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Periapical Lesions Decrease Insulin Signaling in Rat Skeletal Muscle
Basic Research—Biology
Periapical Lesions Decrease Insulin Signaling in Rat Skeletal Muscle Rafael Dias Astolphi, MSc,* Mariane Machado Curbete, BS,* Fernando Yamamoto Chiba, PhD,* Luciano Tavares Angelo Cintra, PhD,† Edilson Ervolino, PhD,* Max Sander de Oliveira da Mota, MSc,* Cristina Antoniali, PhD,* Clea Adas Saliba Garbin, PhD,‡ and Doris Hissako Sumida, PhD* Abstract Introduction: Serum inflammatory cytokines derived from oral inflammation are associated with decreased insulin signaling (IS) and insulin resistance, which is a major risk factor for type 2 diabetes mellitus. This study aimed to investigate IS in the liver and skeletal muscle (SM) and disorders related to the serum lipid profile and glucose and insulin levels of nondiabetic rats with induced chronic periapical lesions (PLs). Methods: Twenty-eight Wistar rats were divided into control and PL groups. PLs were induced by exposing the pulpal tissue to the oral environment. Experiments were conducted in both groups 30 days after pulp exposure. Maxillae were processed for histopathological analysis. IS was evaluated according to insulin receptor substrate (pp185–insulin receptor substrate 1 [IRS-1]/insulin receptor substrate 2 [IRS-2]) tyrosine phosphorylation status, IRS-1 serine phosphorylation status, and IRS-1 and IRS-2 content in the liver and SM by Western blotting. Serum total cholesterol, triglyceride, glucose, and insulin levels were measured enzymatically using a commercial kit. Results: PL rats showed reduced pp185 P-Tyr and increased IRS-1 serine phosphorylation status in the SM but no change in the liver after insulin stimulation. No significant changes in IRS-1 and IRS-2 content, serum total cholesterol, triglyceride, glucose or insulin levels were noted. Conclusions: PLs are associated with decreased insulin signaling in the SM of rats. Because a decrease in insulin signaling is associated with insulin resistance, our results emphasize the importance of preventing local inflammatory diseases such as PLs to prevent alterations in IS in muscle. (J Endod 2015;-:1–6)
Key Words Insulin receptor substrate proteins, lipidemia, periapical lesions, skeletal muscle
T
ype 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin signaling (such as insulin resistance [IR]), or both. Based on recent estimates from the World Health Organization, more than 300 million people worldwide have diabetes. Furthermore, diabetesrelated deaths are expected to double between 2005 and 2030 (1). Insulin regulates cellular metabolism and growth by binding to the insulin receptor, which induces autophosphorylation of numerous tyrosine residues (2) and phosphorylation of tyrosine in cytoplasmic substrates, including a broad band of 165–185 kd cytoplasmic protein called pp185. This protein consists of 2 proteins termed insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) that comigrate during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (3, 4). These tyrosine-phosphorylated substrates may bind with and activate phosphatidylinositol 3 kinase (PI3-kinase), producing several signals (5). One of these signals is the activation of phosphoinositide-dependent kinase 1, which is involved in the phosphorylation and activation of protein kinase B (or Akt) (6). Akt then regulates various responses of insulin, such as glucose transportation to insulin-sensitive tissues such as skeletal muscle (SM) (7). Insulin is also a powerful anabolic hormone whose function in the liver is gluconeogenesis suppression and lipogenesis activation during times of nutrient excess. Thus, the loss of insulin activity in the liver would be predicted to result in unfettered gluconeogenesis and decreased lipogenesis (8). In the IR condition, the liver loses its lipogenic ability, and fatty acids may be released into the circulation in a phenomenon known as dyslipidemia. Under IR, it is also possible to observe abnormalities of triglyceride storage and lipolysis in insulin-sensitive tissues (9). IRS protein expression and phosphorylation are markedly reduced in many IR states although the mechanism for this down-regulation is unclear (10). However, it is known that high levels of tumor necrosis factor alpha (TNF-a) in the plasma of mice can induce IR via the inhibition of IRS-1–associated PI3-kinase activity in SM (11) and/or by decreasing pp185 tyrosine phosphorylation in insulin-sensitive tissues such as SM (12) and white adipose tissue (12, 13). Periapical lesions (PLs) induce oral inflammation and immune responses against microorganisms that invade and destroy the dental pulp (14). TNF-a expression at inflammation sites increases after pulp exposure (15). In addition to acting locally, TNF-a from periapical inflammation diffuses into the systemic circulation (16, 17). Thus, PLs may impair IS in a similar manner to impairment by obesity by enhancing activation of the overall systemic immune response initiated by TNF-a. Studies in our
From the *Department of Basic Sciences, Arac¸atuba Dental School, Universidade Estadual Paulista (UNESP), Arac¸atuba, S~ao Paulo, Brazil; †Department of Endodontics, Arac¸atuba Dental School, Arac¸atuba, S~ao Paulo, Brazil; and ‡Department of Child and Social Dentistry, Arac¸atuba Dental School, UNESP, Arac¸atuba, S~ao Paulo, Brazil. Address requests for reprints to Prof Doris Hissako Sumida, Department of Basic Sciences, Arac¸atuba School of Dentistry, UNESP, Rodovia Marechal Rondom, km 527/528, Arac¸atuba, SP, Brazil 15015-050. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.002
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PLs Decrease Insulin Signaling
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Basic Research—Biology laboratory have shown that PLs can cause IR and impairment of IS in white adipose tissue, probably via elevation of the plasmatic TNF-a concentration (13). It has also been reported that insulin-stimulated tyrosine phosphorylation of the insulin receptor in the skeletal muscle and adipose tissue of TNF-a knockout mice increases by approximately 2-fold compared with that in wild rats, suggesting that insulin receptor signaling is an important target for TNF-a (18). PLs can induce metabolic disorders characterized by increased serum glucose (14–16) and insulin (16) concentrations. Studies have also indicated that other sources of oral inflammation, such as periodontal diseases, which cause increased TNF-a levels in the serum (12), are associated with worsening of the hyperlipidemic state (19). Previous studies have shown that patients with periodontal diseases have higher total cholesterol and triglyceride levels than individuals with good oral hygiene (20). Endodontic infections are very common worldwide, with an incidence similar to that of periodontal disease (21). In addition, T2DM affects the severity of endodontic and periodontal infections (21), and diabetes may be associated with hyperlipidemia (22). Differently from periodontal disease, few studies regarding the potential of pulpal infection to promote hyperlipidemia, hyperglycemia, and hyperinsulinemia are available in the literature (13, 23). Therefore, more studies regarding these associations are necessary. The inflammatory process may promote alteration in IS transduction and IR and increase the risk of T2DM and its complications. A previous study performed in our laboratory using the same experimental model showed alteration in IS in white adipose tissue; therefore, it is essential to investigate whether a PL per se is able to cause altered IS in other tissues, such as the liver and SM of nondiabetic rats. We hypothesize that PLs will cause a decrease in IS in the liver and SM, which may cause dyslipidemia, hyperglycemia, and hyperinsulinemia. This study represents an important step for preventing a decrease in IS, which can negatively influence systemic health as a whole.
