Basic ResearchBiology

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 Bomm, PhD, Edilson Ervolino, PhD,* and Doris Hissako Sumida, PhD*
Abstract
Introduction: Inammatory cytokines are associated with decreased insulin signal transduction. Moreover, local oral inammation, such as that accompanying periodontal 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 sensitivity 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 concentrations of tumor necrosis factor a (TNF-a) were determined 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 Students t test was used. In all tests, P < .05 was considered signicant. 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 inammatory diseases, such as PLs, with regard to the prevention of insulin resistance. (J Endod 2013;39:648652)

Key Words
Diabetes mellitus, insulin resistance, periapical lesions, tumor necrosis factor a

ndodontic infection and periodontal disease are very common conditions worldwide (13). 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 inammatory conditions such as chronic apical periodontitis and systemic health is one of the most interesting areas currently being studied by the medical and dental scientic community (5, 6). Several such studies have now conrmed that the presence of chronic inammation 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 165185 kd cytoplasmic protein called pp185. This band consists of 2 proteins, insulin receptor substrate (IRS)-1 and -2, that comigrate during sodium dodecyl sulfate polyacrylamide 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 inammation, such as that which usually accompanies 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 inammation and immune responses against microorganisms that invade and destroy the dental pulp (18). A number of cytokines are able to induce root bone resorption, including TNF-a and interleukin (IL)-6, and the expression of these cytokines at sites of inammation further increases after pulp exposure (19). In particular, the amounts of TNF-a are signicantly 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 signicantly higher in pregnant or ovariectomized female rats with pulpal

From *Basic Sciences, Child and Social Dentistry, and Endodontics, Arac atuba Dental School, UNESPUniversidade Estadual Paulista, Arac atuba, Sao Paulo, Brazil; atuba Veterinary Medicine School, UNESPUniversidade Estadual Paulista, Arac atuba, Sao Paulo, Brazil. and Clinic and Surgery and Animal Reproduction, Arac 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 Paulista, Rod Marechal Rondom, km 527/528, Arac atuba, SP, Brazil. E-mail address: dorishs@foa.unesp.br 0099-2399/$ - see front matter Copyright 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.031

648

Astolphi et al.

JOE Volume 39, Number 5, May 2013

Basic ResearchBiology
abscesses (21, 22). In addition, pregnant rats with induced PLs have signicantly higher blood glucose and serum insulin concentrations. Moreover, elevated levels of TNF-a and IL-6 can be observed in tissues such as liver and the uterine horns of rats with PLs (21). TNF-a alters insulin signaling by inhibiting pp185 (IRS-1/IRS-2)-mediated PI 3kinase activation (23, 24) and by decreasing pp185 tyrosine phosphorylation status (16, 25). Furthermore, TNF-a induces insulin resistance through serine phosphorylation of pp185 (P-Ser) and, in this way, attenuates insulin receptor signaling (26, 27). The inammatory process may alter insulin signal transduction, and no studies reported in the literature to date have correlated endodontic infections with insulin sensitivity in nondiabetic animals or humans. Therefore, the aim of this study was to determine whether induced PLs are able to increase plasmatic TNF-a and decrease insulin sensitivity and insulin signaling in nondiabetic rats. We hypothesized that PLs alter systemic insulin signaling and insulin sensitivity via elevated plasmatic TNF-a.

