The Effect Of Periodontitis On Adipose Tissue Inflammation In Obese Rat Model And Preliminary Pro-Insulin Resistance Mechanism Study

Background:Obesity, a subclinical state with multiple of lipid stored in adipose tissue, is closely correlated with important physiological parameters such as lipid profile, blood pressure and systemic insulin sensitivity. Obesity is a major contributor to systemic chronic disease, including type2diabetes militus (T2DM) and atherosclerosis. The first and the important pathological changes in obese state is the adipocyte hypertrophy (increased size) and hyperplasia (increased number). It is well established that the role of adipose tissue in obesity is not only a passive energy storage site, but it is understood that adipose tissue plays a much more active role as an endocrine organ. Adipose tissue can secrete a plenty of cytokines, so-called "adipokines". The adipokines can act as mediators and signal factors, and interact with energy metabolism, immune and inflammatory regulation.There are a couple of adipokines that are of particular interest to researchers and TNF-α and IL-6are the ones of those. Previous reports shows that TNF-α and IL-6are closed related with pathological mechanism of insulin resistance and T2DM. Adipose tissue level of TNF-α is correlated with fasting plasma glucose, insulin, triacylglycerol concentrations and insulin resistance in patients with and without T2DM.The expression for IL-6is also increased in obese adipose tissue and is10-fold that in adipose tissue from lean individuals. In obese condition, adipose tissue is a major source of circulating IL-6concentrations, and contributes as much as30%of total body production. The combination of TNF-a and IL-6increases lipolysis and fat oxidation, which induce adipose tissue inflammation and followed by the development of systemic insulin resistance. Recently, leptin and adiponectin has been paid much attention as new adipokines and take part in the regulation of metabolism and inflammation. Previous experimental data indicated that leptin modulates glucose homeostasis. Leptin is secreted by adipocytes into circulation. Leptin acts mainly in the hypothalamus and has an impact on appetite, whereas expression of the leptin receptor has been observed in peripheral insulin-target tissues, including fat, liver and muscle, suggesting potential effects of leptin on peripheral insulin actions. In adipocytes, leptin inhibits insulin signaling by two different ways:as an autocrine signal and through neuroendocrine pathways. In contrast, adiponectin possesses anti-inflammatory or insulin-sensitizing properties. Thus, these adipokines can act as regulating mediators between adipose tissue and systemic disorders including insulin resistance.Chronic periodontitis is one of the most widespread inflammatory diseases, characterized by inflammation and destruction of connective tissue attachment and alveolar bone. Published cross-sectional epidemiologic studies have suggested that periodontal disease, particularly periodontitis is associated with obesity. Finding from a longitudinal study has demonstrated a dose-response relationship between body mass index (BMI) and the development of periodontal disease. Periodontitis has been reported to adversely affect glycemic control in patients with diabetes mellitus and contribute to the development of diabetic complications. It is well established that inflammatory processes as common pathways involve in the pathogenesis of obesity, periodontitis and T2DM.However, the mechanistic relationship between IR and periodontitis in obesity remains unclear. Whether chronic periodontitis can regulate the expression of key adipokines and result in the inflammation of adipose tissue and whether the combination of chronic periodontitis and obesity can exacerbate the inflammatory condition of adipose tissue and systemic insulin resistance. The establishment of experimental animal models is ideal to investigate the potential mechanism between periodontitis and T2DM and thus provide some useful theoretical evidence.Part oneEstablishment Experimental Periodontitis in Obese Rat ModelObjectiveTo establish obese rat model by monosodium glutamate (MSG) inducement and experimental periodontitis by ligature inducement with periodontal pathogens infection; To evaluate obese condition by investigating Lee’s index, blood pressure, and fasting glucose; To evaluate periodontal infection by Micro-CT scanning and histological sections.MethodsAll animal’s procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee.(1) Eighty natal SD male rats were randomly divided into four groups of20rats each, respectively:obese rats with periodontitis (OB+CP group), obese rats without periodontitis (OB group), normal rats with periodontitis (CP group) and normal rats without periodontitis (C group).(2) In the OB group and OB+CP group, rats received subcutaneous injection of MSG (3mg/g of body weight) on alternate days for the first10postnatal days. The animals were weaned on the21st postnatal day and fed normally. At12weeks of age, body weight, body length, blood pressure, fasting glucose were investigated.(3) At3months of age,3/0silk ligatures soaked with periodontal pathogens, were placed in subgingival position of the bilateral maxillary first and second molars for8weeks in the CP group and C group, whereas the control group were free of MSG-injection and ligation.