Leptin and Thyrotropin Relationship Is Modulated by Smoking Status in Euthyroid Subjects

Abstract

Background:

The relationship between thyrotropin (TSH) concentrations and body mass index (BMI) in euthyroid subjects has been demonstrated only in some studies. Leptin regulates TSH secretion and TSH stimulates leptin secretion. The main aims of our study were to assess the relationship between leptin, the thyroid axis, and thyroid autoimmunity in a representative sample of a nonhospitalized euthyroid adult population of Catalonia and to determine whether smoking status could influence this relationship.

Methods:

This cross-sectional population-based study includes 894 euthyroid iodine-sufficient adults (390 men, 44.87±15.03 years old) with BMI 26.19±4.61 kg/m², representative of people living in Catalonia. The study analyzes the relationship between TSH, free thyroxine (FT₄), leptin, thyroperoxidase and/or thyroglobulin antibodies (thyroid autoimmunity), smoking status, and BMI. Measurements also include glycemia and insulinemia to calculate homeostasis model assessment of insulin resistance (HOMA-IR) index as a measure of insulin sensitivity.

Results:

In the univariate analysis and in the overall group, TSH correlated directly with BMI, leptin, and HOMA-IR (p=0.039, p<0.001, and p=0.010, respectively). In all men, TSH correlated directly with leptin (p=0.004), and in all women, directly with leptin (p=0.002) and HOMA-IR (p=0.031) and inversely with FT₄ (p=0.024). Only in men who smoke, TSH correlated directly with leptin (p=0.010) and HOMA-IR (p=0.024). In women, TSH correlated directly with leptin (p=0.004) and in nonsmoking women, inversely with FT₄ (p=0.047). In the multiple regression analysis, age (β=−0.00310, p=0.0265), smoking status (β=−0.24085, p=0.0202), and thyroid autoimmunity (β=0.20652, p=0.0075) were independent predictors of TSH variations. Leptin was a significant independent predictor of TSH variations only in smokers (β=0.16451, p=0.047).

Conclusions:

Leptin is an independent predictor of TSH concentration variations only in euthyroid smoker subjects of both sexes at all ranges of BMI, but not in nonsmokers. Age, smoking status, and positive thyroid autoimmunity also influenced TSH variability.

Introduction

I t has been reported that serum thyrotropin (TSH) concentrations are elevated in obese subjects in comparison to lean controls (1 –4), and a positive correlation between serum concentrations of TSH and body mass index (BMI) has been found by several investigators (5 –7). These data have been interpreted to suggest that thyroid function (even within the normal range) could be a contributing factor to body weight variation in the general population (8,9). However, other studies have given support to the opposite concept, according to which obesity may be responsible for the observed changes in circulating thyroid function parameters (6). The positive correlation found between TSH and BMI may be due to a higher prevalence of hypothyroidism, autoimmune or not, and obesity, two very common disorders, or alternatively, TSH elevation could represent an adaptive process of the hypothalamus–pituitary–thyroid axis in obese individuals, especially those suffering from morbid obesity. This last possibility is supported by the decrease of TSH concentrations after BMI amelioration either by hypocaloric diet or by bariatric surgery observed in obese subjects (10 –13). Marzullo et al. (14) suggested that obesity could be a risk factor for thyroid autoimmunity through the actions of leptin on the autoimmune system (15). However, other studies have provided no evidence for an association between thyroid autoimmunity and hyperthyrotropinemia in obese or morbidly obese patients (1,2). All these controversial results could be explained by the heterogeneity of populations included in the different studies. So far, published data mixed subjects with different degrees of obesity, including also morbid obesity even though it is recognized that this degree of overweight is a distinct condition with a greater risk of alterations in comparison to the other obesity categories (16).

Leptin, the most well-characterized adipocyte-derived hormone, participates in the regulation of energy homeostasis by signaling to the central nervous system as an indicator of adipose tissue reserves (3,17). Secondary to this information, a series of regulatory effects are elicited, among them prominent modifications of the thyroid axis activity (18). Some investigations suggest that leptin regulates, at least partially, TSH secretion in humans (19,20). In addition, leptin plays a role in the peripheral metabolism of thyroid hormones by activation of thyroxine (T₄) to triiodothyronine conversion (3), and leptin administration raises TSH levels, probably through thyrotropin-releasing hormone stimulation, and it normalizes the decreased thyroid hormone levels in euthyroid food-deprived animals (18,21). Conversely, it has also been demonstrated that TSH exerts a direct stimulation on leptin secretion by the adipose tissue (22,23). Moreover, TSH receptors have been identified in human adipose tissue (24). Thus, leptin has been postulated as a potential major link between BMI and TSH concentrations. Moreover, leptin has also been shown to regulate several immune processes involving Th-1 lymphocyte subsets and may therefore act as an immunoregulator (15). In humans, some data have been published showing that modifications in body weight reduce leptin and thyroid hormones (25,26), whereas other reports have described a leptin effect on TSH secretion (19). The influence of smoking and thyroid autoimmunity on the association between serum TSH and anthropometric measures remains unclear (7). Some authors observed an association between serum TSH and BMI only in the presence of thyroid autoimmunity (27), and others, but not all, found that the association of serum TSH with body mass differs between smokers and never-smokers (28,29).