Short Intravenous Insulin Tolerance Test Insulin tolerance tests (ITTs) were conducted on 7 animals from each group (CN and PL) following the protocol described in Chehoud et al (27). A dose of 0.75 U/kg body weight insulin (Novolin; Novo Nordisk, Bagsvaerd, Denmark) was administered through the penile vein. Blood samples (approximately 50 mL) were collected from nicked tails before hormone administration and then at 4, 8, 12, and 16 minutes after insulin administration. Glucose was measured using a glycemia monitor (Accu-Chek Advantage; Roche Diagnostic, Indianapolis, IN). The results were analyzed by comparison of the constant rate for glucose disappearance (Kitt) from time point 0 to the 16-minute time point of the test. The Kitt was calculated using the formula 0.693/t1/2. The glucose t was calculated from the slope of the least-square analysis of the plasma glucose concentrations during the linear decay phase (28). Assessment of the Insulin Receptor Substrate (pp185 IRS-1/IRS-2) Tyrosine and Serine Phosphorylation Status Periepididymal white adipose tissue (WAT) was collected from 7 animals from each group (PL and CN) before and after the administration of 1.5 U regular insulin (intravenously through the portal vein) at 120 seconds for WAT. Tissue samples were prepared according to the method described in Carvalho et al (29) and subjected to Western blotting for the quantication of pp185 (IRS-1/IRS-2) tyrosine phosphorylation status (P-Tyr) using antiphosphotyrosine antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) and the serine phosphorylation status (P-Ser) using antiphosphoserine antibody (EMD Millipore Corp, Billerica, MA). b-Actin antibody (EMD Millipore Corp) was used as the control. Immunoreactive bands were detected by autoradiography using a chemiluminescent substrate system (GE Healthcare, Buckinghamshire, UK) according to the manufacturers instructions. Quantitative analysis of the blots was performed using Scion Image-Release Beta 3b software (National Institutes of Health, Frederick, MD). Statistical Analyses Two analyses were performed:
1. Insulin signaling and glycemia values from the ITT test: the normality of the data set analyzed was veried. Analysis of variance was performed followed by the Tukey post hoc test when analysis of variance suggested a signicant difference between groups (P < .05). Data analysis was performed using a statistics software package (SAS System v.9.2; SAS Institute Inc, Cary, NC). 2. Analysis of TNF-a levels: the Students t test was performed; P < .05 was considered statistically signicant.

Materials and Methods

Forty-four male Wistar albino rats weighing 250280 g were used in the study. The animals were housed in temperature-controlled rooms and were given access to water and food ad libitum. All experiments were approved by the local ethics committee according to protocol number 2011/0471. All efforts were made to minimize the number of animals used and their suffering.

Animal Preparation The animals were divided into 2 groups of 22 animals in each group: the control group (CN) and the PL group. The PL group was anesthetized by intraperitoneal injection with 87 mg/kg ketamine (Ketamina Agener; Agener, Embu-Guac u, SP, Brazil) and 13 mg/kg xylazine (Dorcipec; Vall ee, Montes Claros, MG, Brazil). The pulp of the right upper rst molars was exposed on the mesial surface using a surgical round bur (Long Neck; Dentsply Maillefer, Petr opolis, RJ, Brazil). The pulps were exposed to the oral environment to induce the formation of PLs. At 30 days after pulp exposure, they were subjected to a 14-hour fast followed by anesthetization via intraperitoneal injection with 50 mg/kg sodium thiopental (Thiopentax; Crist alia, Itapira, SP, Brazil) before experiments. Histopathological Analysis For histologic analysis, 5 animals from each group (PL and CN) were randomly selected 30 days after pulp exposure. Their right hemi-maxillae were harvested and soaked for 48 hours in 4% formaldehyde. After demineralization with 18% EDTA, the samples were embedded in parafn. Serial parafn sections of 6-mm thickness were made of the mesial-distal aspects of the whole right upper rst molars and stained with hematoxylin-eosin. Morphologic studies were conducted on the periapical area. Glucose and TNF-a Level Measurement Blood samples (8 mL) from 8 animals from each group were collected via cardiac puncture followed by euthanasia with an overdose of an anesthetic agent. The samples were then distributed into EDTA tubes (1.9 mg/mL; Guangzhou Improve Medical Instruments, Fangcun, Guangzhou, China). The blood samples were centrifuged immediately after collection at 3000 g for 15 minutes at 4 C, and plasma was stored in aliquots at 70 C until used. The plasmatic TNF-a of these samples was measured using an enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, CA). The limit of detection of TNFa was <0.7 pg/mL.
JOE Volume 39, Number 5, May 2013

