminishes hepatic steatosis during total parenteral nutrition in the rat by reducing caecal flora . Moreover, endotoxins exert a strong effect on liver of mice fed a high-fat diet .
Interestingly, some groups  but not all  observed that toll-like receptor 4 (TLR4) 299Gly allele was associated with reduced C-reactive protein levels and a decreased risk of clinical diabetes, suggesting that TLR4 intervenes in the complications of obesity.
We hypothesised that the presence of non-functional TLR4 should modify the development of adipose tissue and liver steatosis during a high-fat diet. We studied the effects of an obesity- and insulin resistance-inducing high-fat diet in endotoxin-resistant C3H/HeJ mice (Tlr4Lps-d), which carry a spontaneous mutation inactivating Tlr4 , and in their respective C3H/HeOuJ control mice. Complementary experiments were performed in Cd14−/− mice.
Materials and methods
Mice and animal care
Adult (12-week-old) male C3H/HeJ and C3H/HeOuJ mice, as well as Cd14−/− and C57Bl/6J control mice, were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed with a 12-h light–dark cycle. Mice had free access to a high-fat diet and water. The diet consisted of either a pelleted (42.7% carbohydrate, 42% fat, 15.3% protein, 18.8 kJ/g; TD88137; Harlan Teklad, Madison, WI, USA) or a liquid diet (47% carbohydrate, 35% fat, 18% protein, 4.186 kJ/ml; Lieber–De Carli Control diet; Bioserv Industries, Frenchtown, NJ, USA). Food intake was measured daily. The use of the liquid diet allowed the constitution of an additional group of pair-fed control C3H/HeOuJ or C57Bl/6J mice (pair-fed group), which were fed the same quantity of the diet as that consumed on the previous day by the C3H/HeJ or Cd14−/− mice. In addition, ten mice of C3H genotype were fed a standard chow diet (60% carbohydrate, 12% fat, 28% protein, 14.2 kJ/g; A03; UAR-SAFE, Epinay sur Orge, France). Body weights were measured weekly. Feeding efficiency was calculated by dividing body weight gain by energy intake over the diet period.
After a 6-h fast, mice were killed under anaesthesia with isoflurane inhalation. After blood collection from the retro-orbital sinus, plasma samples were stored at −20°C. Adipose tissue and liver were collected and weighed. In some experiments, mice were injected with LPS from E. coli serotype 026:B6 (100 mg/kg i.p; Sigma, St Louis, MO, USA). This study protocol was approved by the local ethics committee of the Faculté de Médecine of Marseille (authorisation number 52–23).
Biochemical and immunological analyses
Plasma triacylglycerol and glucose were determined by routine chemical methods. Insulin levels of fasting mice were measured by radioimmunoassay (Linco Research, St Louis, MO, USA). Plasma ghrelin levels were assayed with a rat ghrelin enzyme immunoassay kit (Phoenix Peptide, Belmont, CA, USA). Leptin, TNF, TNF receptor superfamily, member 1b (TNFRSF1B, previously known as TNF receptor type 2 [TNFR2]) and IL6 were measured with ELISA (R&D systems, Abingdon, UK). Murine plasminogen activator inhibitor-1 (PAI-1, now known as serine [or cysteine] peptidase inhibitor, clade E, member 1 [SERPINE1]) was assessed with a home-made ELISA .
Tissue lipid analysis
Lipids from white adipose tissue (WAT) and liver were extracted by the method of Folch et al. . Triacylglycerol was measured using a triacylglycerol and free glycerol reagent (Sigma).
Total DNA content from adipose tissue
WAT was incubated overnight in the presence of proteinase K (100 μg/ml). DNA was then purified using phenol/chloroform and quantified by measuring the absorbance at 260 nm.
Pieces of WAT and liver were embedded into Tissue-Tek OCT (Miles, Elkhart, IL, USA). Cryosections were stained with Nuclear Fast Red. Images of adipocytes were acquired from a Leica DMRB light microscope fitted with a colour CCD camera (Coolsnap; Princeton Instruments, Evry, France). About 300 cell surfaces per fat pad were measured at ×10 magnification using Scion imaging software (Scion Image, Frederick, MD, USA). Liver sections were stained with Oil Red O and observed under the same microscope at ×20 magnification.
