Periapical Lesions Decrease Insulin Signal and Cause Insulin Resistance Rafael Dias Astolphi, MSc,* Mariane Machado Curbete, MSc,* Natalia Helena Colombo, MSc,* Daisy Jaqueline Shirakashi, MSc,* Fernando Yamamoto Chiba, PhD, † Annelise Katrine Carrara Prieto, MSc, ‡ Luciano Tavares Angelo Cintra, PhD, ‡ Suely Regina Mogami Bomfim, PhD, § Edilson Ervolino, PhD,* and Doris Hissako Sumida, PhD* Abstract Introduction: Inflammatory cytokines are associated with decreased insulin signal transduction. Moreover, local oral inflammation, such as that accompanying peri- odontal disease, is associated with insulin resistance and type 2 diabetes mellitus. The aim of this study was to evaluate the effect of periapical lesions (PLs) on insulin signaling and insulin sensitivity in rats. We hypothesized that PLs alter systemic insulin signaling and insulin sensi- tivity via elevated plasmatic tumor necrosis factor a (TNF-a). Methods: Wistar rats were divided into control (CN) and PL groups. PLs were induced by exposing pulpal tissue to the oral environment. After 30 days, insulin sensitivity was measured using the insulin tolerance test. After euthanization, maxillae were processed for histopathology. Plasmatic concen- trations of tumor necrosis factor a (TNF-a) were deter- mined via the enzyme-linked immunosorbent assay. Insulin signal transduction was evaluated using insulin receptor substrate tyrosine phosphorylation status and serine phosphorylation status in periepididymal white adipose tissue via Western blotting. For insulin signaling and insulin tolerance tests, the analyses performed were analysis of variance followed by the Tukey post hoc test. For TNF-a analysis, the Student’s t test was used. In all tests, P < .05 was considered significant. Results: The rats with PLs showed higher plasmatic TNF-a, lower constant rate for glucose disappearance values, and reduced pp185 tyrosine phosphorylation status but no change in serine phosphorylation status in white adipose tissue after insulin stimulation. Conclusions: PLs can cause alterations to both insulin signaling and insulin sensitivity, probably because of elevation of plasmatic TNF-a. The results from this study emphasize the importance of the prevention of local inflammatory diseases, such as PLs, with regard to the prevention of insulin resistance. (J Endod 2013;39:648–652) Key Words Diabetes mellitus, insulin resistance, periapical lesions, tumor necrosis factor a E ndodontic infection and periodontal disease are very common conditions world- wide (1–3). Results from numerous studies have suggested links between periodontal disease and diabetes, but endodontic disease has not been studied extensively in this regard (4). The possible connection between chronic oral inflamma- tory conditions such as chronic apical periodontitis and systemic health is one of the most interesting areas currently being studied by the medical and dental scientific community (5, 6). Several such studies have now confirmed that the presence of chronic inflammation predicts the development of type 2 diabetes mellitus (T2DM) (7). T2DM is a metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action (such as insulin resistance [IR]) (8), or both (9). Based on recent estimates from the World Health Organization, more than 300 million people worldwide have diabetes. Furthermore, diabetes deaths will double between 2005 and 2030 (10). Insulin regulates cellular metabolism and growth by binding to the insulin receptor that induces autophosphorylation of numerous tyrosine residues (11) and phosphorylation of tyrosine in cytoplasmic substrates including a broad band of 165–185 kd cytoplasmic protein called pp185. This band consists of 2 proteins, insulin receptor substrate (IRS)-1 and -2, that comigrate during sodium dodecyl sulfate poly- acrylamide gel (SDS-PAGE) (12, 13). These tyrosine-phosphorylated substrates may bind with and activate the phosphatidylinositol (PI) 3-kinase (14), and this activation is essential for glucose transportation to insulin-sensitive tissues (15). It has been proposed that oral inflammation, such as that which usually accom- panies periodontal disease, may impair insulin sensitivity in a similar manner to obesity by enhancing activation of the overall systemic immune response initiated by cytokines such as TNF-a. This cytokine is related to reduced insulin signaling and IR (7, 16, 17). The periapical lesion (PL) induces oral inflammation and immune responses against microorganisms that invade and destroy the dental pulp (18). A number of cyto- kines are able to induce root bone resorption, including TNF-a and interleukin (IL)-6, and the expression of these cytokines at sites of inflammation further increases after pulp exposure (19). In particular, the amounts of TNF-a are significantly higher in the exposed pulp of mice compared with healthy pulp (20). As well as acting locally, these mediators diffuse into the systemic circulation. Blood glucose, serum TNF-a, and IL-6 are significantly higher in pregnant or ovariectomized female rats with pulpal From *Basic Sciences, † Child and Social Dentistry, and ‡ Endodontics, Arac ¸ atuba Dental School, UNESP–Universidade Estadual Paulista, Arac ¸ atuba, Sao Paulo, Brazil; and § Clinic and Surgery and Animal Reproduction, Arac ¸atuba Veterinary Medicine School, UNESP–Universidade Estadual Paulista, Arac ¸ atuba, Sao Paulo, Brazil. Supported by grants (2011/04255-8 and 2011/13454-4) from the S~ ao Paulo Research Foundation, S~ ao Paulo, SP, Brazil. Address requests for reprints to Associate Professor Doris Hissako Sumida, Department of Basic Sciences, Arac ¸ atuba School of Dentistry, UNESP Univ Estadual Pau- lista, Rod Marechal Rondom, km 527/528, Arac ¸ atuba, SP, Brazil. E-mail address: dorishs@foa.unesp.br 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.031 Basic Research—Biology 648 Astolphi et al. JOE — Volume 39, Number 5, May 2013