Results
Histopathological Analysis Representative hematoxylin-eosinstained sections are shown in Figure 1AD. Thirty days after pulp exposure, the pulp showed total necrosis and PLs were established. The PLs were restricted exclusively to the periapex region with a diameter of approximately 1 mm. Furthermore, PLs were composed primarily of neutrophils (polymorphonuclears) and mononuclear cells. Glucose and TNF-a Level Measurement Compared with CN rats, PL rats showed no glycemic alterations. However, the plasmatic TNF-a was higher in PL rats (Table 1).
Periapical Lesions Cause Insulin Resistance

649

Basic ResearchBiology

Figure 1. Photomicrography of healthy molar (CN) and molar with periapical lesion (PL) after 30 days. (A and B) Normal aspects of periodontal insertion in the periapical region. (C and D) Panoramic views of PL molar showing the crown opening, pulp necrosis, and established periapical lesion with the presence of neutrophils (polymorphonuclears) and mononuclear cells. Scale bars = 1 mm (A and C) and 250 mm (B and D). Arrrowheads indicate inammatory inltrate. ab, alveolar bone; d, dentin; dp, dental pulp; p, periodontal ligament. *Surgically induced crown opening (hematoxylin-eosin staining, A and C, original magnication 25; B and D, original magnication 100).

Short Intravenous Insulin Tolerance Test With regard to the glycemia comparisons at each time point, there was no signicant difference at any of the time points. The glucose disappearance rates (Kitt) during the ITT performed in the rst 16 minutes after the hormone infusion in the CN and PL groups are shown in Table 1. The PL group had a signicantly lower (P < .05) glucose disappearance rate (Kitt) in relation to the CN group, showing decreased insulin sensitivity in PL rats compared with CN rats. Assessment of the Insulin Receptor Substrate (pp185 IRS-1/IRS-2) Tyrosine and Serine Phosphorylation Status Increased pp185 phosphorylation status was observed after insulin stimulation in relation to baseline in both groups in WAT. After insulin stimulation, the pp185 tyrosine phosphorylation status was reduced (P < .05) in WAT in the PL group compared with the CN group (Fig. 2A and B). However, there is no difference in pp185 serine phosphorylation status in WAT between the groups (CN and PL) (Fig. 2C and D).

mental mouse model, Lumeng et al (24) showed in adipocytes that TNF-a can impair insulin action by altering the expression of both signaling and glucose transport proteins. In this case, TNF-a is able to cause IR by inhibiting pp185 (IRS-1/IRS-2)-mediated PI 3-kinase activation and tyrosine phosphorylation (23, 24). It is known that pp185 contains a number of tyrosine and serine phosphorylation sites (12, 30). When pp185 is phosphorylated on tyrosine, the insulin signal is stimulated (15). On the other hand, in the presence of TNF-a, pp185 may be phosphorylated on serine or P-Tyr may be reduced (16, 23, 25, 26). P-Ser of pp185 might associate with the insulin receptor in a manner that blocks the tyrosine autophosphorylation reaction, thereby attenuating the insulin signal (26). Using an experimental model involving mice with inammation derived from sepsis, Yan et al (31) reported decreased IRS-1 P-Tyr and increased IRS-1 P-Ser because of high serum TNF-a. However, although our data showed reduced P-Tyr of pp185, no difference in the pp185 phosphorylation on serine (P-Ser) status was found between CN and PL groups after insulin stimulation (Fig. 2). In
TABLE 1. Glycemia, Plasmatic TNF-a, and Glucose Disappearance Rate (Kitt) of CN and PL Rats Parameters
Glycemia (mg/dL) (n = 8) TNF-a (pg/mL) (n = 8) Glucose disappearance rate (Kitt) (n = 7)
*P < .05 for CN versus PL rats.

