The role of visfatin levels in gingival crevicular fluid as a potential biomarker in the relationship between obesity and periodontal disease

Abstract Objectives Visfatin is an adipokine that plays an important role in immune functions as a growth factor, enzyme, and pro-inflammatory mediator. We aimed to determine the levels of visfatin, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in gingival crevicular fluid (GCF) in both obese/non-obese patients, with/without generalized chronic periodontitis (GCP). Methodology Patients were categorized as obese (O) (n=31) or non-obese (nO) (n=19). Groups were divided into four subgroups according to periodontal conditions: (1) periodontally healthy without obesity (nO-Ctrl); (2) GCP without obesity (nO-CP); (3) periodontally healthy with obesity (O-Ctrl); and (4) GCP with obesity (O-CP). Demographic variables, anthropometric and laboratory data were recorded. Periodontal parameters were measured at baseline and 3rd months after either non-surgical periodontal treatment or calorie -restricted diet therapy. At the same time, GCF samples were taken from patients to analyze TNF-alpha, IL-6,and visfatin levels. Results Periodontal parameters were significantly higher in the O group than in the nO group (P<0.05). IL-6 levels were higher in the O group than in the nO group (P<0.001). The visfatin levels of the obese patients were reduceddecreased following the treatments (P<0.05). Cholesterol levels were higher in the O group than in the nO groups (P<0.05). IL-6 levels were higher in O-CP and O-Ctrl groups than in the nO-Ctrl group (P<0.05). Compared to the other groups, visfatin levels were significantly higher in the O-CP group but decreased following treatment (P<0.05). Conclusions Our findings suggest that visfatin and IL-6 levels in GCF are associated with the pathogenesis of obesity and periodontitis. Within the limits of this study, we considered that there might be an association between the lipid profile and periodontitis on systemically healthy individuals.