Isolation and culture of peritoneal macrophages
Peritoneal macrophages were prepared as described  and then incubated for 18 h with or without LPS from E. coli serotype 026:B6 (50 ng/ml, Sigma). Media were collected and stored at −80°C. Total cell proteins were assayed according to specifications of the bicinchoninic acid protein assay kit from Sigma.
In vivo lipolysis
After a 6-h fast, five mice were injected intraperitoneally with (−)-isoproterenol hydrochloride (10 mg/kg; Sigma). Blood was collected before and 15 min after injection. Plasma NEFA concentrations were measured using NEFA C kit (Wako, Neuss, Germany). These mice were not used for further experiments.
Glucose turnover studies and in vivo glucose utilisation index in individual tissues
In vitro glucose uptake
Primary adipocytes (10 days post-differentiation)  were cultured overnight in serum-free DMEM with low glucose (1 g/l). After Krebs–Ringer phosphate buffer (KRP) wash, cells were incubated at 37°C for 30 min in the presence or absence of 100 nmol/l of insulin in KRP containing 1% BSA. Then 18.5 kBq per well 2-deoxy[3H]glucose (Amersham Biosciences, Orsay, France) was added and incubated for 10 min at 37°C. The cells were washed with cold KRP and solubilised in 0.1% SDS. The radioactivity of a 200-μl aliquot was determined in a scintillation counter. Glucose uptake was expressed as the degree of increase relative to that of basal C3H/HeOuJ cells.
Real-time quantitative PCR
Total RNAs were prepared using Trizol reagent (Invitrogen, Cergy Pontoise, France). cDNA was synthesised using MMLV transcriptase (Invitrogen) from 1 μg of total RNA and stored at −80°C until use. PCRs were performed under standard conditions . The relative amounts of each studied mRNA were normalised to acidic ribosomal phosphoprotein P0 (Arbp, also known as 36b4) mRNA levels and expressed relatively to the mean value of the control mice group (User Bulletin no. 2; Applied Biosystems, Foster City, CA, USA). Primer express software (Applied Biosystems) was used to design primers (available upon request from firstname.lastname@example.org).
Data and statistics
Data are expressed as mean±SEM. Differences between independent variables were tested using the Mann–Whitney test. Differences between paired variables were tested using the paired Wilcoxon test. The Kolmogorov–Smirnov Z test was used to test differences in adipocyte surface. A p value < 0.05 was considered significant.
C3H/HeJ mice do not respond to LPS
Inflammatory marker production by peritoneal macrophages from C3H/HeOuJ and C3H/HeJ mice incubated with or without LPS stimulation for 18 h
TNF (pg/mg protein)
951 ± 251
9,058 ± 1,781
585 ± 99
475 ± 119
IL6 (pg/μg protein)
51 ± 22
253 ± 38
3.2 ± 0.3*
3.1 ± 0.5
TNFRSF1B (pg/μg protein)
13.6 ± 2.2
44.8 ± 3.3
6.0 ± 1.1*
6.5 ± 2.3
PAI-1 (pg/mg protein)
255 ± 63
1,111 ± 243
56.6 ± 6.6*
59.8 ± 9.0
Plasma TNF levels in C3H/HeOuJ mice increased at 1.5 h after LPS injection to 771 ± 335 pg/ml (n = 6), but remained undetectable in C3H/HeJ mice (n = 5).
C3H/HeJ mice exhibit a higher feeding efficiency than control mice
Characteristics of C3H/HeOuJ and C3H/HeJ mice with free access to a pelleted high-fat diet for 22 weeks
Body weight (g)
46.8 ± 0.4
44.6 ± 0.6
12.6 ± 1.6
12.0 ± 2.3
663 ± 69.7
383 ± 35
42.9 ± 7.0
38.4 ± 8.0
1.10 ± 0.38
0.96 ± 0.18
1.20 ± 0.07
1.26 ± 0.04
0.67 ± 0.11
0.73 ± 0.10
Cumulative energy intake (kJ/mouse)
12,060 ± 184
9,222 ± 92
Feeding efficiency (g/100 kJ)a
0.33 ± 0.007
0.44 ± 0.026
To ensure that food intake did not differ between the groups, food consumption was summed over the diet period (Table 2). Unexpectedly, although the C3H/HeJ reached the same body weight, they consumed less energy than C3H/HeOuJ control mice. As a result, feeding efficiency was increased in mutant mice (Table 2).