Materials and Methods Twenty-eight male Wistar albino rats weighing 250–280 g were used in the study. The animals were housed in temperaturecontrolled 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.
with hematoxylin-eosin. Histopathological studies were conducted on the periapical area by a certified histologist (E.E.).
Assessment of IRS (pp185–IRS-1/IRS-2) Tyrosine Phosphorylation Status, IRS-1 Serine Phosphorylation Status, and IRS-1 and IRS-2 Content Samples from the liver and gastrocnemius SM were collected from 7 animals from each group (PL and CN) before and after the administration of 1.5 U regular insulin into the portal vein using an insulin syringe according to the methodology found in previous studies (12, 13, 24) at different times (30 seconds for the liver and 90 seconds for SM). Tissue samples were prepared according to the method described by Carvalho et al (24) and subjected to Western blotting for quantification of pp185 (IRS-1/IRS-2) tyrosine phosphorylation status (P-Tyr) using an antiphosphotyrosine antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA), anti–IRS-1 phosphoserine (EMD Millipore Corporation, Billerica, MA), anti–IRS-1 (Santa Cruz Biotechnology, Inc), or anti–IRS-2 (Santa Cruz Biotechnology, Inc). A beta-actin antibody (EMD Millipore Corporation) was used as a control. Immunoreactive bands were detected by autoradiography using a chemiluminescent substrate system (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. Quantitative analysis of the blots was conducted using Scion Image software (Scion ImageRelease Beta 3b; National Institutes of Health, Frederick, MD). Serum Lipid Profile and Glucose and Insulin Concentrations To determine the serum lipid profile and glucose and insulin concentrations, venous blood samples (50 mL) were collected via cardiac puncture after the rats had fasted overnight for 8 to 12 hours. The blood samples were centrifuged immediately after collection at 1800g for 15 minutes at 4 C to obtain serum (25). Serum total cholesterol, triglyceride, and glucose levels were measured enzymatically by using a commercial kit (Colesterol Liquiform Labtest, Triglicerides Liquiform Labtest, and Glicose Liquiform Labtest, respectively; Labtest Diagnostica Ind e Com Ltda, Lagoa Santa, MG, Brazil) (23, 26). Insulinemia was measured using a radioimmunoassay (Coat-A-Count; DPC Diagnostics Products, Los Angeles, CA). The total assessed values were tabulated for each experimental group, and a single calibrated operator analyzed the data in a blinded manner.
Animal Preparation The animals were divided into 2 groups with 14 animals in each group: the control (CN) group and the PL group. The PL group was anesthetized by intraperitoneal injection with 87 mg/kg ketamine (Ketamina Agener; Embu-Guac¸u, SP, Brazil) and 13 mg/kg xylazine (Dorcipec; Vallee, Montes Claros, MG, Brazil). To induce PLs, the dental pulp of the right upper first molar was exposed to the oral environment on the mesial surface using a surgical round burr (Long Neck Maillefer; Dentsply, Petropolis, RJ, Brazil). At 30 days after pulp exposure, the rats were subjected to a 14-hour fast followed by anesthetization via intraperitoneal injection with 50 mg/kg sodium thiopental (Thiopentax; Cristalia, Itapira, SP, Brazil) before experiments.
1. For insulin signaling assays: the normal distribution of the data set analyzed was verified. Analysis of variance was performed followed by the Tukey post hoc test when the analysis of variance suggested a significant difference between groups (P < .05). Data analysis was performed using a statistical software package (SAS System v.9.2; SAS Institute Inc, Cary, NC). 2. For serum lipid profile, glycemia, insulinemia, and IRS-1 and IRS-2 content: the Student t test was performed; P < .05 was considered statistically significant.
Histopathological Analysis For histologic analysis, 5 animals from each group (PL and CN) were randomly selected 30 days after pulp exposure. The right maxillae were dissected and fixed in 4% formaldehyde for 48 hours. The specimens were demineralized with 18% EDTA and embedded in paraffin. Serial paraffin sections (6 mm) were obtained in the mesial-distal aspects of the whole right upper first molars, and the sections were stained
Histopathological Analysis Representative hematoxylin-eosin–stained sections are shown in Figure 1A–D. The CN group had dental and periodontal tissue with normal histologic features. The PL group had complete pulp necrosis and apical periodontitis. PLs were round, restricted to the periapical
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Statistical Analyses Two analyses were performed:
Results
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Basic Research—Biology
Figure 1. Histologic appearance of the periapical area of the right upper first molar in the (A and B) CN and (C and D) PL groups. Photomicrographs showing the periapical area related to (A and C) mesiobuccal and (B and D) distobuccal roots with feature normal in the (A and B) CN group and the periapical lesion established in the (C and D) PL group after 30 days of the pulp exposure. Scale bars: A and C, 200 mm; B and D, 100 mm. *Infiltrate inflammatory in the periapical region. ab, alveolar bone; ce, cementum; dp, dental pulp; n, necrotic tissue remnants; pdl, periodontal ligament. Hematoxylin-eosin staining.
tissues, and approximately 1 mm in size. A moderately dense connective tissue circumscribed a wide intense inflammatory infiltrate composed primarily of mononuclear cells and some polymorphonuclear neutrophils.
