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Abstract

Background:

This study aims to assess the levels of visfatin in children and adolescents with autoimmune thyroiditis (AIT) and its relationship with disease-related variables.

Methods

The study included 84 children and adolescents with AIT and 84 healthy subjects. Measurements of fasting glucose, insulin, thyroid-stimulating hormone (TSH), free thyroxin (FT4), antithyroid peroxidase antibodies (TPOAb) and antithyroglobulin antibody (TgAb) were completed, with assessment of visfatin levels.

Results:

Overt hypothyroidism was diagnosed in 58 cases, while subclinical hypothyroidism was diagnosed in 26 of the studied patients. Total studied cases, overt and subclinical subgroups had significantly higher levels of TSH, HOMA-IR, TPOAb, TgAb and visfatin and significantly lower FT4 level compared with the control. Spearman correlation analysis revealed that visfatin was correlated positively with BMI, homeostasis model assessment (HOMA-IR), TSH and TPOAb, while inversely correlation with FT4. In logistic regression analysis, visfatin was correlated only with TPOAb.

Conclusions:

This preliminary study provides evidence of significantly higher level of visfatin in children and adolescents with AIT. Visfatin might have a potential role in the pathogenesis of AIT, which needs to be validated by measuring immunological responses in children and adolescents with AIT.

Keywords

Visfatin Autoimmune thyroiditis Thyroid-stimulating hormone Overt hypothyroidism Subclinical hypothyroidism

Introduction

Autoimmune thyroiditis (AIT) is the most common cause of acquired thyroid dysfunction in children and adolescents, and has a wide spectrum of clinical manifestations and a variable clinical course.¹ Although both genetic and environmental factors have important roles in autoimmune thyroid disease, the underlying pathogenesis of these disorders remains unclear.² While scientists continue to investigate these causes, children and adolescents continue to suffer from AIT. To this end, an increased interest has developed in the connection between adipose tissue-secreted proteins that influence inflammation and the onset and perpetuation of autoimmunity.³ AIT is characterized by progressive thyroid cell destruction caused by cell apoptosis. The percentage of in situ apoptotic thyrocytes increases in AIT, suggesting that apoptosis plays an important role in function regulation and cell proliferation.⁴ Visfatin is an adipocytokine with suggested enzymatic, immunological and metabolic properties.⁵ Visfatin level has been found to be elevated in many chronic inflammatory autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases and psoriasis.⁶ Few studies evaluating visfatin in adults with AIT have been carried out in recent years; however, the available results are conflicting.^7,8 In view of the possible association of visfatin with inflammation and apoptosis, we therefore aimed to assess visfatin in Egyptian children and adolescents with AIT and its relationship with disease-related variables.

Patients and method

This case-control study included 84 children and adolescents with newly diagnosed AIT. Diagnosis of AIT had been made by elevated antithyroid peroxidase antibodies (TPOAb) and/or antithyroglobulin antibodies (TgAb), as well as typical hypoechogenicity of the thyroid in high-resolution sonography.⁹ Exclusion criteria consisted of other autoimmune diseases, diabetes mellitus, infection, which were reported to alter visfatin level, and cases of L thyroxine treatment. The study also included 84 healthy children and adolescents matched for age, gender, pubertal status and socioeconomic status as control subjects for statistical comparisons. Healthy controls had no goiter or clinical, laboratory evidence of thyroid disease or family history of any autoimmune disease. Patients were recruited during their regular follow-up appointments every 4–6 months in the outpatient endocrinology clinic of Assiut University Children Hospital, Assiut, Egypt. Control subjects were recruited from the general population. All participants were subjected to full medical histories and complete physical examination for signs of hypothyroidism and the presence of goiter was performed. Weight was measured to the nearest 0.1 kg on a standard beam scale, with the subject dressed only in light underwear and without shoes. Height was measured without shoes using a Harpenden stadiometer (Holtain Ltd., Crosswell, UK) to the nearest 0.1 cm. BMI was calculated using this formula: BMI = weight (kg)/height(m)². BMI was expressed as standard deviation scores (SDSs) using the Egyptian Growth Reference Data.¹⁰ Pubertal stage was assessed according to the Tanner criteria.¹¹

Laboratory analysis

Thyroid-stimulating hormone (TSH) serum level was determined by ultra-sensitive immunoassays (Immulite TM 2000 Third Generation, Diagnostic Products Corporation, Los Angeles, CA, USA). Free thyroxin (FT4) was determined by radioimmunoassay (RIA) using an automated system (Roche Diagnostics, Mannheim, Germany). The reference range for TSH was 0.4–4.0 mU/L, and for FT4 10.0–26.0 pmol/L. The coefficients of variation (CVs) were 5.0 and 5.1% at TSH concentrations of 4.0 and 10.0 mU/L, respectively and for FT4, the CV was 6.5% at 10.0 pmol/L.

