1. Retinol Binding Protein-4 levels in thyroid overt and subclinical dysfunction Kokkinos Spiridon, Tiaka Elisavet, Manolakis Anastasios, Papanas Nikolaos, Papatheodorou Konstatntinos, Christakidou Paraskevi, Maltezos Efstatios, Papazoglou Dimitrios Second Department of Internal Medicine, University General Hospital of Evros, Alexandroupolis, Greece Medical School of Democritus University of Thrace, Alexandroupolis, Greece

2. Background Adipokines : –are a variety of active biological substances –are secreted by adipose tissue –act in an autocrine, paracrine and endocrine manner –have been implicated in appetite control, thermogenesis, thyroid and reproductive functions Thyroid dysfunction : – is accompanied by changes in intermediary metabolism (alterations in body weight, insulin resistance and lipid profile) –has an impact on liver, muscle and adipose tissue metabolism as a consequence of the cellular effect of thyroid hormones

3. Retinol binding protein-4 (RBP4) : –is a novel adipokine –is expressed and secreted by adipose tissue and liver –induces insulin resistance Elevated RBP4 values are reported in subjects with insulin resistance states (obesity, type 2 diabetes, metabolic syndrome, cardiovascular disease, non-alcoholic fatty liver disease) Circulating RBP4 decreases during medical interventions that result in amelioration of the metabolic profile.

4. Aim Examination of serum RBP4 levels in clinical and subclinical hyperthyroid and hypothyroid subjects as compared to controls. Examination of serum RBP4 concentrations in clinical forms of thyroid dysfunction before therapy and after normalization of thyroid hormone concentrations with appropriate therapy.

5. Subjects All subjects were ambulatory and were studied as outpatients during visits to the Outpatient Clinic of the Second Department of Internal Medicine. 150 patients: –overt hyperthyroidism (n=38) –subclinical hyperthyroidism (n=27) –overt hypothyroidism (n=32) –subclinical hypothyroidism (n=53) 28 euthyroid individuals (control group)  Hyperthyroidism  Graves’ disease  toxic adenoma  toxic multinodular goitre  Hypothyroidism  chronic autoimmune (Hashimoto’s) thyroiditis  postoperative hypothyroidism  postradiation hypothyroidism  idiopathic hypothyroidism Exclusion criteria: –heart, liver or renal disease, known history of diabetes or any acute or chronic infectious disease. –medication known to affect glucose, lipid or hormone levels.

6. Study design All patients were evaluated at the time of diagnosis. Patients with clinical thyroid dysfunction were evaluated again after normalization of thyroid function following appropriate therapy. Healthy subjects were tested once. Evaluation: –detailed clinical history –complete physical examination –Body composition [Skylark Body Fat analyzer (model BT-905), Gima, Italy] –measurement of routine biochemical and haematological parameters –Serum concentrations of FT4, FT3, TSH, insulin and RBP-4, thyroid peroxidise autoantibodies, thyroid-stimulating immunoglobulins

7. Hormone and adipokines assays Serum RBP4 levels were measured using a commercial ELISA kit (RnD Systems Europe, Ltd., Abingdon, UK). Hormones (TSH, FT4, FT3 and insulin) were measured by RIA. Subclinical hyperthyroidism and hypothyroidism were defined as a TSH level less than 0.3 or higher than 4mIU/L respectively, with normal FT4 and FT3 levels. The homeostasis model insulin resistance index (HOMA-IR) was calculated using fasting insulin and glucose concentrations.

8. Statistical analysis Results are given as mean±SD. Mean RBP-4 levels between the five groups were compared using one-way ANOVA with Bonferroni's post-hoc test. For paired data, statistical analysis before and after therapy were carried out with the paired Student’s t-test. Correlations between variables were assessed using Pearson’s correlation analysis. All statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL). P<0.05 was considered statistically significant.