Assessment of IRS (pp185–IRS-1/IRS-2) Tyrosine Phosphorylation Status, IRS-1 Serine Phosphorylation Status, and IRS-1 and IRS-2 Content Increased pp185 phosphorylation was observed after insulin stimulation in relation to the baseline in both groups (CN and PL) and tissues (SM and the liver). After insulin stimulation, pp185 phosphorylation was lower (P < .05) in the SM in the PL group relative to the CN group. However, this alteration in insulin signaling was not observed between groups for the liver (Fig. 2A–F). After insulin infusion, the LP group showed an increase in IRS-1 serine phosphorylation status in SM but not in the liver compared with the CN group (Fig. 2). There was no difference in IRS-1 and IRS-2 content between the CN and LP groups in both tissues (Fig. 3A–F). Serum Lipid Profile and Glucose and Insulin Concentrations No statistically significant differences were observed in the serum total cholesterol, triglyceride, glucose, and insulin levels between groups (Table 1).
Discussion In the present study, induced chronic PLs in rats decreased insulin signaling by reducing the pp185 (IRS-1/IRS-2) P-Tyr and increasing the IRS-1 serine phosphorylation in SM after insulin JOE — Volume -, Number -, - 2015
stimulation (Western blot analysis). This impairment of insulin signaling in the SM is relevant because this tissue is the major site for insulin-stimulated glucose disposal (27). We emphasize that, to our knowledge, no study has examined the relationship between pulpal infection and the effects on IS in the SM in the literature thus far. pp185 is a major protein that may be phosphorylated on tyrosine in the liver, SM, and adipose tissue under the effect of insulin. When pp185 is phosphorylated at tyrosine, insulin signaling is stimulated (3). Plomgaard et al (28) showed that serum infusion of TNF-a reduced tyrosine phosphorylation and increased serine phosphorylation of IRS-1 in muscles, thus negatively regulating insulin signaling and whole-body glucose uptake in healthy humans. A previous study (13) performed in Wistar rats showed that PLs caused increases in serum TNF-a. It is important to identify the particular phosphorylated serine residues of IRS-1 that trigger the degradation of this IRS and the kinases responsible for phosphorylation (10). According to Nolte et al (27), TNF-a may inhibit insulin signaling in SM through effects similar to those of the serine phosphatase inhibitor okadaic acid. Additionally, it has been shown recently that TNF-a activates SK6 (p70S6k, a serine kinase), which directly phosphorylates IRS-1 at multiple serine residues (29). However, in isolated rat skeletal muscle, TNF-a had no effect on insulin signaling, even at very high doses (27). Colombo et al (12) reported reduced pp185 P-Tyr levels in the SM in an experimental model (in vivo) involving rats with periodontal disease, another source of oral inflammation. Previous studies reported that PL develops during the initial 2 weeks after pulp exposure in rat molar teeth (15). After 2 weeks, the abscesses enter a chronic phase (16). PLs are highly complex and usually result from a persistent inflammatory response induced
PLs Decrease Insulin Signaling
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Basic Research—Biology
Figure 2. Evaluation of pp185 (IRS-1/IRS-2) tyrosine phosphorylation (P-Tyr) and IRS-1 serine phosphorylation (P-Ser) before ( ) and after (+) insulin stimulation in the (A–C) liver and (D–F) gastrocnemius skeletal muscle of control (CN) and periapical lesion (PL) rats after 30 days of the pulp exposure. A and D show typical autoradiography where similar quantities of protein (185 mg) were subjected to SDS-PAGE. Beta-actin was used as a control. B and E show P-Tyr levels (expressed in arbitrary units) in the (B) liver and (E) SM, respectively. C and F show P-Ser levels (expressed in arbitrary units) in the (C) liver and (F) SM, respectively. Data are presented as the mean standard error of the mean (n = 7).
by prolonged exposure of periapical tissues to various microbial agents, thus evoking an immunologic reaction. In this local defense mechanism, various inflammatory mediators, particularly inflammatory cytokines such as TNF-a, play a complex and central role in the regulation of the immune response (21, 30, 31). Previous studies, including those performed in our laboratory using the same experimental model, showed that TNF-a from chronic PL reaches the systemic circulation and can cause IR (13, 16). It has been recognized that PLs are a potential public health problem that requires further studies (16) to clarify their relationship with metabolic disorders, such as hyperlipidemia (23), hyperglycemia, and hyperinsulinemia (16). PLs present a challenge to health care professionals providing comprehensive care to patients in relation to pulpal disease and systemic diseases because PLs are among the most frequent infections in humans (1–3). Our study showed no changes in the serum total cholesterol and triglyceride concentration of nondiabetic rats with induced chronic PLs (30 days). Our findings agree with those of Cintra et al (23), who found no differences in the lipid profile between rats with induced chronic pulp inflammation (30 days) and healthy rats. Although the relationship of diabetes and periapical inflammation has been previously investigated (30, 32, 33), only a few studies have 4
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reported the influence of PLs on blood cholesterol and triglyceride levels in nondiabetic rats. In our study, the differences in the values of glycemia and insulinemia for the groups (PL and CN) were not found to be statistically significant. These findings are in accordance with those of Cintra et al (14), who reported no difference in serum glucose concentration between rats with periapical inflammation and control rats. However, our results do not corroborate the findings of Bain et al (16), who affirmed that blood glucose and insulin concentrations were significantly higher in pregnant rats with induced chronic pulpal abscesses. The ability of inflammation in PL rats to decrease insulin signaling by reducing P-Tyr in pp185 (IRS-1/IRS-2) and increasing IRS-1 serine phosphorylation is unaffected in the liver. These results are in agreement with those of Colombo et al (12) and Chiba et al (31, 34), who observed reduced pp185 phosphorylation in SM but not in the liver in an experimental model involving periodontal disease and fluoride intake, respectively. This finding may explain the lack of glycemia alteration in rats even with reduced pp185 P-Tyr levels in SM tissue. Interestingly, Cho et al (35) suggested that the liver may compensate for IR in muscle tissues to prevent hyperglycemia. It is known that the alteration of PI3K in the liver results in a marked reduction in insulin-stimulated PI3K activity in JOE — Volume -, Number -, - 2015
Basic Research—Biology
Figure 3. Evaluation of IRS-1 and IRS-2 content in the (A–C) liver and (D–F) gastrocnemius skeletal muscle of CN and PL rats after 30 days of the pulp exposure. A and D show typical autoradiography results in the (A) liver and (D) SM, respectively, where similar quantities of protein (185 mg) were subjected to SDS-PAGE. Beta-actin was used as a control. B and E show values of IRS-1 content (expressed in arbitrary units) in the (B) liver and (E) SM, respectively. C and F show values of IRS-2 content (expressed in arbitrary units) in the (C) liver and (F) SM, respectively. Data are presented as the mean standard error of the mean (n = 7).
