Epidemiological and cross-sectional studies indicate a strong association among periodontitis, obesity and type 2 diabetes mellitus (T2DM), with chronic inflammation as a possible common denominator (1–5). However, the precise mechanism responsible for this association has not been established because of the difficulties in investigating a cause-and-effect relationship in human subjects. Temporal, and also cause-and-effect, relationships can be more effectively controlled in animal models to study associations between periodontitis/inflammation and diabetes. To demonstrate a causal effect of periodontitis on the development of insulin resistance (IR) and T2DM, we recently used Zucker diabetic fatty (ZDF) rats as a ligature-induced periodontitis model system (6). ZDF rats are prone to obesity as a result of the presence of a mutation in the leptin receptor, and female ZDF rats develop diabetes when provided a high-fat (HF) diet (7). We reported that periodontitis influences the onset of IR and hyperglycemia in female ZDF rats fed a HF diet, but not in rats fed a low-fat (LF) diet (6). These findings indicate that periodontitis, in combination with a HF diet, promotes the development of hyperglycemia and IR in diabetic-prone animals. However, the mechanism accounting for this effect on glucose homeostasis is currently unknown. Toll-like receptor 4 (TLR4) is a pattern recognition receptor expressed by various cell types, including macrophages, hepatocytes and pancreatic β-cells (8–14). TLR4 recognizes certain bacterial lipopolysaccharides (LPS) (15), including certain LPS originating from periodontal pathogens (16–18). The activation of TLR4 by LPS results in increased signal transduction that upregulates the transcription of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β (15,19–23). Interestingly, certain fatty acids (FAs) also activate TLR4 signaling, thereby increasing the production of proinflammatory cytokines (20). There are several mechanisms by which proinflammatory cytokines induce IR. For example, TNF-α can phosphorylate insulin receptor substrate-1 and -2 on a serine residue and impair normal insulin signal transduction (24). Having demonstrated that induced periodontitis in diabetic-prone rats, in combination with a HF diet, affects plasma glucose levels (6), we now test the hypothesis that disruption of TLR4 in animals with periodontitis fed a HF diet will result in improved glucose homeostasis. We therefore induced periodontitis in mice fed a HF diet and which have a TLR4 loss-of-function (LOF) mutation rendering them resistant to stimulation with certain types of LPS and FAs via this receptor. To assess glucose homeostasis, we measured fasting plasma glucose and insulin levels and performed glucose tolerance tests. To evaluate a possible involvement of TLR4 (15) in the development of insulin resistance, and thus glucose homeostasis, we assessed insulin signaling (phosphorylation of Akt) by Western blotting (22), and measured TNF-α mRNA levels using quantitative real-time PCR in the liver, which is a major target of insulin action and plays a central role in the regulation of glucose homeostasis.