Discussion
The present study showed that induced PLs decreased insulin signaling in rats by reducing the pp185 (IRS-1/IRS-2) phosphorylation of tyrosine (P-Tyr) status in WAT after insulin stimulus (Fig. 2). This result is in agreement with the ndings of Colombo et al (16), who reported reduced pp185 P-Tyr status in the WAT in an experimental model involving male rats with periodontal disease, another source of oral inammation. Furthermore, using an insulin-resistant experi650

CN (mean SEM) PL (mean SEM)

115.4 2.71 461. 0.55 3.04 0.26 10.5 13.23 7.19 0.62* 1.98 0.3*

Astolphi et al.

JOE Volume 39, Number 5, May 2013

Basic ResearchBiology

Figure 2. Evaluation of pp185 (IRS-1/IRS-2) P-Tyr and P-Ser before () and after (+) insulin stimulation in periepididymal WAT of CN and PL rats after 30 days. A and C show typical autoradiography indicating P-Tyr and P-Ser of pp185, respectively, where similar quantities of protein (185 mg) were subjected to SDS-PAGE. b-actin was used as the control. B and D show P-Tyr and P-Ser status values (expressed in arbitrary units), respectively, which are presented as means standard error of the mean (n = 7). *P < .05; ***P < .001.

agreement with our results, Lumeng et al (24) observed decreased insulin sensitivity via reduced pp185 P-Tyr but no difference in pp185 P-Ser status in adipocytes under the inuence of macrophagederived factors. Therefore, considering our data and that of Lumeng et al, it is possible to conclude that a decrease in P-Tyr and an increase in P-Ser of pp185 do not necessarily occur simultaneously to cause IR. We suggest that TNF-a may activate another inhibitory insulin pathway that is not only related to P-Ser of pp185 but also causes impairment of insulin signaling. PLs are highly complex and usually result from a persistent inammatory response induced by prolonged exposure of periapical tissues to various microbial agents, thus evoking an immunologic reaction. In this local defense mechanism, various inammatory mediators, in particular inammatory cytokines such as IL-6 and TNF-a, play a complex and central role in the regulation of the immune response (21, 32, 33). It is likely that these cytokines are released into the systemic circulation because animal models indicate that proinammatory cytokine concentrations are higher within the serum of animals with PLs (21). As shown by previous studies (19, 20), our data have shown an increase in plasmatic TNF-a in PL rats compared with controls (Table 1). However, Zhang et al (22) reported no difference on TNF-a plasmatic concentration between male rats 28 days after the induction of PLs compared with that in control animals. In mice with another kind of oral inammation, which is induced by periodontal disease, the concentrations of proinammatory cytokines are also reportedly elevated in the serum. This may contribute to a systemic hyperinammatory state, which is a risk factor for several types of systemic diseases such as diabetes (16). Although it is known that proinammatory cytokines are important for the pathogenesis of PLs, how their functions are balanced and systemically controlled is not well understood (34). One of the major links between oral and systemic health is the relationship between periodontal disease and diabetes mellitus caused by inammation (16, 29). Besides periodontal disease, which causes localized inammation, there is periapical inammation, which is considered a potential public health problem that requires additional
JOE Volume 39, Number 5, May 2013