the liver and significant defects in lipid and glucose homeostasis (8). This finding may also explain the absence of changes in the lipid profile and glycemia because the initial step of insulin function in the liver was preserved; therefore, the lipidic and glycidic metabolism of the liver was not affected. The reduced pp185 P-Tyr levels in the skeletal muscle of PL rats evident in the present study may have resulted from the elevation of plasmatic cytokine levels, as observed in earlier work performed in our laboratory using the same experimental model (13). It should be noted that the decrease in the pp185 P-Tyr levels in the SM of PL rats was not because of the reduction of IRS-1 and IRS-2 content, showing TABLE 1. Serum Lipid Profile and Glucose and Insulin Concentrations of Control (CN) Rats and Periapical Lesion (PL) Rats Parameters Total cholesterol (n = 7) Triglyceride (n = 7) Glucose (mg/dL) (n = 7) Insulin (n = 7)
CN (mean ± SEM) PL (mean ± SEM) 61.71 2.25a 77.29 8.98a 121.4 7.46a 9.824 3.20a
68.57 3.78a 61.14 12.76a 119.2 5.13a 9.48 4.96a
SEM, standard error of the mean. Same letters indicate the absence of statistical difference among the groups (P > .05).
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that there was no parallel relationship. Moreover, these data suggest that additional studies in both rats and humans should be performed to establish potential links among PLs, systemic diseases, and inflammation as a risk factor. Our data suggest that 30-day PLs may cause impairment in insulin signaling in the SM of nondiabetic rats by decreasing pp185 tyrosine phosphorylation and increasing IRS-1 serine phosphorylation in this tissue. However, 30-day PLs did not affect serum total cholesterol, triglyceride, glucose, or insulin levels. Considering that SM is a major site of insulin-stimulated glucose disposal, the present study emphasizes the importance of preventing local inflammatory diseases such as PLs to prevent insulin signal impairment in SM, which could damage systemic health.
Acknowledgments Supported by grants from the S~ao Paulo Research Foundation (FAPESP) (grant no. 2012/08722-2), Pro-Rector for Research of UNESP (PROPe-UNESP), and Foundation for the Development of UNESP (FUNDUNESP), S~ao Paulo, SP, Brazil. The authors deny any conflicts of interest related to this study. PLs Decrease Insulin Signaling
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Basic Research—Biology References 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2012;35(Suppl 1):S64–71. 2. White MF. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 1998;182:3–11. 3. Rothenberg PL, Lane WS, Karasik A, et al. Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase. J Biol Chem 1991;266:8302–11. 4. Sun XJ, Rothenberg P, Kahn CR, et al. Structure of the insulin receptor substrate IRS1 defines a unique signal transduction protein. Nature 1991;352:73–7. 5. Backer JM, Myers MG, Shoelson SE, et al. Phosphatidylinositol 3’-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J 1992;11:3469–79. 6. Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 1996;15:6541–51. 7. 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(Pt 3):631–5. 8. Taniguchi CM, Kondo T, Sajan M, et al. Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta. Cell Metab 2006;3:343–53. 9. Lewis GF, Carpentier A, Adeli K, Giacca A. Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev 2002;23:201–29. 10. Pederson TM, Kramer DL, Rondinone CM. Serine/threonine phosphorylation of IRS1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes 2001;50:24–31. 11. Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 2002;8:731–7. 12. Colombo NH, Shirakashi DJ, Chiba FY, et al. Periodontal disease decreases insulin sensitivity and insulin signaling. J Periodontol 2012;83:864–70. 13. Astolphi RD, Curbete MM, Colombo NH, et al. Periapical lesions decrease insulin signal and cause insulin resistance. J Endod 2013;39:648–52. 14. Cintra LT, Samuel RO, Facundo AC, et al. Relationships between oral infections and blood glucose concentrations or HbA1c levels in normal and diabetic rats. Int Endod J 2014;47:228–37. 15. Stashenko P, Wang CY, Tani-Ishii N, Yu SM. Pathogenesis of induced rat periapical lesions. Oral Surg Oral Med Oral Pathol 1994;78:494–502. 16. Bain JL, Lester SR, Henry WD, et al. Effects of induced periapical abscesses on rat pregnancy outcomes. Arch Oral Biol 2009;54:162–71. 17. Zhang H, Bain JL, Caskey CP, et al. Effects of gender on serum biomarkers of systemic inflammation coincident to experimentally-induced periapical lesions. Arch Oral Biol 2011;56:168–76. 18. Hotamisligil GS. Mechanisms of TNF-alpha-induced insulin resistance. Exp Clin Endocrinol Diabetes 1999;107:119–25. €, K€oroglu BK, Hic¸yılmaz H, et al. Pro-inflammatory cytokine levels in as19. Fentoglu O sociation between periodontal disease and hyperlipidaemia. J Clin Periodontol 2011;38:8–16.
