F. Cecoli 1, G. Andraghetti 1, C. Ghiara 2, L. Briatore 1, D. Cavallero 1,M.Mussap 2,F.Minuto 1, and M. Giusti 1

Transcription

1 J. Endocrinol. Invest. 31: , 28 Absence of thyrotropin-induced increase in leptin levels in patients with history of differentiated thyroid carcinoma undergoing recombinant human thyrotropin testing* F. Cecoli 1, G. Andraghetti 1, C. Ghiara 2, L. Briatore 1, D. Cavallero 1,M.Mussap 2,F.Minuto 1, and M. Giusti 1 1Department of Endocrinological and Metabolic Sciences; 2 Department of Laboratory Medicine, San Martino Hospital and University of Genoa, Genoa, Italy ABSTRACT. Background: Some extra-thyroid effects of TSH have been described in vitro and in vivo. TSH has recently been suggested to induce interleukin-6 secretion by adipocytes. Leptin is the main protein secreted by adipose tissue. Objective: The aim of our study was to evaluate the acute effect of the recombinant human TSH (rhtsh)-induced TSH surge on serum leptin levels in thyroidectomized patients undergoing levothyroxine (L-T 4 ) suppressive therapy for differentiated thyroid carcinoma (DTC). Design: A cohort of 15 female DTC patients was evaluated. Standard rhtsh testing was performed. Leptin, TSH, thyroid hormones, and thyroglobulin were measured before and 3, 6, and 9 days after rhtsh testing. Some metabolic parameters were also evaluated at the baseline. Results: Baseline leptin levels were 12.2±3.2 μg/l. Only body mass index (BMI) correlated significantly (p<.5) with leptin levels. After rhtsh administration, TSH levels increased significantly (p<.1), while thyroid hormones remained unchanged. Twenty hours after the last rhtsh administration, leptin (11.8±3. μg/l) levels were unchanged. The maximal TSH level was negatively related with BMI (p<.5), but no correlation between maximal TSH and leptin levels after rhtsh was noted. Conclusions: Our in vivo experimental model suggests that an acute TSH increase after rhtsh testing is ineffective in changing circulating leptin levels. (J. Endocrinol. Invest. 31: , 28) 28, Editrice Kurtis *The present paper was presented in part at the 9 th European Congress of Endocrinology, Budapest, Hungary, 28 th April- 2 nd May, 27. Key-words: Differentiated thyroid carcinoma, leptin, rhtsh. Correspondence: M. Giusti, MD, Dipartimento di Scienze Endocrinologiche e Metaboliche. UO Clinica Endocrinologica. Ospedale-Università San Martino Genova, Viale Benedetto XV, 6 I-161 Genoa, Italy. magius@unige.it Accepted February 3, 28. INTRODUCTION Thyroid hormones, as well as leptin, are considered important signals of energy balance (1). Since alterations in thyroid function are associated with changes in body weight and energy expenditure, much research has been devoted to studying the mutual roles of thyroid hormones and leptin in this respect (2). Reduced body weight has been associated with a significant decrease in serum levels of leptin and thyroid hormones. In one study, this effect was reversed by the administration of recombinant leptin, but no significant changes in TSH levels were observed (3). Another study, however, has shown that high TSH and free T 3 (f-t 3 ) levels are reduced after body-weight loss induced by calorie restriction in obese women (4). Moreover, a positive correlation has been observed between fluctuations in 24-h TSH release and the decline of circulating leptin (4). Iacobellis et al. (5) found a positive correlation between TSH and leptin adjusted for body mass index (BMI) in euthyroid obese women; they therefore suggested that TSH may be a marker of energy balance in this kind of subject. However, another study showed that in humans with morbid obesity there was a high prevalence of subclinical hypothyroidism, but that TSH was not related to serum leptin levels (6). Contrasting results have been observed with regard to TSH and leptin in diffuse toxic goiter before and after euthyroidism induced by pharmacological treatment. A positive correlation between TSH and leptin, regardless of the state of thyroid function, was found by one group (7), but not by another (8). Both hyperthyroid and hypothyroid patients have shown low serum leptin levels, but only in the latter has an increase been observed after therapy (9). Another study demonstrated that serum leptin levels were correlated with BMI and TSH in both hypothyroid and hyperthyroid patients, and concluded that serum leptin levels were affected in thyroid disorders and that the correlation of leptin with TSH was independent of thyroid hormones (1). These findings contrast with other results, which suggested that there was no correlation between leptin and thyroid hormones in hypothyroid males or in euthyroid lean and obese control subjects (11). Some effects of TSH on adipose tissue have been described in vitro and in vivo. TSH seems to stimulate leptin secretion through a direct effect on adipocytes in vitro (12). In a recent paper, Antunes et al. (13) showed that interleukin-6 (IL-6) release from human abdominal adipose cells is regulated by TSH: the stage of differentiation and the origin of the fat depot affect basal and TSH-stimulated IL-6 release from adipose cells in culture. Furthermore, rhtsh elevates serum IL-6 response in thyroidectomized patients, indicating an extrathyroidal site of TSH action in vivo (13). Although the link between leptin and the thyroid axis has been extensively studied, there are conflicting data on the possible correlation between serum leptin levels and TSH. TSH may exert a critical role in the differentiation and fat depot of human adipose tissue (14) by 888