C3H/HeJ mice exhibit a higher body weight than pair-fed C3H/HeOuJ control mice
Fat distribution and food intake of C3H/HeOuJ and C3H/HeJ mice with free access to a liquid high-fat diet for 10 weeks
Body weight (g)
36.4 ± 1.9
37.3 ± 0.7
27.0 ± 0.9
Epididymal fat pad
1.23 ± 0.11
1.85 ± 0.13
0.50 ± 0.08
% Body weight
3.4 ± 0.7
5.0 ± 0.4
1.8 ± 0.2
Subcutaneous fat pad
0.82 ± 0.09
0.96 ± 0.08
0.25 ± 0.05
% Body weight
2.2 ± 0.4
2.6 ± 0.2
0.9 ± 0.2
Cumulative energy intake (kJ/mouse)
4,496 ± 218
3,830 ± 88
3,784 ± 105
Feeding efficiency (g/100 kJ)
0.23 ± 0.04
0.35 ± 0.02
0.03 ± 0.04
TLR4 inactivation does not increase whole-body insulin sensitivity after 22 weeks of high-fat diet
TLR4 inactivation supports higher insulin sensitivity and lipogenesis in WAT after 22 weeks of high-fat diet but not in skeletal muscle
We hypothesised that TLR4 inactivation could affect insulin sensitivity of insulin target tissues in different ways. Indeed, it has recently been demonstrated that inflammation is regulated differently in adipose tissue than in muscle , as is insulin sensitivity . Insulin-stimulated glucose utilisation was significantly higher in subcutaneous adipose tissue of C3H/HeJ than in that of C3H/HeOuJ mice (Fig. 2b). To further investigate the effects of TLR4 signalling on insulin action, we evaluated whether TLR4 activation by LPS could affect insulin-dependent glucose uptake by adipocytes. While insulin increased glucose uptake in adipocytes from both genotypes, LPS significantly decreased the ability of insulin to stimulate glucose uptake, this inhibitory effect being less pronounced in C3H/HeJ adipocytes (Fig. 2d). In contrast, the insulin-stimulated glucose utilisation rate in vastus lateralis was lower in C3H/HeJ mice than in C3H/HeOuJ mice (Fig. 2c). A trend towards a decrease, albeit not significant, was also observed in tibialis anterior (data not shown). Hence, these results demonstrate that obese TLR4-deficient mice have an adipose tissue-specific increase in insulin sensitivity.
Non-functional TLR4 does not modify isoproterenol-induced lipolysis after 22 weeks of high-fat diet
Inflammation is known to increase adipocyte lipolysis . We thus examined whether C3H/HeJ mice exhibited a lower lipolytic potential. Fasting plasma NEFA levels were not different between both strains (C3H/HeOuJ: 0.79 ± 0.04 vs C3H/HeJ: 0.82 ± 0.08 mmol/l). Isoproterenol administration led to a similar increase in plasma NEFA concentrations in the two groups (C3H/HeOuJ: 1.72 ± 0.07 vs C3H/HeJ: 1.70 ± 0.09 mmol/l).
TLR4 inactivation supports a lower adipose tissue inflammatory state and attenuates obesity-induced changes in adipose tissue gene expression
We then studied whether these changes in the inflammatory pattern of CH3/HeJ adipose tissue were associated with higher mRNA levels of genes involved in insulin action and sensitivity, glucose and triacylglycerol metabolism, and adipocyte differentiation. Adipose tissue of obese TLR4 mutant mice expressed higher levels of Irs1, adiponectin and solute carrier family 2 (facilitated glucose transporter), member 4 (Slc2a4, previously known as Glut4) mRNA (Fig. 3b,e). The mRNA levels of two key lipogenic factors, sterol regulatory element binding factor 1 (Srebf1, previously known as Srebp1c) and fatty acid synthase (Fasn, previously known as Fas), were also higher (Fig. 3c,f). Further, peroxisome proliferator-activated receptor γ (Pparg) mRNA followed a similar pattern (Fig. 3c,f). These data suggest that the pro-inflammatory changes that occur in adipose tissue during the development of obesity are in part TLR4-dependent. The lower inflammatory state observed in C3H/HeJ adipose tissue may support the possibility of a higher insulin sensitivity.