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20. Majithiya JB, Balaraman R, Giridhar R, Yadav MR. Effect of bis[curcumino]oxovanadium complex on non-diabetic and streptozotocin-induced diabetic rats. J Trace Elem Med Biol 2005;18:211–7. 21. Peters LB, Lindeboom JA, Elst ME, Wesselink PR. Prevalence of apical periodontitis relative to endodontic treatment in an adult Dutch population: a repeated crosssectional study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111: 523–8. 22. Taleghani F, Shamaei M. Association between chronic periodontitis and serum lipid levels. Acta Med Iran 2010;48:47–50. 23. Cintra LT, da Silva Facundo AC, Azuma MM, et al. Pulpal and periodontal diseases increase triglyceride levels in diabetic rats. Clin Oral Investig 2013;17: 1595–9. 24. 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;137:151–9. 25. Finamor IA, Saccol EM, Gabriel D, et al. Effects of parboiled rice diet on oxidative stress parameters in kidney of rats with streptozotocin-induced diabetes. J Med Food 2012;15:598–604. 26. Trinder P. Quantitative determination of triglyceride using GPO-PAP method. Ann Clin Biochem 1969;6:24–7. 27. Nolte LA, Hansen PA, Chen MM, et al. Short-term exposure to tumor necrosis factoralpha does not affect insulin-stimulated glucose uptake in skeletal muscle. Diabetes 1998;47:721–6. 28. Plomgaard P, Bouzakri K, Krogh-Madsen R, et al. Tumor necrosis factor-alpha induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 2005;54:2939–45. 29. Zhang J, Gao Z, Yin J, et al. S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2. J Biol Chem 2008;283:35375–82. 30. Marotta PS, Fontes TV, Armada L, et al. Type 2 diabetes mellitus and the prevalence of apical periodontitis and endodontic treatment in an adult Brazilian population. J Endod 2012;38:297–300. 31. Chiba FY, Colombo NH, Shirakashi DJ, et al. NaF treatment increases TNF-a and resistin concentrations and reduces insulin signal in rats. J Fluor Chem 2012;136:5. 32. Bender IB, Bender AB. Diabetes mellitus and the dental pulp. J Endod 2003;29: 383–9. 33. Lopez-Lopez J, Jane-Salas E, Estrugo-Devesa A, et al. Periapical and endodontic status of type 2 diabetic patients in Catalonia, Spain: a cross-sectional study. J Endod 2011;37:598–601. 34. Chiba FY, Colombo NH, Shirakashi DJ, et al. Insulin signal decrease in muscle but not in the liver of castrated male rats from chronic exposure to fluoride. Fluoride 2010;43:6. 35. Cho Y, Ariga M, Uchijima Y, et al. The novel roles of liver for compensation of insulin resistance in human growth hormone transgenic rats. Endocrinology 2006;147: 5374–84.
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Periapical Lesions Decrease Insulin Signaling in Rat Skeletal Muscle Rafael Dias Astolphi, MSc,* Mariane Machado Curbete, BS,* Fernando Yamamoto Chiba, PhD,* Luciano Tavares Angelo Cintra, PhD,† Edilson Ervolino, PhD,* Max Sander de Oliveira da Mota, MSc,* Cristina Antoniali, PhD,* Clea Adas Saliba Garbin, PhD,‡ and Doris Hissako Sumida, PhD* Abstract Introduction: Serum inflammatory cytokines derived from oral inflammation are associated with decreased insulin signaling (IS) and insulin resistance, which is a major risk factor for type 2 diabetes mellitus. This study aimed to investigate IS in the liver and skeletal muscle (SM) and disorders related to the serum lipid profile and glucose and insulin levels of nondiabetic rats with induced chronic periapical lesions (PLs). Methods: Twenty-eight Wistar rats were divided into control and PL groups. PLs were induced by exposing the pulpal tissue to the oral environment. Experiments were conducted in both groups 30 days after pulp exposure. Maxillae were processed for histopathological analysis. IS was evaluated according to insulin receptor substrate (pp185–insulin receptor substrate 1 [IRS-1]/insulin receptor substrate 2 [IRS-2]) tyrosine phosphorylation status, IRS-1 serine phosphorylation status, and IRS-1 and IRS-2 content in the liver and SM by Western blotting. Serum total cholesterol, triglyceride, glucose, and insulin levels were measured enzymatically using a commercial kit. Results: PL rats showed reduced pp185 P-Tyr and increased IRS-1 serine phosphorylation status in the SM but no change in the liver after insulin stimulation. No significant changes in IRS-1 and IRS-2 content, serum total cholesterol, triglyceride, glucose or insulin levels were noted. Conclusions: PLs are associated with decreased insulin signaling in the SM of rats. Because a decrease in insulin signaling is associated with insulin resistance, our results emphasize the importance of preventing local inflammatory diseases such as PLs to prevent alterations in IS in muscle. (J Endod 2015;-:1–6)
Key Words Insulin receptor substrate proteins, lipidemia, periapical lesions, skeletal muscle
T
ype 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin signaling (such as insulin resistance [IR]), or both. Based on recent estimates from the World Health Organization, more than 300 million people worldwide have diabetes. Furthermore, diabetesrelated deaths are expected to double between 2005 and 2030 (1). Insulin regulates cellular metabolism and growth by binding to the insulin receptor, which induces autophosphorylation of numerous tyrosine residues (2) and phosphorylation of tyrosine in cytoplasmic substrates, including a broad band of 165–185 kd cytoplasmic protein called pp185. This protein consists of 2 proteins termed insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) that comigrate during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (3, 4). These tyrosine-phosphorylated substrates may bind with and activate phosphatidylinositol 3 kinase (PI3-kinase), producing several signals (5). One of these signals is the activation of phosphoinositide-dependent kinase 1, which is involved in the phosphorylation and activation of protein kinase B (or Akt) (6). Akt then regulates various responses of insulin, such as glucose transportation to insulin-sensitive tissues such as skeletal muscle (SM) (7). Insulin is also a powerful anabolic hormone whose function in the liver is gluconeogenesis suppression and lipogenesis activation during times of nutrient excess. Thus, the loss of insulin activity in the liver would be predicted to result in unfettered gluconeogenesis and decreased lipogenesis (8). In the IR condition, the liver loses its lipogenic ability, and fatty acids may be released into the circulation in a phenomenon known as