Gut microbiota is a key modulator of insulin resistance in TLR 2 knockout mice

Abstract

Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Metabolic parameters of TLR2 knockout (TLR2−/−) and WT mice during 16 wk.

(A) Weight gain after 16 wk. (B) Food intake after 8 and 16 wk. (C) Epididymal fat pad weight after 8 and 16 wk. (D) WT and TLR2−/− mice after 20 wk. (E) Glucose tolerance test. (F) Serum insulin concentration. (G) Glucose uptake obtained from euglycaemic hyperinsunaemic clamp. (H) Oxygen consumption and (I) respiratory exchange rate. (J) UCP-1 expression in the brown adipose tissue. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (J, lower panel). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. *p<0.05 between TLR2−/− mice and their controls.

Figure 2. TLR2 KO mice exhibit taxonomical alterations in gut microbiota.

Untreated WT (A) and TLR2 knockout (TLR2−/−) mice (B) stools were analyzed via 16S rRNA analysis. Data are presented from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow.

Figure 3. Measurements of cytokines, adipokines, and LPS.

Serum concentration of IL-6 (A), TNF-α (B), adiponectin (C), leptin (D), and LPS (E). Proportion of Bifidobacterium was obtained by 16S rRNA analysis of stools (F). Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2 KO mice and their controls; all evaluations were made with mice on standard chow.

Figure 4. Evaluation of pathways involved in the impairment of insulin signaling.

Phosphorylation of JNK in muscle (A), liver (B), and white adipose tissue (WAT) (C). Phosphorylation of PERK in muscle (D), liver (E), and WAT (F). Serine 307 phosphorylation of IRS-1 from muscle (G), liver (H), and WAT (I). Activation of TLR4 (studied by the immunoprecipitation of MyD88 and blotting with TLR4) in muscle (J), liver (K), and WAT (L). JNK, PERK, and IRS-1 protein expression in muscle, liver, and white adipose tissue (A–I, lower panels). Expression of IκB-α in muscle (M), liver (N), and WAT (O). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (M–O, lower panels). NFκB activation in muscle (P), liver (Q), and WAT (R). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2−/− mice and their controls.

Figure 5. TLR2 knockout (TLR2−/−) mice present decreased insulin signaling.

Tyrosine 941 phosphorylation of the insulin receptor substrate (IRS)-1 in muscle (A), liver (B), and WAT (C). Serine phosphorylation of AKT in muscle (D), liver (E), and WAT (F). IRS-1 and AKT protein expression in muscle, liver, and white adipose tissue (A–F, lower panels). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. *p<0.05 between WT with and without insulin stimulus; ** p<0.05 between TLR2−/− mice and their controls with insulin stimulus.

Figure 6. Insulin sensitivity and signaling after treatment with selective inhibitors.

Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp from TLR2−/− mice treated or not with the drugs: SP600125 (SP), JNK inhibitor; 4-phenil butyric acid (PBA), endoplasmic reticulum stress inhibitor; TLR4 antisense oligonucleotide (ASO); (A) TAK-242, inhibitor of TLR4. (B) Serine phosphorylation of AKT after the treatment with SP600125 and PBA. (C) Serine phosphorylation of AKT after the treatment with TLR4 ASO and TAK-242. (D) Phosphorylation of JNK after the treatment with the drugs mentioned. Fasted TLR2 knockout mice and WT mice were gavaged by LPS (1.08, 10−8 g) diluted in water (100 µL) or without LPS. (E) Blood was collected from the cava vein 60 min after gavage and serum LPS was determined. (F) Zonula occludens (ZO)-1 expression in the ileum. (G) Frequency of CD4+Foxp3+ regulatory T cells in WT mice. (H) Frequency of CD4+Foxp3+ regulatory T cells in TLR2−/− mice. All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between WT mice with or without insulin stimulus; ** p<0.05 between WT and TLR2−/− mice with insulin stimulus ; $ p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with SP; # p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with PBA; § p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with ASO; & p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with TAK-242; ap<0.05 between WT mice with or without LPS stimulus; ° p<0.05 between WT and TLR2−/− mice with LPS stimulus.

Figure 7. Alterations in the metabolic parameters and in insulin signaling and sensitivity after treatment with antibiotics.

(A) Epididymal fat pad weight. (B) Visceral adipose tissue weight. (C) Glucose tolerance test. (D) Oxygen consumption. (E) Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp. (F) UCP-1 expression in the brown adipose tissue. (G) Serine phosphorylation of AKT after the treatment with AB. (H) Phosphorylation of JNK after the treatment with AB. (I) Zonula occludens (ZO)-1 expression in the ileum. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). Data are presented as means ± S.E.M from six to eight mice per group, from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. # p<0.05 between WT mice with or without insulin stimulus; * p<0.05 between WT and TLR2−/− mice with insulin stimulus; ** p<0.05 between TLR2−/− and TLR2−/− treated with AB, with insulin stimulus.

Figure 8. WT mice reproduce TLR2 knockout (TLR2−/−) mice after cecal microbiota transplantation.

(A) Epididymal fat pad weight. (B) Visceral adipose tissue weight. (C) Weight gain of transplanted mice. (D) Serum glucose. (E) Glucose tolerance test. (F) Incremental area under curva (IAUC) obtained from the glucose tolerance test. (G) Oxygen consumption. (H) Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp. (I) UCP-1 expression in the brown adipose tissue. (J) Serine phosphorylation of AKT after the treatment with AB. (K) Phosphorylation of JNK after the treatment with AB. AKT and JNK protein expression in the liver of transplanted mice (J, K, lower panels). (L) Zonula occludens (ZO)-1 expression in the ileum. (M) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice. (N) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice transplanted with WT microbiota. (O) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice transplanted with TLR2−/− microbiota. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between Bacillus-associated mice transplanted with TLR2−/− microbiota (MA+TLR2−/−) and those transplanted with WT microbiota (MA+WT); ** p<0.05 between Bacillus-associated mice transplanted with WT microbiota (MA+WT) and Bacillus-associated mice (MA); # p<0.05 between Bacillus-associated mice with or without insulin stimulus.

Figure 9. Metabolic parameters of TLR2 KO (TLR2−/−) mice fed a high-fat diet.

(A) Weight gain after 10 wk of high-fat diet (HFD). (B) Food intake. (C) Epididymal fat pad weight. (D) Glucose tolerance test. (E) Serum insulin concentration. (F) Glucose uptake obtained from the euglycaemic hyperinsulinaemic clamp. (G) Oxygen consumption and (H) respiratory exchange rate. (I) UCP-1 expression in the brown adipose tissue. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between TLR2−/− mice and their controls.

Figure 10. Insulin signaling is impaired in TLR2 knockout (TLR2−/−) mice fed on a high-fat diet.

Phosphorylation of AKT in muscle (A), liver (B), and white adipose tissue (WAT) (C). AKT protein expression in muscle, liver, and WAT (A–C, lower panels). Phosphorylation of JNK in muscle (D), liver (E), and WAT (F). JNK protein expression in muscle, liver, and WAT (D–F, lower panels). IκB-α expression in muscle (G), liver (H), and WAT (I). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2−/− mice and their controls, with insulin stimulus; ** p<0.05 between WT with and without insulin stimulus.