TLR4 inactivation attenuates high-fat diet-induced liver steatosis
Effect of TLR4 deficiency on liver weight
Pelleted high-fat diet
Liver weight (g)
3.39 ± 0.27
2.3 ± 0.21
Liver (% body weight)
7.22 ± 0.34
5.11 ± 0.24
Liquid high-fat diet
Liver weight (g)
1.38 ± 0.07
1.17 ± 0.04
0.99 ± 0.03
Liver (% body weight)
3.78 ± 0.11
3.16 ± 0.07
3.67 ± 0.08
Significant fibrosis is associated with non-alcoholic steatohepatitis and has been considered a factor in prognosis . We thus investigated mRNA levels of Tgfb1 and Pai-1, key effectors of the activity of TGFB1. Both Pai-1 and Tgfb1 mRNA levels were lower in the liver of C3H/HeJ mice than in that of C3H/HeOuJ (Fig. 4b). These data argue for an effect of the TLR4 pathway on the evolution of fibrosis associated with fatty liver.
Cd14−/− mice develop a phenotype close to that of C3H/HeJ mice
To confirm the contribution of the LPS pathway to the observed results, we completed our study by phenotyping Cd14−/− mice and their controls after 10 weeks of free access to a high-fat diet. High-fat feeding for 10 weeks led to a significant weight gain in both Cd14−/− and control mice. Similarly to C3H/HeJ mice, feeding efficiency was higher in Cd14−/− mice than in controls given free access to food (data not shown). In subcutaneous adipose tissue, Cd14 inactivation was associated with 5- to 25-fold higher mRNA levels of genes involved in insulin action (Slc2a4, Irs1, adiponectin) and lipogenesis (Pparg, Fasn) (ESM Fig. 1). Despite the same liver mass (C57B1/6J: 4.3 ± 0.1, Cd14−/−: 4.5 ± 0.4% of body weight), the hepatic triacylglycerol content tended to be higher in C57Bl/6J than in Cd14−/− mice (176 ± 13 vs 139 ± 11 mmol/g; p = 0.08).
Our data demonstrate that TLR4 exerts an important role on obesity-associated phenotypes. When mice were fed a high-fat diet, absence of TLR4 modulated the feeding behaviour and increased WAT insulin sensitivity and lipogenesis. Importantly, TLR4 mutant mice were protected from developing hepatic steatosis.
We found that TLR4 inactivation improved insulin-induced glucose uptake in WAT. This protection from high-fat diet-induced adipose tissue insulin resistance was associated with a failure of the high-fat diet to induce expression of inflammatory mediators in WAT. This finding was recently supported by a report that NEFA stimulate proinflammatory pathways and reduce insulin sensitivity in WAT through TLR4 activation . The attenuated effect of LPS on insulin-induced glucose uptake in mutant adipocytes may reflect a direct contribution of TLR4 present in adipocytes. Accordingly, TLR4 activation by LPS in 3T3-L1 adipocytes prevented insulin-dependent glucose uptake .
Our data may also result from the inhibition of TLR4-mediated inflammatory pathways in macrophages, with a resultant decrease in stimulatory action on adipocytes. In support of this hypothesis, TLR4 protein levels were much lower in adipocytes than in macrophages . Remarkably, the attenuation of inflammation was not accompanied by a reduction of macrophage infiltration into WAT. Indeed, we did not find a decrease in Emr1 mRNA levels in WAT of mutant mice. Consistent with this, Suganami et al.  showed that co-culture of 3T3-L1 adipocytes and C3H/HeJ macrophages was associated with an attenuation of Tnf and Il1B mRNA expression, but also with a significant increase in mRNA expression levels of the powerful monocyte chemoattractant, monocyte chemoattractant protein 1.
TNF is known to reduce adipose tissue mass by decreasing PPARG production, stimulating lipolysis and repressing genes involved in the uptake and metabolism of lipids and glucose [37, 38, 39, 40, 41]. Considering all this, the higher insulin sensitivity that we observed in our study in WAT and that was associated with higher expression of genes mediating insulin sensitivity (Irs1, Slc2a4, adiponectin) and lipogenesis (Srebf1, Fasn, Pparg) may be secondary to the tissue TNF and IL1B depletion, and might contribute to the increase in adipocyte volume seen in C3H/HeJ mice despite lower food intake. This notion is consistent with our finding that insulin sensitivity was increased in WAT from C3H/HeJ mice during euglycaemic–hyperinsulinaemic clamp conditions. Greater expression of Srebf1 and Fasn could be consecutive to the increased insulin sensitivity of the adipocytes, since insulin regulates the expression of both.