2 Leptin after rhtsh in DTC patients interacting with specific receptors expressed in adipocytes (14). The acute increase in serum TSH levels produced by recombinant human TSH (rhtsh) administration can be exploited in an in vivo model to evaluate the huge effect exerted by TSH on serum leptin levels. Our study was designed to further evaluate the acute effect of rhtsh on serum leptin levels in a well-characterized group of differentiated thyroid carcinoma (DTC) patients and to extend the evaluation of other clinical and laboratory parameters of lipid metabolism. To our knowledge, this is the first study in humans on this topic. MATERIALS AND METHODS Subjects The study was conducted during scheduled yearly rhtsh testing of 15 female outpatients (mean age±sd: 49.2±12.6 yr) with a history of stage 1-3 DTC. Some clinical data of the subjects studied are reported in Table 1. Patients were divided into premenopausal (no.=8; yr) and post-menopausal (no.=7; yr) females on the basis of their gonadal status. Total thyroid ablation by near-total thyroidectomy and subsequent radioiodine therapy was our standard treatment for all subjects. Histology revealed papillary cancer in 13 cases; 1 patient had follicular variant of papillary cancer and 1 had follicular cancer. All patients were on an levothyroxine (L-T 4 ) regimen at the time of examination. Therapy was TSH-suppressive in the majority of patients. In 4 patients (cases no. 4, 5, 8, 14) rhtsh testing was performed 2 months after their scheduled L-T 4 regimen had been reduced by about 25%, in accordance with our protocol of sensitizing the thyroid to rhtsh administration (15). Case no. 9 did not show suppressed TSH despite receiving elevated L-T 4 dosages, probably because of inadequate absorption of the drug (Table 1). Protocol All patients underwent a clinical examination comprising pharmacological history, BMI evaluation, neck palpation, and ultrasonography. Biochemical evaluation, which was performed in the fasting condition in the morning after 12-h abstinence from smoking, comprised serum leptin, free-thyroid hormones, TSH and thyroglobulin (Tg) measurement. Samples were collected before and 3, 6, and 9 days after rhtsh testing. Patients received two consecutive daily (days 1 and 2) im doses of rhtsh (.9; Genzyme Co., Cambridge, MA, USA). Baseline (day ) serum total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), triglycerides (TG), insulin concentration and glucose concentration were also evaluated in fasting patients. Insulin sensitivity was estimated in accordance with the homeostasis model assessment for insulin resistance (HOMA-IR). Anti-Tg autoantibody was evaluated to identify sera in which a Tg-recovery test was necessary to obtain reliable Tg values (16). Serum samples were frozen at 7 C before assays, which were performed within 6 months of collection. Assays BMI was calculated as weight (in kg) divided by height squared (m 2 ). In accordance with international guidelines, all subjects with a BMI above 3 kg/m 2 were classified as obese. Serum leptin was assayed by immunoreactivity by means of a commercially available radioimmunoassay kit (DRG Instruments GmbH, Germany). The normal range is μu/l for females with a normal BMI (18-25 kg/m 2 ). In our laboratory, the intra-assay coefficient of variation (CV) was 4%. Serum Tg was assayed by chemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). Assays were standardized against the certified reference for human Tg (CRM 457) of the Community Bureau of References of the European Commission. The lower analytic limit and functional sensitivity of the Table 1 - Some clinical and serum biochemical data from the differentiated thyroid carcinoma patients studied at the baseline time of recombinant human TSH testing. Case Age BMI L-T 4 TSH HOMA-IR HDL-C LDL-C TG No. (yr) (kg/m 2 ) (μg/week) (miu/l) Ratio (mmol/l) (mmol/l) (mmol/l) Pre-menopausal Post-menopausal n.a. n.a. n.a BMI: body mass index; L-T 4 : levothyroxine; HOMA-IR: homeostasis model assessment for insulin resistance; HDL-C: HDL cholesterol; LDL-C: LDL cholesterol; TG: triglycerides; n.a.: not available. 889