Subclinical hypothyroidism is defined as a serum TSH concentration above the statistically defined upper limit of the reference range when serum free T4 concentration is within its reference range. Overt hypothyroidism was defined as an elevated TSH together with low free T4 levels.¹²

Serum antithyroid peroxidase antibodies (TPOAb) and antithyroglobulin (TgAb) were measured by rapid enzyme-linked immunosorbent assay (ELISA) (Genesis Diagnostics, Littleport, UK). TgAb and TPOAb concentrations more than 100 and 75 IU/mL, respectively, were considered positive. Positivity of at least one antibody was considered as having autoimmunity of the thyroid (AIT).

Visfatin plasma levels was measured with an ELISA assay kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The intra- and interassay CVs were <10%.

Glucose level was assessed with the use of Hitachi Cobas e601 chemiluminescent analyzer (Roche Diagnostics) and insulin concentration was assessed using an ELISA kit from Phoenix Pharmaceuticals. The estimate of insulin resistance by homeostasis model assessment (HOMA-IR) was calculated.

The study was approved by the ethics committee of the Children Hospital and Faculty of Medicine, Assiut University, and was performed in accordance with the standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from the legal guardians of participants before enrollment in the study.

Statistical analysis

Calculations were done with the statistical package SPSS for windows, version 16.0 (SPSS Inc., Chicago, IL, USA). Quantitative data were expressed as mean ± SD and qualitative data as absolute number (%). Student’s t test was used to compare means. ANOVA post-hoc test was applied for multiple comparisons. The Spearman correlation test was used to look for association between visfatin and different clinical and laboratory data. Variables that showed significant association with increased visfatin levels with Spearman analysis (p < 0.05) were used in a logistic regression model to determine their independent relation to visfatin. Results were given as odds ratio with 95% confidence interval. For all tests, values of p < 0.05 were considered statistically significant.

Results

The most relevant characteristics of total studied cases of AIT patients and subgroups (overt and subclinical hypothyroidism) are shown in Table 1. Total studied cases, overt and subclinical subgroups had significantly higher levels of TSH, HOMA-IR, TPOAb, TgAb and visfatin and significantly lower FT4 levels compared with the controls. Spearman correlation analysis revealed that visfatin was positively correlated with BMI SDS (r: 434, p = 0.05), HOMA-IR (r: 654, p = 0.001), TSH (r: 769, p = 0.001) and TPOAb (r: 498, p = 0.001), while inversely correlated with FT4 (r: 562, p = 0.001) (Table 2). Logistic regression analysis revealed that visfatin was correlated only with TPOAb (OR:4.1, CI: 3.2–8.7, p = 0.001) (Table 3).

Table 1.

Demographic, anthropometric and laboratory data of the studied cases and subgroups.

	Total cases (n = 84)	Overt hypothyroidism (n = 58)	Subclinical hypothyroidism (n = 26)	Control (n = 84)
Age	15.2 ± 2.7	14.5 ± 3.6	10.2 ± 2.8*	14.5 ± 2.2
Sex female/male	50/34	42/16	16/10	62/22
BMI, SDS	0.87 ± 0.67**	1.02 ± 0.81**	0.96 ± 0.62*	0.45 ± 1.4
TSH (μU/mL)	56.8 ± 7.6**	88.4 ± 17.4***	16.1 ± 5.5*	3.04 ± 1.1
FT4 (pmol/L)	7.2 ± 1.04*	5.2 ± 2.3**	18.3 ± 2.45	18.5 ± 3.55
Fasting glucose (mg/dl)	89.6 ± 9.2	79.2 ± 12.8	82.4 ± 10.3	84.6 ± 6.8
Fasting insulin (μIU/ml)	7.9 ± 6.1**	9.9 ± 2.1**	6.8 ± 4.7*	4.3 ± 2.7
HOMA-IR	1.74 ± 1.26*	2.44 ± 0.43**	1.52 ± 0.45*	0.89 ± 0.59
TPOAb (IU/mL)	546.5 ± 67.7**	765.6 ± 88.7***	348.7 ± 43.9*	16.7 ± 4.5
TgAb (IU/mL)	299.4 ± 32.2**	314.8 ± 54.1***	199.4 ± 23.8*	28.1 ± 14.6
Visfatin (ng/ml)	72.3 ± 12.5**	99.2 ± 23.1***	53.4 ± 16.6 *	13.4 ± 4.6

Data are expressed as mean ± SD. BMI, body mass index; FT4, free thyroxine; SDS, standard deviation score, HOMA-IR, the homeostasis model assessment of insulin resistance; TgAb, antithyroglobulin antibody; TPOAb, thyroid peroxidase antibody; TSH, thyroid-stimulating hormone

Significance versus control subjects: *p < 0.05; **p < 0.01; *** p < 0.001.