study (21). It becomes a challenge to health care professionals to give comprehensive care to the patient in relation to pulpal disease and systemic diseases such as TDM2 because periodontal disease and PLs are the most common infections in humans (13). We also conrmed reduced insulin sensitivity in the PL group compared with control rats (Table 1). These results are in agreement with the ndings of Bain et al (21), who studied pregnant female rats with PLs. In our studies, insulin sensitivity was measured via the glucose disappearance rate (Kitt) during the 16-minute ITT. The Kitt test reects the combination of suppression of hepatic glucose output and the stimulation of peripheral glucose uptake by insulin. Insulin sensitivity is determined by calculating the rate of decline of the log-transformed glucose concentrations estimated by linear regression. Therefore, it is possible to derive the rate constant Kitt, which is expressed as the percentage decline in glucose per minute (35). Kitt is suitable as a simple and rapid estimation of in vivo insulin action (16, 27, 28). Despite reduced insulin sensitivity, our PL rats did not exhibit glycemic alteration (Table 1). No studies were found in the literature reporting glucose level measurement in nondiabetic male rats with PLs. However, it has been shown in female rats that PLs lead to increased blood glucose (21, 22) compared with animals without PLs. Using a periodontal disease experimental model, another source of oral inammation, Colombo et al (16) veried no glycemic differences between the control and experimental groups. The pathophysiology of T2DM is not restricted to insulin production or insulin effects in tissues. Innate immune metabolic pathways may be activated in adipose tissue, liver, bone, and muscle in parallel with mechanisms involved in the ght against infection and injury. It appears that chronic activation of these innate pathways can lead to T2DM. An understanding of all these processes will lead to a more complete picture of the pathophysiology of insulin resistance and T2DM (36). The reduced pp185 P-Tyr and insulin sensitivity in PL rats evident in the present study was likely caused by the elevation of plasmatic TNFa levels. Moreover, these data suggest that additional studies in both rats and humans should be performed in order to establish potential links between PLs, systemic diseases, and inammation as a risk factor. 651

Periapical Lesions Cause Insulin Resistance

Basic ResearchBiology
Conclusions
PLs are able to cause alterations in both insulin signaling and insulin sensitivity, probably via elevation of the plasmatic TNF-a. Thus, the results of the present study emphasize the importance of preventing local inammatory diseases such as PLs in an effort to prevent insulin resistance, which is a principal characteristic of T2DM.
16. Colombo NH, Shirakashi DJ, Chiba FY, et al. Periodontal disease decreases insulin sensitivity and insulin signaling. J Periodontol 2012;83:86470. 17. Katz J. Elevated blood glucose levels in patients with severe periodontal disease. J Clin Periodontol 2001;28:7102. 18. Bergenholtz G. Effect of bacterial products on inammatory reactions in the dental pulp. Scand J Dent Res 1977;85:1229. 19. Stashenko P, Wang CY, Tani-Ishii N, et al. Pathogenesis of induced rat periapical lesions. Oral Surg Oral Med Oral Pathol 1994;78:494502. 20. Kawashima N, Stashenko P. Expression of bone-resorptive and regulatory cytokines in murine periapical inammation. Arch Oral Biol 1999;44:5566. 21. Bain JL, Lester SR, Henry WD, et al. Effects of induced periapical abscesses on rat pregnancy outcomes. Arch Oral Biol 2009;54:16271. 22. Zhang H, Bain JL, Caskey CP, et al. Effects of gender on serum biomarkers of systemic inammation coincident to experimentally-induced periapical lesions. Arch Oral Biol 2011;56:16876. 23. del Aguila LF, Claffey KP, Kirwan JP. TNF-alpha impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells. Am J Physiol 1999;276: E84955. 24. Lumeng CN, Deyoung SM, Saltiel AR. Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins. Am J Physiol Endocrinol Metab 2007;292:E16674. 25. Chiba FY, Colombo NH, Shirakashi DJ, et al. NaF treatment increases TNF-a and resistin concentrations and reduces insulin signal in rats. J Fluorine Chem 2012; 136:5. 26. Hotamisligil GS, Peraldi P, Budavari A, et al. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science 1996;271:6658. 27. Chehoud KA, Chiba FY, Sassaki KT, et al. Effects of uoride intake on insulin sensitivity and insulin signal transduction. Fluoride 2008;41:2705. 28. Bonora E, Moghetti P, Zancanaro C, et al. Estimates of in vivo insulin action in man: comparison of insulin tolerance tests with euglycemic and hyperglycemic glucose clamp studies. J Clin Endocrinol Metab 1989;68:3748. 29. Carvalho CR, Brenelli SL, Silva AC, et al. Effect of aging on insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of rats. Endocrinology 1996;137:1519. 30. Greene MW, Sakaue H, Wang L, et al. Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation. J Biol Chem 2003;278:8199211. 31. Yan XW, Li WQ, Wang XD, et al. [Effects of insulin receptor substrate-1 and its serine phosphorylation and tyrosine phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis: experiment with rats]. Zhonghua Yi Xue Za Zhi 2006;86:29227. 32. Stashenko P, Teles R, DSouza R. Periapical inammatory responses and their modulation. Crit Rev Oral Biol Med 1998;9:498521. 33. Prso IB, Kocjan W, Simi c H, et al. Tumor necrosis factor-alpha and interleukin 6 in human periapical lesions. Mediators Inamm 2007;2007:38210. 34. Coli c M, Gazivoda D, Vucevi c D, et al. Proinammatory and immunoregulatory mechanisms in periapical lesions. Mol Immunol 2009;47:10113. 35. Wallace TM, Matthews DR. The assessment of insulin resistance in man. Diabet Med 2002;19:52734. 36. Fern andez-Real JM, Pickup JC. Innate immunity, insulin resistance and type 2 diabetes. Diabetologia 2012;55:2738.