3 F. Cecoli, G. Andraghetti, C. Ghiara, et al. method are.1 μg/l and.5 μg/l, respectively. In our laboratory the intra-assay CV were 5% and 8%. On the basis of the functional sensitivity of the methods, we selected.5 μg/l as the cut-off value to discriminate undetectable from detectable Tg levels. Tg-antibodies were measured by commercial assay (Dia Sorin, Saluggia, Italy). A concentration of 1 miu/l IgG to Tg was taken as the cut-off value. Serum free-thyroid hormones and TSH were measured by ultra-sensitive chemiluminescence immunoassay (Roche Diagnostics). Normal ranges are miu/l for TSH, pmol/l and pmol/l for f-t 3 and free-t 4 (f-t 4 ), respectively. Serum glucose concentration was measured by an enzymatic method (normal values: mmol/l) on an automatic analyzer (Modular P8 Roche Diagnostics, Mannheim, Germany) and insulin concentration by a standard sandwich enzyme immunoassay (Immunotech, Prague, Czech Republic). The HOMA index was calculated as fasting insulin concentration (μu/ml) fasting glucose concentration (mmol/l)/22.5 (17). Serum TC, HDL-C, LDL-C and TG were evaluated by means of an enzymatic method on an automatic analyzer (Modular P8 Roche Diagnostics). In our laboratory, the normal ranges are mmol/l for TC, mmol/l for HDL-C, mmol/l for LDL-C, and mmol/l for TG. Statistical analysis Data were analysed by means of the Prism 4. software (Graph- Pad Software, San Diego CA, USA). The leptin, TSH, f-t 3, f-t 4, and Tg changes under rhtsh testing were analysed by means of the non-parametric Kruskal-Wallis test, followed when appropriate by Dunn s multiple comparison test. Baseline data were compared with those of the reference population by Mann-Whitney test. Correlation analyses between variables were carried out by Spearman correlation. All values quoted are means±sem. Data below the functional sensitivity of the assay were analysed for statistical purposes by means of the functional sensitivity value. Exact p values ranging from.2 to.1 are reported. Significance was taken as p<.5. At least 1 yr after primary therapies, the best predictor of cure in DTC patients is considered to be an undetectable Tg (<.5 μg/l) level after rhtsh testing, combined with negative neck ultrasonography (18, 19). RESULTS L-T 4 regimen ranged from 525 to 122 μg/week (892.1±52.3 μg/week). Free-T 3 was in the normal range in all subjects; f-t 4 levels ranged from 1.5 to 24 pg/ml. Under L-T 4, TSH ranged from undetectable (<.3 mu/l) to 5.96 mu/l (median.4 mu/l; 1.22±.47 mu/l). The inverse correlation between L-T 4 dosage and TSH suppression was not significant (rs.28; p=.3). Undetectable baseline Tg levels were found in all but one patient (Table 1, case no. 3). According to BMI data, 4 patients were obese (BMI>3 kg/m 2 ) (Table 1, cases no. 5, 9, 1, 15). Three patients presented a high HOMA-IR (>4) (Table 1, cases no. 1, 14, 15). LDL-C levels were above 3.36 mmol/l in 5% of patients. Baseline leptin levels were 12.2±3.2 μg/l. No correlation between age and BMI, HOMA-IR or lipid parameters was found. A significant (p<.5) correlation between BMI and baseline leptin levels was observed, but no correlation between BMI and HOMA-IR or lipid parameters was found. Free-T 3 and f- T 4 concentrations were unaffected by rhtsh administration (data not reported). A significant (p<.1) increase in serum TSH following rhtsh administration was observed on day 3 (151.3±5.7 mu/l) and day 6 (1.3±1.3 mu/l), but these values returned to baseline levels by day 9 (2.7±.6 mu/l). According to Tg-stimulated levels (<.5 μg/l) and neck sonography (negative), all but two patients were considered disease-free. Two patients (Table 1, cases no. 3 and 7) were considered partially ablated after post-surgical radioiodine therapy. A significant negative correlation was observed between maximal TSH level and BMI (p<.5) (Fig. 1). Figure 2 reports the behavior of leptin after rhtsh administration. Mean leptin levels did not significantly change during rhtsh administration, and were 11.8±3. μg/l 24 h after the last rhtsh administration, 12.5±3.5 μg/l on day 6 and 12.5±3.4 on day 9 (Fig. 2). No correlation was observed between the peak value of TSH on day 3 and the maximal percentage change in leptin (11.81±2.99%) after rhtsh administration. A Leptin (μg/l) B TSH (mu/l) no.=15, rs.69, p= BMI kg/m 2 no.=15, rs.55, p= BMI kg/m 2 Fig. 1 - A) the significant (p<.5) positive correlation between body mass index (BMI) and baseline leptin levels. B) the significant (p<.5) negative correlation between maximal TSH level and BMI. 89