dyslipidemia. Under IR, it is also possible to observe abnormalities of triglyceride storage and lipolysis in insulin-sensitive tissues (9). IRS protein expression and phosphorylation are markedly reduced in many IR states although the mechanism for this down-regulation is unclear (10). However, it is known that high levels of tumor necrosis factor alpha (TNF-a) in the plasma of mice can induce IR via the inhibition of IRS-1–associated PI3-kinase activity in SM (11) and/or by decreasing pp185 tyrosine phosphorylation in insulin-sensitive tissues such as SM (12) and white adipose tissue (12, 13). Periapical lesions (PLs) induce oral inflammation and immune responses against microorganisms that invade and destroy the dental pulp (14). TNF-a expression at inflammation sites increases after pulp exposure (15). In addition to acting locally, TNF-a from periapical inflammation diffuses into the systemic circulation (16, 17). Thus, PLs may impair IS in a similar manner to impairment by obesity by enhancing activation of the overall systemic immune response initiated by TNF-a. Studies in our
From the *Department of Basic Sciences, Arac¸atuba Dental School, Universidade Estadual Paulista (UNESP), Arac¸atuba, S~ao Paulo, Brazil; †Department of Endodontics, Arac¸atuba Dental School, Arac¸atuba, S~ao Paulo, Brazil; and ‡Department of Child and Social Dentistry, Arac¸atuba Dental School, UNESP, Arac¸atuba, S~ao Paulo, Brazil. Address requests for reprints to Prof Doris Hissako Sumida, Department of Basic Sciences, Arac¸atuba School of Dentistry, UNESP, Rodovia Marechal Rondom, km 527/528, Arac¸atuba, SP, Brazil 15015-050. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.002
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Basic Research—Biology laboratory have shown that PLs can cause IR and impairment of IS in white adipose tissue, probably via elevation of the plasmatic TNF-a concentration (13). It has also been reported that insulin-stimulated tyrosine phosphorylation of the insulin receptor in the skeletal muscle and adipose tissue of TNF-a knockout mice increases by approximately 2-fold compared with that in wild rats, suggesting that insulin receptor signaling is an important target for TNF-a (18). PLs can induce metabolic disorders characterized by increased serum glucose (14–16) and insulin (16) concentrations. Studies have also indicated that other sources of oral inflammation, such as periodontal diseases, which cause increased TNF-a levels in the serum (12), are associated with worsening of the hyperlipidemic state (19). Previous studies have shown that patients with periodontal diseases have higher total cholesterol and triglyceride levels than individuals with good oral hygiene (20). Endodontic infections are very common worldwide, with an incidence similar to that of periodontal disease (21). In addition, T2DM affects the severity of endodontic and periodontal infections (21), and diabetes may be associated with hyperlipidemia (22). Differently from periodontal disease, few studies regarding the potential of pulpal infection to promote hyperlipidemia, hyperglycemia, and hyperinsulinemia are available in the literature (13, 23). Therefore, more studies regarding these associations are necessary. The inflammatory process may promote alteration in IS transduction and IR and increase the risk of T2DM and its complications. A previous study performed in our laboratory using the same experimental model showed alteration in IS in white adipose tissue; therefore, it is essential to investigate whether a PL per se is able to cause altered IS in other tissues, such as the liver and SM of nondiabetic rats. We hypothesize that PLs will cause a decrease in IS in the liver and SM, which may cause dyslipidemia, hyperglycemia, and hyperinsulinemia. This study represents an important step for preventing a decrease in IS, which can negatively influence systemic health as a whole.
Materials and Methods Twenty-eight male Wistar albino rats weighing 250–280 g were used in the study. The animals were housed in temperaturecontrolled 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.
with hematoxylin-eosin. Histopathological studies were conducted on the periapical area by a certified histologist (E.E.).
Assessment of IRS (pp185–IRS-1/IRS-2) Tyrosine Phosphorylation Status, IRS-1 Serine Phosphorylation Status, and IRS-1 and IRS-2 Content Samples from the liver and gastrocnemius SM were collected from 7 animals from each group (PL and CN) before and after the administration of 1.5 U regular insulin into the portal vein using an insulin syringe according to the methodology found in previous studies (12, 13, 24) at different times (30 seconds for the liver and 90 seconds for SM). Tissue samples were prepared according to the method described by Carvalho et al (24) and subjected to Western blotting for quantification of pp185 (IRS-1/IRS-2) tyrosine phosphorylation status (P-Tyr) using an antiphosphotyrosine antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA), anti–IRS-1 phosphoserine (EMD Millipore Corporation, Billerica, MA), anti–IRS-1 (Santa Cruz Biotechnology, Inc), or anti–IRS-2 (Santa Cruz Biotechnology, Inc). A beta-actin antibody (EMD Millipore Corporation) was used as a control. Immunoreactive bands were detected by autoradiography using a chemiluminescent substrate system (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. Quantitative analysis of the blots was conducted using Scion Image software (Scion ImageRelease Beta 3b; National Institutes of Health, Frederick, MD). Serum Lipid Profile and Glucose and Insulin Concentrations To determine the serum lipid profile and glucose and insulin concentrations, venous blood samples (50 mL) were collected via cardiac puncture after the rats had fasted overnight for 8 to 12 hours. The blood samples were centrifuged immediately after collection at 1800g for 15 minutes at 4 C to obtain serum (25). Serum total cholesterol, triglyceride, and glucose levels were measured enzymatically by using a commercial kit (Colesterol Liquiform Labtest, Triglicerides Liquiform Labtest, and Glicose Liquiform Labtest, respectively; Labtest Diagnostica Ind e Com Ltda, Lagoa Santa, MG, Brazil) (23, 26). Insulinemia was measured using a radioimmunoassay (Coat-A-Count; DPC Diagnostics Products, Los Angeles, CA). The total assessed values were tabulated for each experimental group, and a single calibrated operator analyzed the data in a blinded manner.