Table 2.

Spearman correlation between visfatin and various confounding variables in patients with AIT.

Confounding variables	Univariate analysis(r and p values)
Age	0.122, 0.245
BMI SDS	+0.434, 0.05*
HOMA-IR	+0.654,0.001**
TSH	+0.769,0.001**
FT4	-0.562,0.001**
TPOA b	+0.498,0.001**
TgAb	0.123,0439

BMI, body mass index; FT4, free thyroxine; HOMA-IR, the homeostasis model assessment of insulin resistance; TGAb, thyroglobulin antibody; TPOAb, thyroid peroxidase antibody; TSH, thyroid-stimulating hormone.

p < 0.05; ** p < 0.01.

Table 3.

Multiple logistic regression analysis between visfatin and various confounding variables in patients with AIT.

Risk factors	Multivariate analysis
Risk factors	Odd’s ratio	95% confidence interval	p-value
BMI SDS	0.786	0.769–1.018	0.395
HOMA-IR	0.558	0.345–1.647	0.352
TSH	0.777	0.765–1.168	0.455
TPOAb	4.1	3.2–8.7	0.001**
TgAb	0.639	0.432–1.34	0.436

BMI, body mass index; HOMA-IR, the homeostasis model assessment of insulin resistance; TGAb, thyroid globulin antibody; TPOAb, thyroid peroxidase antibody; TSH, thyroid-stimulating hormone.

p < 0.05; ** p < 0.01.

Discussion

In the present study, total studied cases, overt and subclinical subgroups had significantly higher levels of visfatin compared with the control (Table 1). No similar studies had been done in this age group as all previous studies have been conducted in adults. Caixas and colleagues⁷ reported elevated level of visfatin in hypothyroidism, with further increase after restoration of thyroid function. Ozkaya and colleagues observed that visfatin levels decreased after recovery.⁸ In those studies the etiology of hypothyroidism varied from chronic autoimmune thyroiditis and postpartum thyroiditis to thyroid function insufficiency after radioiodine treatment or after thyroidectomy. Autoimmune status of studied patients has not been taken into consideration. The controversial findings might result from the heterogeneity of study groups. Cytokines are involved in the pathogenesis of thyroid diseases working in both the immune system and directly targeting the thyroid follicular cells. They are involved in the induction and effector phase of the immune response and inflammation, playing a key role in the pathogenesis of AIT disease. Visfatin promotes the development of both T- and B-lymphocytes and stimulates leukocytes for production of pro-inflammatory cytokines such as IL-6, TNF-α and IL-1b.¹³ Its pro-inflammatory effect is also associated with the activation of T-cells by upregulation of co-stimulatory molecules (CD40, CD54, CD80) on monocytes.¹⁴ Cytokines are involved in the pathogenesis of thyroid diseases working in both the immune system and directly targeting the thyroid follicular cells. They are involved in the induction and effector phase of the immune response and inflammation, playing a key role in the pathogenesis of AIT disease. The presence of multiple cytokines has been demonstrated; IL-1alpha, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, TNF-alpha and IFN-gamma within the inflammatory cells and thyroid follicular cells. Finally, cytokines derived from T-cells can directly damage thyroid cells, leading to functional disorders and may also stimulate the production of nitric oxide and prostaglandin, thus increasing the inflammatory response in AIT.¹⁵ Unfortunately, the immunological markers of AIT were not studied, which is considered one of the limitations of the study. Visfatin plays an important role in the regulation of cell apoptosis and inflammation, and promotes cell survival in macrophages subjected to endoplasmic reticulum stress¹⁶ and confers to immune system cells the ability to survive during stresses such as inflammation.¹⁷ Apoptosis of thyrocytes is an important feature of AIT and is induced by the extrinsic pathway involving binding of the Fas cell-death receptor (APO-1/CD-95) ligand with the Fas receptor.¹⁸ Expression of Fas and Fas ligand are increased in thyrocytes during the active phase of AIT.¹⁹

