Involvement of toll-like receptor 4 in alveolar bone loss and glucose homeostasis in experimental periodontitis

Abstract

Background and objective: There is general agreement that certain fatty acids and lipopolysaccharides (LPS) promote inflammation through toll-like receptor 4 (TLR4), and that inflammation promotes insulin resistance. We therefore hypothesized that mice with periodontitis and a TLR4 loss-of-function (LOF) mutation fed a high-fat (HF) diet would develop improved glucose homeostasis compared with wild-type (WT) animals with periodontitis fed a HF diet.

Material and methods: Wild-type and TLR4 mutant mice fed a HF diet were divided into four groups (n = 6/group): WT; WT with periodontitis (WT/P); mutant (Mut); and mutant with periodontitis (Mut/P). Periodontitis was induced by placing LPS soaked ligatures around maxillary second molars. Fasting insulin and glucose levels were measured weekly for 10 wk. Glucose tolerance was evaluated at baseline (week 1) and at 9 wk. Insulin signaling (phosphorylation of Akt) and tumor necrosis factor-α (TNF-α) mRNA levels in liver were determined when the mice were killed at week 10.

Results: Mut/P mice developed less alveolar bone loss compared with WT/P mice (p < 0.05). Fasting glucose levels were improved after 8 wk of feeding a HF diet (weeks 9 and 10) in Mut/P mice compared with Mut, WT and WT/P mice (p < 0.05). Glucose tolerance was impaired in all groups compared with baseline (p < 0.05), except for the Mut/P group. Insulin signaling was improved (p < 0.05), and expression of TNF-α was decreased (p < 0.05) in the liver of Mut/P mice compared with the liver of WT/P mice.

Conclusion: The TLR4 LOF mutation partially protects against alveolar bone loss and improves glucose homeostasis in mice with periodontitis fed a HF diet.

Fig. 1

(A) Alveolar bone loss captured by stereomicroscopy of defleshed mouse maxilla. (a) Maxilla from a control wild-type (WT) mouse without periodontitis; (b) maxilla from a WT mouse with periodontitis (WT/P); (c) maxilla from a toll-like receptor 4 (TLR4) mutant mouse without periodontitis (Mut); (d) maxilla from a Mut mouse with periodontitis (Mut/P). Red lines are drawn on the mesial and distal transition line angles. The area within the line was calculated as bone loss. The areas were measured from the buccal and palatal sides, and the total area of bone loss was calculated per tooth. (B) Average bone loss per tooth calculated using software (ImageJ). Data are presented as mean ± SEM. Statistical analysis was performed using a Mann–Whitney U-test. *p < 0.05 between direct comparison groups.

Fig. 2

Composite graph showing temporal relationships between fasting glucose (dashed line) and insulin (solid line) levels in wild-type mice without periodontitis (WT) (A), in WT mice with periodontitis (WT/P) (B), in toll-like receptor 4 (TLR4) mutant mice without periodontitis (Mut) (C) and in Mut mice with periodontitis (Mut/P) (D) over a 10 wk period. Left y-axis: fasting insulin levels (μg/L). Right y-axis: fasting glucose levels (mg/dL).

Fig. 3

(A) Comparison of intraperitoneal glucose tolerance (ipGTT) test results obtained at weeks 1 (baseline; dashed line) and 9 (solid line) within a group in wild-type mice without periodontitis (WT) (a), WT mice with periodontitis (WT/P) (b), toll-like receptor 4 (TLR4) mutant mice without periodontitis (Mut) (c) and Mut mice with periodontitis (Mut/P) (d). (B) Comparison of ipGTT results obtained at week 9 between the following groups: WT vs. WT/P (e), WT vs. Mut (f), Mut vs. Mut/P (g) and WT/P vs. Mut/P (h). Values for each time-point are expressed as mean ± SEM. The x-axis indicates min following intraperitoneal dextrose injection. The y-axis indicates glucose levels in mg/dL. Statistical analysis was performed using the Wilcoxon rank sum test with *p < 0.05. p = 0.064 at the 120-min time-point in Fig. 3B, e.

Fig. 4

(A) Akt phosphorylation in liver as determined by Western blot analysis. Upper panel: Western blot (representative of four independent experiments). Lower panel: pAkt/Akt ratio (y-axis) determined from densitometric scanning of four independent western blot experiments. (B) Tumor necrosis factor-α (TNF-α) mRNA levels in liver, determined by real-time quantitative (q)PCR. P indicates periodontitis. (A), (B) Statistical analysis was performed using a Mann–Whitney rank sum test. *p < 0.05 between groups.