IL1RN is an anti-inflammatory cytokine . Human and rodent WAT is a major source of IL1RN [43, 44]. Il1rn−/− mice, in which excess IL1 signalling may be induced, show a lean phenotype . This phenotype has been associated with a defect in adipogenesis, a reduction of genes involved in lipogenesis, an increase in energy expenditure and a reduction in diurnal food intake . The increase in IL1RN production in WAT may be directly involved in the phenotype obtained in TLR4 mutant mice, which exhibited both increased peripheral lipogenesis and lower thermogenesis. Note that in our study, Il1rn expression was increased in a situation where inflammation was dampened, suggesting that IL1RN may not only represent a counter-regulatory mechanism that limits inflammation, but may also be produced independently of an inflammatory process during fat accumulation.
We observed contrasting behaviour with regard to insulin sensitivity in skeletal muscle and adipose tissue. This could be related to the low inflammation level in skeletal muscle during obesity . The skeletal muscle produces low levels of CD68 and monocyte chemoattractant protein 1 and neither pioglitazone nor metformin has any effect on production of these two . Thus, a modification of peripheral glucose disposal under inflammation manipulation is more likely to be due to changes in adipose tissue and then to secondary effects on muscle. Another plausible explanation is that the ability of TLR4 deficiency to improve insulin resistance in adipose tissue and not muscle is regulated through PPARG , whose levels are up to 50-fold higher in adipose tissues than in skeletal muscles . IL1RN may be involved in the described phenotype by virtue of its ability to reduce insulin sensitivity in muscle through a specific decrease in glucose uptake as recently described in rats . Alternatively, it remains possible that plasma factor(s) dominantly affect insulin signalling in muscles by mechanisms that are independent of TLR4.
We found a reduction in total brown adipose tissue fat mass in C3H/HeJ associated with decreased Ucp1 expression. This finding shows that TLR4 inactivation may play a role in brown adipose tissue development.
Our data provide evidence that TLR4 deficiency slows down non-alcoholic liver steatosis. This result should be seen together with those obtained in alcohol-induced steatosis. C3H/HeJ mice fed ethanol do not develop fatty liver but C3H/HeOuJ do. Chronic alcohol administration increases gut-derived endotoxin in the portal blood, partly due to gut leakiness , which activates Kupffer cells and causes liver injury. Interestingly, we found that the protective effect of TLR4 inactivation was maintained throughout the diet period. Even after 22 weeks of high-fat diet the expression levels of key lipogenic markers still differed. Tgfb1 and Pai-1 expression levels in liver were decreased in CH3/HeJ mice, arguing for an effect of the TLR4 pathway, known as a key determinant of non-alcoholic steatohepatitis , on the evolution of hepatic fibrosis. Several studies have shown an inverse relationship between hepatic triacylglycerol stores and hepatic insulin sensitivity . The observation that TLR4 mutant animals have a much lower insulin level at similar plasma glucose concentrations suggests that the liver responds more sensitively to insulin-induced inhibition of glucose output.
One limitation of our study could be the use of non-littermate controls. However, we obtained similar findings in Cd14-deficient mice, demonstrating that two approaches to blunting the LPS pathway lead to the same modifications: (1) in feeding behaviour; (2) in adipose tissue expression levels of genes involved in glucidolipidic metabolism; and (3) in the development of liver steatosis.
Our results indicate that, apart from an acute LPS challenge, TLR4 contributes to the production of key cytokines in WAT during obesity. They also highlight the contribution of the TLR4 pathway to the control of obesity and its related complications. We propose that interventions known to reduce endotoxin toxicity  or to modulate the TLR4 pathway may help control some key elements of the metabolic syndrome.
This work was supported by: (1) the Programme National de Recherche sur les Maladies Cardiovasculaires-Inserm (National Research Program on CardioVascular Disease): “Vascular risk and metabolic syndrome: Haemostasis, inflammation and metabolism interplay” (no. A04046AS); and (2) the Fondation de France (French Foundation): “Vascular risk and metabolic syndrome: interaction between innate immunity, haemostasis and glucido-lipidic metabolism.” M. Poggi received financial support from “Groupe de Réflexion sur la Recherche Cardio-vasculaire.”
Duality of interest
The authors declare that they have no duality of interest.