Introduction
Overweight and obesity are defined as the accumulation of fat in body tissues that might impair overall health. 1 Adults are considered overweight if their body mass index [BMI, calculated as (weight in kg)/(height in meters) 2 ] is ≥25 and obese if BMI≥30 kg/m 2 . The prevalence of overweight and obesity has increased worldwide during recent decades. 1 Obesity is usually related to a chronic low-grade systemic inflammation resulting in significant changes in the concentrations of cytokines and hormones that subsequently leads to the development of obesitylinked disorders, including insulin resistance, type 2 diabetes, cardiovascular diseases, dyslipidemia, and metabolic syndrome. 1 Since the host response is among the most crucial factors affecting the pathogenesis of periodontal disease, multiple studies have addressed the possible associations between BMI, overweight, obesity, diabetes, the serum level of lipids, cholesterol, and periodontal breakdown, with mixed results. 1,2 Many studies have demonstrated a positive association between obesity and periodontitis and suggested that obesity-related inflammation might promote periodontitis by secretion of inflammatory markers by the adipose tissue, which might subsequently increase gingival inflammation. 2,3 The association between obesity and periodontal disease is based on the amassing of white adipose tissue (WAT) and increased secretion levels of adipokines from WAT.
WAT is an energy storage organ with some metabolic activities, participating in the endocrine and secretory systems. 4 It secretes several immune-modulator adipokine molecules, such as adiponectin, leptin, visfatin, resistin, chemerin, tumor necrosis factoralpha (TNF-α), interleukin-1β (IL-1β), and IL-6. 4 It has been found that these molecules are involved in a wide range of physiologic and pathological processes, including immunity and inflammation. 5 Thus, cytokines and hormones released from adipose tissue might play a role in the destruction of periodontal tissue by inducing hyper-inflammatory responses.
Visfatin is identified as a new adipokine, which is involved in the early development of the B-cell growth factor and cytokine-like effects. 6 Visfatin is a 52-kDa protein that increases pre-B-cell colony release from lymphocytes. 6 It is a multi-potential mediator that functions as a growth factor, cytokine, an enzyme with a role in energy metabolism, and as a pro-inflammatory mediator. 6 Visfatin is mainly released from adipose tissue, especially by macrophages, and can also be released from lymphocytes, dendritic, muscle, periodontal ligament and bone marrow cells. 7,8 Visfatin has an important role in the regulation of immune response. Visfatin inhibits neutrophil apoptosis during infection and inflammation, and increases TNF-α, IL-1β, and IL-6 levels. 6 Visfatin is also known as nicotinamide phosphoribosyltransferase. 6 The expression of visfatin is increased under inflammatory conditions, such as rheumatoid arthritis, cardiovascular diseases, type 2 diabetes mellitus, and periodontal disease. 9 Several studies have investigated the link between periodontitis and obesity, and these investigations have also shown that obesity and periodontitis are also related to gingival crevicular fluid (GCF) levels of adipokines. [2][3][4]9 Increased adipocyte levels, such as from visfatin, cause secretion of cytokines, which are known to play an important role in periodontitis and might trigger periodontitis formation and development. 9 Therefore, the main objective of this study was to analyze the levels of visfatin, IL-6, and TNF-α in obese and non-obese individuals, with or without generalized chronic periodontitis (GCP). Secondarily, we aimed to evaluate metabolic and clinical periodontal parameters, as well as to clarify the relationship between these parameters and adipocytokines.    The assay was performed using recombinant human standards according to the manufacturer's instructions.
Optical density was measured at 450 to 490 nm. GCF results were reported as the total amount (pg/ml).         10 (2005) reported that women have a higher prevalence of body fat than men, regardless of age, from puberty onwards. Nevertheless, Saxlin, et al. 11 (2011) suggest that gender does not influence on the association between obesity and periodontitis. Based on the current study design, it is not possible to explain the influence of gender on the association between obesity and the development of periodontitis. We suggest that biological and hormonal changes may play a role in this difference between genders. It is known that female patients are influenced by hormonal changes in the process from puberty to menopause. This may be explained by the fact that the female patients of this study have more than one pregnancy, their daily activities are limited and they are not active in their working life. One of the other reasons is that many of our socio-cultural traditions are easily able to over-eat high-calorie foods and promote more carbohydrate intake. The fact that the total cholesterol, HDL, LDL, and triglyceride values we obtained in our study did not differ between the groups supports this view. With age, the percentage of body fat increases for both men and women. In addition, the obese patients were older than the non-obese participants in this study. As mentioned earlier, obesity and aging are associated with periodontitis. 12  were higher in obese than non-obese subjects. 13,15,16 In the present study, PI, GI, PD, CAL, and BOP Additionally, Altay, et al. 16 (2013) demonstrated that obesity increased PI compared to non-obese subjects, but there was no significant difference in GI, PD, and BOP values. 16 In contrast, Gursoy, et al. 14 (2006) and Zuza, et al. 17  Adipocytes cause hyperinflammatory responses by over expressing adipokines and adipocytokines 9 .
Accordingly, it has been proposed that the increased amount of adipose tissue in obese individuals triggers the development of periodontitis.
Visfatin is thought to have insulin-like effects, particularly those that lower plasma glucose levels. 7,18 Also, it has been suggested that visfatin release in fat cells is independently associated with obesity and metabolic syndrome. 8,18 The abundance of adipose tissue in these patients is responsible for elevated levels of visfatin. 18,19 In our study, the mean GCF visfatin level was 11.71 pg in the obese group and 8.81 pg in the nO group. However, this difference was not statistically significant. The authors reported that the level of visfatin was higher in obese women than in non-obese participants. 20 The same study also reported a correlation with plasma visfatin and body weight reduction using an exercise program. 20 Similarly, in the present study, the total amount of visfatin in to systemically healthy individuals with periodontal disease. 8 Mohamed, et al. 22 (2015) reported a positive correlation between PD and visfatin levels in GCF, and they found that visfatin increased significantly the number of diseased sites. Türer, et al. 23 (2015) showed that GCF and serum visfatin levels are higher in patients with chronic periodontitis than those with gingivitis and healthy controls. They also reported that visfatin levels were significantly decreased following periodontal treatment. Ghallab, et al. 21 (2015) detected significantly higher visfatin mRNA and protein expression in patients with CP+T2DM compared to controls, which is in agreement with a previous study.
It has been reported that salivary visfatin levels increase in patients with periodontal disease and that there is a relationship between salivary visfatin concentration and periodontal infection. 24  This study has some limitations. First, in calculating the sample size, the low number of participants should be sufficient to reach significant results, but it is not possible to define clinically important differences in all parameters. Therefore, multicenter studies with larger populations are needed to validate the present study findings. Secondly, most obese participants included in this clinical trial were moderately obese. In addition, depth of PD and CAL were not categorized due to of sample size. Future interventional studies are needed to define the levels of adipocytokines in more severe cases of obesity (obese class-III), including those with and without periodontitis, and categorized according to PD and CAL depth (e.g., shallow, mild to moderate, and deep).

Conclusion
In conclusion, the results of this study suggest that visfatin and IL-6 levels might play a role in the pathogenesis of obesity and periodontal disease and that these can be used as reliable markers for monitoring the progress of both diseases. Moreover, our findings demonstrate that, in addition to obesity, lipid profiles can be associated with periodontitis in systemically healthy people.