4 Leptin after rhtsh in DTC patients DISCUSSION In recent years, it has been shown that, as well as thyrocytes, many other tissues, including lymphocytes, fibroblasts, the pituitary, thymus, testes, kidney, and brain tissue express TSH receptors (TSHR). Moreover, some extra-thyroid effects of TSH have been described both in vitro and in vivo: in vivo, the stimulation of thyroid angiogenesis (2) and the production of nitric oxide (21) and, in vitro, the production of cyclic adenosine monophosphate (22) in human coronary artery smooth muscle cells. It has also been suggested that TSH may exert a critical role in skeletal remodelling by interacting with a specific receptor expressed on bone cells (23) and that TSH could modulate the production of many parameters of bone turnover (24). In a recent paper, we excluded an acute direct effect of TSH on osteoprotegerin and on the receptor activator of the nuclear factor-kb ligand system in the bone remodeling observed in altered states of thyroid function (25). Moreover, some effects of TSH on adipose tissue have been described in vitro and in vivo.tsh seems to stimulate leptin secretion by human adipose tissue in vitro through a direct action on the TSHR of adipocytes (12). In a recent paper, Antunes et al. (13) showed that differentiated adipocytes derived from the abdominal subcutaneous tissue respond to TSH by releasing IL-6. Furthermore, IL-6 release into the circulation occurs in thyroidectomized patients receiving rhtsh, indicating a probable extra-thyroid site of TSH action in vivo (13). There is a dual and complex relationship between leptin and TSH, which show coordinated pulsatility, with similar circadian rhythms (26), and seem to regulate each other. To investigate this relationship, we used an in vivo model constituted by thyroidectomized patients treated with L-T 4 suppressive therapy during rhtsh testing. In this way, we were able to evaluate the acute effect on serum leptin levels of the TSH surge induced by rhtsh administration. To our knowledge, this is the first study, in humans, on this topic. Although leptin modulation of the thyroid axis is well accepted, it remains unclear whether Leptin (μg/l) rhtsh Days Fig. 2 - Time-course of serum leptin levels before and after recombinant human TSH (rhtsh) administration. thyroid dysfunction can in itself regulate circulating leptin levels. It has been acknowledged that leptin itself directly stimulates TRH expression in the hypothalamic paraventricular nucleus, and subsequently TSH and thyroid hormones (2). Many studies have tried to explain how serum leptin levels change in both hypo- and hyperthyroidism, but the results have been conflicting. Thyrotoxicosis is characterized by a catabolic condition associated with an enhanced protein break-down, which is manifested by a weight loss which is very similar to that seen in the fasting state. In this condition, a chronic reduction in leptin levels, such as in starvation, has been described (2). In our study, most of the DTC patients were in a condition of relative thyrotoxicosis because of the high doses of L-T 4 administered as a suppressive therapy. In this condition, no correlation was observed between TSH and leptin levels either at the baseline or during rhtsh testing. Therefore, our data do not support an acute direct effect on leptin in patients with a history of DTC on L-T 4 regimen, and thus contrast with the view that adipocytes constitute an extra-thyroid site of TSH action in vivo. However, we cannot exclude the possibility that longer stimulation with rhtsh might increase leptin production by adipocytes, which is inhibited by a chronic condition of suppressed endogenous TSH and therefore not very responsive to an important but transient TSH peak. We can also hypothesize that TSH acts on adipocytes through a mechanism which is different from that of leptin production, and that it elicits the production of IL-6 through a specific receptor (13). According to most literature reports, there is a positive correlation between leptin and BMI. Indeed, leptin levels reflect whole-body adipose tissue mass and increase in parallel with the amount of adipose tissue in a curvilinear manner (27). In addition, we found a significant negative correlation between maximal TSH levels after rhtsh and BMI. This observation is in agreement with a recent study, which has suggested that the dosage of rhtsh should be personalised on the basis of body composition in patients weighing more than 8 kg (28). In conclusion, our in vivo experimental model suggests that an acute TSH increase after rhtsh testing is ineffective in changing the level of circulating leptin. These results are in contrast with some literature data, which report an in vivo correlation between leptin and TSH in hypothyroid (7), hyperthyroid (5) and obese (6) subjects. In addition, our finding of a correlation between maximal TSH levels after rhtsh and BMI confirms that an individually-tailored rhtsh dosage may be necessary in some DTC patients undergoing rhtsh testing. REFERENCES 1. Dhillo WS. Appetite regulation: an overview. Thyroid 27, 17: Feldt-Rasmussen U. Thyroid and leptin. Thyroid 27, 17: Rosenbaum M, Murphy EM, Heymsfield SB, Matthews DE, Leibel RL. 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