(4) At5months of age, rats were sacrificed under general anesthesia. Samples of the bilateral maxillary molar regions were resected from each rat and immediately fixed in10%neutral formalin for2days, and stored in70%ethanol for scanning by micro-CT. Maxillary samples were further decalcified with10%tetrasodium-EDTA equeous solution (PH7.0) for4weeks at4℃after scaning. The tissue blocks of periodontal samples were embedded in paraffin and sections (thickness:5um) were stained with hematoxylin and eosin (HE). Serum samples were also collected and stored in-80℃.ResultsIn this study, we employed postnatal administration of MSG to rats and induced central obesity in adulthood. Ligatures soaked with periodontal pathogen were used to induce chronic periodontitis for8weeks. Rats in the OB+CP group and CP group showed apparent inflammatory cellular infiltration, collagenous fibers destruction and alveolar bone resorption, which were similar to features of human chronic periodontitis. This suggests that an ideal model of obesity and chronic periodontitis was established.ConclusionsAdministration of MSG can induce obese rat model. Besides, MSG-treated rats also have metabolic state of prediabetes with hypertention and hyperglycemia. This method is simple, effective and can be well repeated. In this obese state, ligature-induced periodontitis can result in typical periodontal destruction and sustain a low-grade chronic inflammation. The combination of periodontitis and obesity were successfully established in rat model. Part twoThe Effect of periodontitis on the Inflammation of Adipose Tissue in Rat ModelObjectiveTo investigate the effect of experimental periodontitis on the morphological changes and inflammation of adipose tissue, and to explore the addictive effect of periodontitis and obesity with the aim of providing theoretical basis on the interrelationships between periodontitis and systemic inflammatory disorders.MethodsAt the time of sacrifice, visceral adipose tissues were fixed in10%neutral formalin. After dehydration with increasing concentrations of ethanol, samples were incubated in xylenes and embedded in paraffin at60℃. Six-micron sections were cut and stained with HE, toluidine blue O or CD68/CD14immunohistochemistry. Quantification of adipocytes was done on HE-stained sections using Image-ProPlus software. Stromal-vascular fractions were observed on toluidine blue O-stained sections. Macrophage infiltration was detected on CD68/CD14immunohistochemical stained sections.ResultsAdipocyte size and amount were measured on HE-stained sections. The numbers of adipocytes in the OB+CP and OB groups were significantly higher than that in the CP and C groups (P<0.05). However, there were no significant differences in the number of adipocytes between the CP group and the C group (t=0.215, P=0.830) and that between the OB+CP group and the OB group (t=0.503, P=0.616). The cell diameter changes in the OB and OB+CP groups were striking. Enlarged cells were prevalent and mean diameters of adipocytes were167.21um and170.02um, respectively, which were higher than that in the CP and C groups (P<0.05). However, adipocyte diameters between CP and C groups were similar (t=0.480, P=0.632), and there was also no difference between OB+CP group and OB group (t=0.773, P=0.441).Toluidine blue O-stained sections described the presence of SVF and nucleated stromal cells. The stromal multinucleated cells were greatly increased in the OB and OB+CP groups when compared to the CP and C groups. The presence of nucleated stromal cells in the CP group was similar to those in the C group.In the visceral fat tissue, immunohistochemical analysis showed that the protein expression for CD68in the OB+CP was significantly higher than that in the OB group (t=3.944, P=0.000); and the expression in the CP group was also significantly higher than that in the C group (t=8.332, P=0.000). Furthermore, the protein expression for CD14in the OB+CP group was significantly higher than that in the OB group (t=5.051, P=0.000).And the difference between the CP group and the C group was statistically significant (t=3.382, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the expression for CD14in the adipose tissue.ConclusionsObesity can induce adipocyte hypertrophy (increased size) and hyperplasia (increased number), enlarged stromal vascular fraction and macrophage infiltration in rat model. In contrast, experimental periodontitis can barely induce similar morphological changes in adipose tissue. However, periodontitis can result in slightly inflammatory disorder of adipose tissue, with characteristic of early enlarged stromal vascular fraction and several macrophage infiltrations. When combined with obesity, periodontitis can induce further hypertrophy and hyperplasia of adipocytes; furthermore, significant enlargement of stromal vascular fraction and macrophage infiltration can be observed in the OB+CP group. Based on the data, we accept that experimental periodontitis has a little impact on the morphological changes of adipose tissue but has remarkable effects on the inflammatory condition of adipose tissue. Periodontitis can exacerbate this inflammatory condition with the presence of obesity. Part threeThe Effect of Periodontitis on Protein Expression of Adipokines and the Relationships with Insulin ResistanceObjectiveTo investigate the impact of experimental periodontitis on the protein expression for leptin and adiponectin protein in adipose tissues and the concentrations in circulation; and further to explore the relationships between the expression of leptin and adiponectin locally and systemically and the systemic insulin resistance.MethodsImmunofluorescence analysis of leptin and adiponectin expression of adipose tissue in rat model was employed and semi-quantitative method was used. Concentrations of serum leptin, adiponectin and insulin were determined using enzyme-linked immunosorbent assay (ELISA) kit; glucose levels were measured by glucose oxidase method. IR was calculated