Animal Preparation The animals were divided into 2 groups with 14 animals in each group: the control (CN) group and the PL group. The PL group was anesthetized by intraperitoneal injection with 87 mg/kg ketamine (Ketamina Agener; Embu-Guac¸u, SP, Brazil) and 13 mg/kg xylazine (Dorcipec; Vallee, Montes Claros, MG, Brazil). To induce PLs, the dental pulp of the right upper first molar was exposed to the oral environment on the mesial surface using a surgical round burr (Long Neck Maillefer; Dentsply, Petropolis, RJ, Brazil). At 30 days after pulp exposure, the rats were subjected to a 14-hour fast followed by anesthetization via intraperitoneal injection with 50 mg/kg sodium thiopental (Thiopentax; Cristalia, Itapira, SP, Brazil) before experiments.
1. For insulin signaling assays: the normal distribution of the data set analyzed was verified. Analysis of variance was performed followed by the Tukey post hoc test when the analysis of variance suggested a significant difference between groups (P < .05). Data analysis was performed using a statistical software package (SAS System v.9.2; SAS Institute Inc, Cary, NC). 2. For serum lipid profile, glycemia, insulinemia, and IRS-1 and IRS-2 content: the Student t test was performed; P < .05 was considered statistically significant.
Histopathological Analysis For histologic analysis, 5 animals from each group (PL and CN) were randomly selected 30 days after pulp exposure. The right maxillae were dissected and fixed in 4% formaldehyde for 48 hours. The specimens were demineralized with 18% EDTA and embedded in paraffin. Serial paraffin sections (6 mm) were obtained in the mesial-distal aspects of the whole right upper first molars, and the sections were stained
Histopathological Analysis Representative hematoxylin-eosin–stained sections are shown in Figure 1A–D. The CN group had dental and periodontal tissue with normal histologic features. The PL group had complete pulp necrosis and apical periodontitis. PLs were round, restricted to the periapical
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Statistical Analyses Two analyses were performed:
Results
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Figure 1. Histologic appearance of the periapical area of the right upper first molar in the (A and B) CN and (C and D) PL groups. Photomicrographs showing the periapical area related to (A and C) mesiobuccal and (B and D) distobuccal roots with feature normal in the (A and B) CN group and the periapical lesion established in the (C and D) PL group after 30 days of the pulp exposure. Scale bars: A and C, 200 mm; B and D, 100 mm. *Infiltrate inflammatory in the periapical region. ab, alveolar bone; ce, cementum; dp, dental pulp; n, necrotic tissue remnants; pdl, periodontal ligament. Hematoxylin-eosin staining.
tissues, and approximately 1 mm in size. A moderately dense connective tissue circumscribed a wide intense inflammatory infiltrate composed primarily of mononuclear cells and some polymorphonuclear neutrophils.
Assessment of IRS (pp185–IRS-1/IRS-2) Tyrosine Phosphorylation Status, IRS-1 Serine Phosphorylation Status, and IRS-1 and IRS-2 Content Increased pp185 phosphorylation was observed after insulin stimulation in relation to the baseline in both groups (CN and PL) and tissues (SM and the liver). After insulin stimulation, pp185 phosphorylation was lower (P < .05) in the SM in the PL group relative to the CN group. However, this alteration in insulin signaling was not observed between groups for the liver (Fig. 2A–F). After insulin infusion, the LP group showed an increase in IRS-1 serine phosphorylation status in SM but not in the liver compared with the CN group (Fig. 2). There was no difference in IRS-1 and IRS-2 content between the CN and LP groups in both tissues (Fig. 3A–F). Serum Lipid Profile and Glucose and Insulin Concentrations No statistically significant differences were observed in the serum total cholesterol, triglyceride, glucose, and insulin levels between groups (Table 1).
Discussion In the present study, induced chronic PLs in rats decreased insulin signaling by reducing the pp185 (IRS-1/IRS-2) P-Tyr and increasing the IRS-1 serine phosphorylation in SM after insulin JOE — Volume -, Number -, - 2015
stimulation (Western blot analysis). This impairment of insulin signaling in the SM is relevant because this tissue is the major site for insulin-stimulated glucose disposal (27). We emphasize that, to our knowledge, no study has examined the relationship between pulpal infection and the effects on IS in the SM in the literature thus far. pp185 is a major protein that may be phosphorylated on tyrosine in the liver, SM, and adipose tissue under the effect of insulin. When pp185 is phosphorylated at tyrosine, insulin signaling is stimulated (3). Plomgaard et al (28) showed that serum infusion of TNF-a reduced tyrosine phosphorylation and increased serine phosphorylation of IRS-1 in muscles, thus negatively regulating insulin signaling and whole-body glucose uptake in healthy humans. A previous study (13) performed in Wistar rats showed that PLs caused increases in serum TNF-a. It is important to identify the particular phosphorylated serine residues of IRS-1 that trigger the degradation of this IRS and the kinases responsible for phosphorylation (10). According to Nolte et al (27), TNF-a may inhibit insulin signaling in SM through effects similar to those of the serine phosphatase inhibitor okadaic acid. Additionally, it has been shown recently that TNF-a activates SK6 (p70S6k, a serine kinase), which directly phosphorylates IRS-1 at multiple serine residues (29). However, in isolated rat skeletal muscle, TNF-a had no effect on insulin signaling, even at very high doses (27). Colombo et al (12) reported reduced pp185 P-Tyr levels in the SM in an experimental model (in vivo) involving rats with periodontal disease, another source of oral inflammation. Previous studies reported that PL develops during the initial 2 weeks after pulp exposure in rat molar teeth (15). After 2 weeks, the abscesses enter a chronic phase (16). PLs are highly complex and usually result from a persistent inflammatory response induced
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Figure 2. Evaluation of pp185 (IRS-1/IRS-2) tyrosine phosphorylation (P-Tyr) and IRS-1 serine phosphorylation (P-Ser) before ( ) and after (+) insulin stimulation in the (A–C) liver and (D–F) gastrocnemius skeletal muscle of control (CN) and periapical lesion (PL) rats after 30 days of the pulp exposure. A and D show typical autoradiography where similar quantities of protein (185 mg) were subjected to SDS-PAGE. Beta-actin was used as a control. B and E show P-Tyr levels (expressed in arbitrary units) in the (B) liver and (E) SM, respectively. C and F show P-Ser levels (expressed in arbitrary units) in the (C) liver and (F) SM, respectively. Data are presented as the mean standard error of the mean (n = 7).