Acknowledgments
The authors deny any conicts of interest related to this study.

References
1. Chala S, Abouqal R, Abdallaoui F. Prevalence of apical periodontitis and factors associated with the periradicular status. Acta Odontol Scand 2011;69:3559. 2. Awuti G, Younusi K, Li L, et al. Epidemiological survey on the prevalence of periodontitis and diabetes mellitus in Uyghur adults from rural Hotan area in Xinjiang. Exp Diabetes Res 2012;2012:758921. 3. Peters LB, Lindeboom JA, Elst ME, et al. Prevalence of apical periodontitis relative to endodontic treatment in an adult Dutch population: a repeated cross-sectional study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:5238. 4. Fouad AF. Diabetes mellitus as a modulating factor of endodontic infections. J Dent Educ 2003;67:45967. 5. Demmer RT, Desvarieux M, Holtfreter B, et al. Periodontal status and A1C change: longitudinal results from the study of health in Pomerania (SHIP). Diabetes Care 2010;33:103743. 6. Segura-Egea JJ, Castellanos-Cosano L, Machuca G, et al. Diabetes mellitus, periapical inammation and endodontic treatment outcome. Med Oral Patol Oral Cir Bucal 2012;17:e35661. 7. Dandona P, Aljada A, Bandyopadhyay A. Inammation: the link between insulin resistance, obesity and diabetes. Trends Immunol 2004;25:47. 8. Nolan CJ, Damm P, Prentki M. Type 2 diabetes across generations: from pathophysiology to prevention and management. Lancet 2011;378:16981. 9. Association AD. Diagnosis and classication of diabetes mellitus. Diabetes Care 2012;35(Suppl 1):S6471. 10. World Health Organization. Diabetes. Fact sheet N 312. World Health Organization; 2012. Available at http://www.who.int/mediacentre/factsheets/fs312/en/. Accessed June 20, 2012. 11. White MF. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 1998;182:311. 12. Rothenberg PL, Lane WS, Karasik A, et al. Purication and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase. J Biol Chem 1991;266:830211. 13. Sun XJ, Rothenberg P, Kahn CR, et al. Structure of the insulin receptor substrate IRS1 denes a unique signal transduction protein. Nature 1991;352:737. 14. Backer JM, Myers MG, Shoelson SE, et al. Phosphatidylinositol 3-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J 1992;11:346979. 15. Clarke JF, Young PW, Yonezawa K, et al. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J 1994;300:6315.

652

Astolphi et al.

JOE Volume 39, Number 5, May 2013