using the homeostasis model assessment (HOMA-IR), where IR=In[(Fasting glucose[mmol/l]×fasting insulin[mU/l])/22.5]. Pearson’s correlation coefficient was used to determine the correlation between the levels of leptin and adiponectin and IR.ResultsIn the visceral fat tissue, immunofluorescence analysis showed that the protein expression for leptin in the OB+CP, CP and OB groups were significantly higher than in the C group (P<0.05). Furthermore, the expression for leptin in the OB+CP group was significantly higher than that in the OB group (t=2.046, P=0.044). And the difference between the CP group and the C group was statistically significant (t=5.237,P=0.000). In contrast, the protein expressions for adiponectin in the OB+CP, OB and CP groups were decreased. Weak protein expression was detected in the OB+CP group and was significantly lower than that in the OB group (t=-2.932, P=0.004). In the CP group, the adiponectin expression level was decreased relative to C group (t=-3.661,P=0.000)The mean levels of serum leptin in the OB+CP, OB and CP groups were significantly greater than that of the C group (P<0.01). Serum leptin in the OB+CP group was significantly higher than that in the OB (t=11.197, P=0.004). And serum leptin in the CP group was higher than in the C group (t=7.156, P=0.000). In adipose tissue, the expression for adiponectin was lower in the OB+CP group than that in the OB group (t=-2.932, P=0.004); and adiponectin in the CP group was also lower than that in the C group (t=-3.661, P=0.000). The circulating level of adiponectin in the OB+CP group was significantly higher than each of the three groups (P<0.01). In the OB+CP group, the levels of adiponectin were significantly higher than that in the OB group(t=3.624, P=0.003).However, adiponectin level was declined in the CP group, but there was no significant difference between CP group and C group (t=-1.323,P=0.207). A synergistic interaction between obesity and periodontitis was evident to the expression for leptin in the adipose tissue and the circulating levels of leptin and adiponectin.Fasting glucose level in the OB+CP group was significantly higher than that in the OB group (t=7.694,P=0.000),and glucose level in the CP group was also higher than that in C groups (t=3.369,.P=0.005). In contrast, there was no difference between OB+CP and OB groups in the insulin levels (t=0.658, P=0.521) and the insulin level in the CP group was higher than that in the C group (t=6.462, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the fasting glucose level (F=17.669, P=0.000).HOMA-IR analysis indicated that MSG-induced obesity was associated with an increase in IR in the OB and OB+CP groups compared to non-obesity rats. Further, HOMA-IR was significantly greater in OB+CP group compared to the OB group (t=5.662,P=0.000). In contrast, there was an increase in IR in the CP group, and the difference between CP group and C group was statistically significant (t=9.992, P=0.000). Significant positive relationships were observed between IR and leptin expression levels in adipose tissue (r=0.844, P=0.000), serum leptin (r=0.875, P=0.000) and adiponectin level (r=0.735, P=0.000). And negative relationship was observed between IR and adiponectin level in the adipose tissue (r=-0.886, P=0.000)ConclusionsExperimental periodontitis can affect expression for leptin and adiponectin protein directly in adipose tissues:upregulation of leptin and downregulation of adiponectin. When combined with systemic pathological changes such as obesity, periodontitis can exacerbate dysregulation of leptin and adiponectin in adipose tissue. Expremental periodontitis can also regulate circulating levels of leptin and adiponectin. When combined with obesity, periodontitis aggravates hyperleptinemia and hyperadiponectinemia and contribute to the development of IR. Part fourThe Effect of Periodontitis on Receptors Expression of Adipokines in Skeletal MuscleObjectiveTo invesitgate the protein expression for leptin receptor and adiponectin receptor1protein in skeletal muscle, with the aim of exploring the effect of experimental periodontitis on adipokines receptors in insulin-targeting tissues and underlying mechanisms of skeletal muscle insulin resistance.MethodsImmunohistochemical analysis of leptin receptor and adiponectin receptor1expression in skeletal muscle was employed and semi-quantitative method was used.ResultsIn the skeletal muscle, the protein expressions for leptin receptor in the OB+CP, OB and CP groups were decreased, but in the C group, the protein expression were popular and strong positive. Fewer protein expression was detected in the OB+CP group when compared with OB group (t=-3.490, P-0.001). In the CP group, the protein expression level were decreased relative to C group but not significant difference (t=0.272, P=0.786). In contrast, the expression for adiponectin receptor1was greatly lower than that in the OB group (t=-3.470, P=0.001); and the expression in the CP group was similar to the C group (t=0.543, P=0.598). A synergistic interaction between obesity and periodontitis was evident to the expression for the leptin receptor and adiponectin receptor1.ConclusionsChronic low-grade inflammation induced by experimental periodontitis can barely down-regulate the protein expression for leptin receptor and adiponectin receptor1in the skeletal muscle. However, periodontitis can significantly down-regulate the protein expressions with the presence of obesity. This may contribute to the binding disruption of these two adipokines and their receptors in skeletal muscle samples, and may further decrease the insulin sensitivity, which accelerate the onset and development of insulin resistance in the skeletal muscle.