In the present study, TPOAb, which is considered the best serological marker of AIT, was significantly higher in total studied cases, overt and subclinical subgroups compared with the control (Table 1). In addition, Spearman correlation analysis showed positive correlations between visfatin levels and TPOAb, which was confirmed by logistic regression analysis (Tables 2 and 3). Our results were in agreement with previous research by Sawicka-Gutaj and colleagues,⁶ who reported the same finding in adults with AIT. TPOAb and thyroglobulin-Ab are recognized by CD8-positive T-cells and through antibody- and complement-dependent cell-cytotoxicity in addition to T-cells and cytokine-mediated apoptosis, which might be involved in the pathogenesis of cAIT^6,20 and thyroid destruction process leading to clinical disease manifestations.²¹ This makes it an attractive target for novel therapeutic strategies in AIT.²¹

In the present study, total studied cases, overt and subclinical subgroups had significantly higher BMI SDS compared with the control (Table 1). In addition, a significant correlation was observed between visfatin and BMI SDS. In contrast, Berndt and colleagues²² reported that visfatin gene expression in visceral adipose tissue (adipose tissue macrophages) is correlated with plasma visfatin levels but not with BMI. In these cases, plasma visfatin concentration seems to be a marker for visceral fat accumulation in obese children. Visfatin binds to insulin receptors directly and phosphorylates IRS-1/-2 and Akt by that pathway, thereby exerting insulin-like action. Moreover, in the original study of Fukuhara and colleagues,²³ visfatin was identified as secreted from visceral adipocytes and plasma visfatin levels were found to be significantly correlated with visceral fat tissue in adults. The results concerning visfatin upregulation in obesity are contradictory.^24,25 Most previous studies have reported elevated visfatin concentrations in obesity,²⁵ though Pagano and colleagues²⁶ have reported decreased plasma visfatin and its messenger RNA in subcutaneous adipose tissue of obese subjects. In fact, obesity is a low-grade chronic inflammatory disease associated with an increased number of macrophages. Thus, adipose tissue macrophages are the major source of visfatin, but not the adipocytes.²⁷

In the present study, HOMA-IR was higher in AIT than controls, which is more obvious with overt hypothyroidism compared with subclinical hypothyroidism. Significant positive correlation was reported between visfatin and HOMA-IR, which suggests the detrimental effect of high visfatin levels in the development of insulin resistance in patients with AIT. Other researchers reported an increase in serum visfatin concentration in different insulin-resistant conditions, such as type 2 diabetes²⁸ and obesity,²⁵ although opposing observations were also reported.²⁶ The correlation observed in our study indicates a possibility that in insulin resistance conditions, visfatin cannot exert its potential beneficial metabolic actions or its increase is a secondary event in order to prevent further development of insulin resistance. On the other hand, insulin was shown to inhibit visfatin release from adipocytes,²⁹ so the observed correlation might simply reflect insulin’s inability to suppress visfatin production in the event of insulin resistance. However, this issue cannot be resolved on the basis of our cross-sectional study.

We also reported that visfatin plasma levels were significantly higher in the subclinical subgroup compared with the control group, correlated positively with TSH and negatively with FT4 and FT3. This is in agreement with Ozkaya and colleagues,⁸ who reported the same findings. This result may add to the possibility that visfatin is involved in the pathogenesis of AIT, but due to the cross-sectional design of this study it cannot describe the longitudinal effect of visfatin. However, controversial results regarding whether thyroid hormones stimulate or downregulate the production of visfatin were found. Ozkaya and colleagues⁸ showed a significant negative correlation between visfatin and FT3. Caixas and colleagues did not find any relationship between visfatin and free thyroid hormones.⁷ Regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis. Thus, visfatin has been recognized as a cytokine with a broad range of immune and inflammatory activities, including induction of inflammatory cytokines and regulation of macrophage and lymphocyte proliferation.²⁷ Changes in visfatin secretion with AIT hypothyroidism may also represent adaptive mechanisms for the decrease or increase in basal energy expenditure and in energy substrate requirements in hypothyroidism. Visfatin secretion imbalance may be involved in the interactions between thyroid hormones and visfatin. Moreover, hypothyroidism could affect the clearance of visfatin.²⁹

This study has some limitations. First: the cross-sectional analysis of this study precludes any temporal or cause–effect conclusions. Second: immunological markers that may be involved in the pathogenesis of AIT were not studied. However, we think that it is of interest for providing the first data regarding the visfatin level in children with AIT. In conclusion, this preliminary study provides evidence of significantly higher levels of visfatin in children and adolescents with AIT. Visfatin might have a potential role in the pathogenesis AIT, which needs to be validated by measuring immunological responses in children and adolescents with AIT.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

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