9. Results The main clinical, biochemical and hormonal data of the patients and controls are summarized in the following table HyperthyroidismSubclinical Hyperthyroidism HypothyroidismSubclinical Hypothyroidids m Controls N3827325328 Gender (m, %)8 (21.0%)8 (29.6%)7 (21.8%)13 (24.5%)8 (28.5%) Age (years)36.5 ± 8.939.9 ± 13.241.0 ± 12.943.2 ±6.137.0 ± 10.2 BMI26.7 ± 6.031.2 ± 6.531.3 ± 6.029.9 ± 6.027.9 ± 4.2 Fat mass (%)30.5 ± 9.738.6 ± 5.835.2 ± 7.135.7 ± 9.133.2 ± 8.0 Glucose (mg/dl)96.0 ± 15.388.5 ± 13.793.8 ±16.689.4 ± 15.189.7 ± 14.4 CRP (mg/l)0.41 ± 0.450.29 ± 0.270.55 ± 0.820.32 ± 0.280.33 ± 0.37 Insulin (mU/l)12.5 ± 3.510.2 ± 4.613.2 ± 3.911.9 ± 6.59.8 ± 2.9 HOMA index2.6 ±1.72.5 ± 1.13.0 ± 1.52.5 ±1.52.3 ± 1.1 FT4 (pg/ml)32.8 ± 8.015.4 ± 2.94.8 ± 2.810.3 ± 2.212.5 ± 2.9 FT3 (pg/ml)8.3 ± 1.94.2 ± 0.62.8 ± 1.03.9 ± 0.73.9 ± 0.6 TSH (mU/l)0.052 ± 0.0530.13 ± 0.1027.70 ± 17.2112.75 ± 11.131.73 ± 0.95 RBP-4 (ng/ml)29.0 ± 10.225.1 ± 12.638.8 ± 16.531.9 ± 13.220.4 ± 8.2

10. RBP-4 baseline levels between groups The mean levels of RBP-4 were significantly higher in patients with clinical hypothyroidism than those in the four other groups. RBP-4 levels tended to be also higher in the three other groups of patients in comparison with controls but these differences did not reach statistical significance.

11. RBP-4 levels in the five groups before (plain) and on (shaded) therapy

12. RBP-4 levels before and on therapy Although RBP-4 levels fell significantly in both groups with clinical thyroid disease after normalization of thyroid hormone levels, still remained higher than those in the control group. RBP-4 levels fell from 29.0±10.2 to 24.9±8.4 ng/ml (p=0.003) in patients with clinical hyperthyroidism and from 38.8±16.5 to 29.0±10.8 ng/ml (p=0.001) in patients with clinical hypothyroidism.

13. RBP-4 levels in the five groups before (plain) and on (shaded) therapy

14. RBP-4 Hyperthyroidism r (p) Hypothyroidism r (p) BMI-0.11 (0.50)0.12 (0.42) Fat mass0.14 (0.41)0.07 (0.57 CRP0.13 (0.46)0.20 (0.82) LDL0.17 (0.32)0.15 (0.24) HDL0.01 (0.97)0.32 (0.06) Triglycerides0.02 (0.91)0.17 (0.18) HOMA0.16 (0.46) 0.21 (0.03) FT40.18 (0.32)-0.22 (0.87) FT3-0.01 (0.95)-0.06 (0.61) TSH-0.05 (0.76) 0.5 (<0.001) Correlation between anthropometric, biochemical and hormonal parameters with RBP-4 (A) and adiponectin (B) levels in the hyper- and hypothyroidism groups RBP-4: Correlation analysis

15. Correlation of RBP4 levels with TSH in the hypothyroid group (clinical and subclinical)

16. Summary patients with clinical hypothyroidism have high serum levels of RBP-4 the normalization of thyroid hormone levels with appropriate treatment in both clinical hypo- and hyperthyroidism results in a statistically significant reduction of RBP4 concentration Plasma RBP4 levels are positively correlated with TSH and HOMA index in hypothyroid patients.

17. These findings suggest that RBP-4 may be implicated in insulin resistance development and associated with increased cardiometabolic risk in hypothyroid patients Future studies should elucidate :  the mechanism through which RBP-4 levels are increased in subjects with hypothyroidism  whether increased RBP-4 levels are a cause or a result of hypothyroidism.