by prolonged exposure of periapical tissues to various microbial agents, thus evoking an immunologic reaction. In this local defense mechanism, various inflammatory mediators, particularly inflammatory cytokines such as TNF-a, play a complex and central role in the regulation of the immune response (21, 30, 31). Previous studies, including those performed in our laboratory using the same experimental model, showed that TNF-a from chronic PL reaches the systemic circulation and can cause IR (13, 16). It has been recognized that PLs are a potential public health problem that requires further studies (16) to clarify their relationship with metabolic disorders, such as hyperlipidemia (23), hyperglycemia, and hyperinsulinemia (16). PLs present a challenge to health care professionals providing comprehensive care to patients in relation to pulpal disease and systemic diseases because PLs are among the most frequent infections in humans (1–3). Our study showed no changes in the serum total cholesterol and triglyceride concentration of nondiabetic rats with induced chronic PLs (30 days). Our findings agree with those of Cintra et al (23), who found no differences in the lipid profile between rats with induced chronic pulp inflammation (30 days) and healthy rats. Although the relationship of diabetes and periapical inflammation has been previously investigated (30, 32, 33), only a few studies have 4
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reported the influence of PLs on blood cholesterol and triglyceride levels in nondiabetic rats. In our study, the differences in the values of glycemia and insulinemia for the groups (PL and CN) were not found to be statistically significant. These findings are in accordance with those of Cintra et al (14), who reported no difference in serum glucose concentration between rats with periapical inflammation and control rats. However, our results do not corroborate the findings of Bain et al (16), who affirmed that blood glucose and insulin concentrations were significantly higher in pregnant rats with induced chronic pulpal abscesses. The ability of inflammation in PL rats to decrease insulin signaling by reducing P-Tyr in pp185 (IRS-1/IRS-2) and increasing IRS-1 serine phosphorylation is unaffected in the liver. These results are in agreement with those of Colombo et al (12) and Chiba et al (31, 34), who observed reduced pp185 phosphorylation in SM but not in the liver in an experimental model involving periodontal disease and fluoride intake, respectively. This finding may explain the lack of glycemia alteration in rats even with reduced pp185 P-Tyr levels in SM tissue. Interestingly, Cho et al (35) suggested that the liver may compensate for IR in muscle tissues to prevent hyperglycemia. It is known that the alteration of PI3K in the liver results in a marked reduction in insulin-stimulated PI3K activity in JOE — Volume -, Number -, - 2015
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Figure 3. Evaluation of IRS-1 and IRS-2 content in the (A–C) liver and (D–F) gastrocnemius skeletal muscle of CN and PL rats after 30 days of the pulp exposure. A and D show typical autoradiography results in the (A) liver and (D) SM, respectively, where similar quantities of protein (185 mg) were subjected to SDS-PAGE. Beta-actin was used as a control. B and E show values of IRS-1 content (expressed in arbitrary units) in the (B) liver and (E) SM, respectively. C and F show values of IRS-2 content (expressed in arbitrary units) in the (C) liver and (F) SM, respectively. Data are presented as the mean standard error of the mean (n = 7).
the liver and significant defects in lipid and glucose homeostasis (8). This finding may also explain the absence of changes in the lipid profile and glycemia because the initial step of insulin function in the liver was preserved; therefore, the lipidic and glycidic metabolism of the liver was not affected. The reduced pp185 P-Tyr levels in the skeletal muscle of PL rats evident in the present study may have resulted from the elevation of plasmatic cytokine levels, as observed in earlier work performed in our laboratory using the same experimental model (13). It should be noted that the decrease in the pp185 P-Tyr levels in the SM of PL rats was not because of the reduction of IRS-1 and IRS-2 content, showing TABLE 1. Serum Lipid Profile and Glucose and Insulin Concentrations of Control (CN) Rats and Periapical Lesion (PL) Rats Parameters Total cholesterol (n = 7) Triglyceride (n = 7) Glucose (mg/dL) (n = 7) Insulin (n = 7)
CN (mean ± SEM) PL (mean ± SEM) 61.71 2.25a 77.29 8.98a 121.4 7.46a 9.824 3.20a
68.57 3.78a 61.14 12.76a 119.2 5.13a 9.48 4.96a
SEM, standard error of the mean. Same letters indicate the absence of statistical difference among the groups (P > .05).
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that there was no parallel relationship. Moreover, these data suggest that additional studies in both rats and humans should be performed to establish potential links among PLs, systemic diseases, and inflammation as a risk factor. Our data suggest that 30-day PLs may cause impairment in insulin signaling in the SM of nondiabetic rats by decreasing pp185 tyrosine phosphorylation and increasing IRS-1 serine phosphorylation in this tissue. However, 30-day PLs did not affect serum total cholesterol, triglyceride, glucose, or insulin levels. Considering that SM is a major site of insulin-stimulated glucose disposal, the present study emphasizes the importance of preventing local inflammatory diseases such as PLs to prevent insulin signal impairment in SM, which could damage systemic health.
Acknowledgments Supported by grants from the S~ao Paulo Research Foundation (FAPESP) (grant no. 2012/08722-2), Pro-Rector for Research of UNESP (PROPe-UNESP), and Foundation for the Development of UNESP (FUNDUNESP), S~ao Paulo, SP, Brazil. The authors deny any conflicts of interest related to this study. PLs Decrease Insulin Signaling
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