The aims of our study were to assess the relationship between leptin, the thyroid axis, and thyroid autoimmunity in a representative sample of nonhospitalized, iodine-sufficient, and euthyroid adult population of Catalonia, with a wide range of body weight, and to determine whether smoking status could influence this relationship.

Materials and Methods

This cross-sectional study was performed in a sample of individuals participating in the Health Survey of Catalonia (Encuesta de Salud de Catalunya, ESCA 2002) carried out in 2002. This survey studied a representative sample of the nonhospitalized adult population of Catalonia, the northeastern region of Spain, and has been published in detail (30). Briefly, the random sort method was used to select a representative sample population (31). The Health Examination Study of Catalonia was conducted in a randomly selected subsample of the participants in the ESCA 2002. The data obtained were weighted to ensure the representativeness of the sex and age groups. The Health Survey of Catalonia (based on interviewer-directed questionnaires with 165 items) aimed to collect information on different markers of health status in the Catalonia population (total population 6,343,110 inhabitants). A total of 8000 subjects aged from 18 to 74 participated in the ESCA 2002. Of these, 1173 subjects participated in a second-phase study, the Health Examination Study of Catalonia, in which additional health information and blood samples were obtained by the research team of the Health Plan Evaluation for Catalonia. In the health survey, a specific question on the current or previous existence of thyroid disorders was posed. Forty-four subjects answered affirmatively (previously diagnosed thyroid dysfunction) and were excluded. Five pregnant women were also excluded. Sixty additional subjects were found to have undiagnosed thyroid dysfunction according to the results of the thyroid function test (TSH values above and below the normal range of 0.3–4 mIU/L) and were also excluded from the present study. Leptin concentrations were not available in 110 individuals, and in 60, anthropometric measures were not completed. Finally, this cross-sectional study was conducted in 894 euthyroid iodine-sufficient adults (390 men, 44.87±15.03 years old) with BMI 26.19±4.61 kg/m² (17.01–52.70). The Health Survey and Examination Studies were approved by the Ethics Committee of the Department of Health of the Autonomous Government of Catalonia. All the subjects who participated in the study gave their informed written consent.

Serum leptin, TSH, and free T₄ (FT₄) concentrations, the presence of thyroid antibodies (thyroid peroxidase antibodies and thyroglobulin antibodies) and urine iodine excretion were analyzed. Blood samples obtained in fasting conditions were immediately centrifuged, and serum was stored at −70°C until assayed. Samples were assayed in batches in three consecutive days by commercially available kits to measure concentrations of FT₄ (Immulite FT₄ Chemiluminescent Enzyme Immunoassay DPC, Los Angeles, CA) and TSH (Immulite Third-Generation TSH Chemiluminescent Enzyme Immunoassay DPC). Reference ranges were as follows: FT₄ 10.29–24.4 pmol/L (0.8–1.9 ng/dL) and TSH 0.3–4 mIU/L. Thyroid peroxidase antibodies and thyroglobulin antibodies were assayed with an enzyme immunoassay (Orgentec Diagnostica, Mainz, Germany). Values above 75 and 150 IU/mL, respectively, were considered positive. A single urine iodine measurement was used to assess iodine status in this population (modified Benotti method) (32). Leptin was measured by a commercial radioimmunoassay (Linco Research Inc., St. Charles, MO) with intra- and interassay coefficient of variation (CV) of 3.4–8.3% and 3.6–6.2%, respectively. The sensitivity of the assay was 0.5 μg/L. The reference range (median 5th–95th percentiles) for leptin obtained in our laboratory for normal-weight women was 10.4 μg/L (2.8–37.6 μg/L) and in normal-weight men 3.7 μg/L (1.2–9.2 μg/L). Immunoreactive insulin concentrations were measured using an automated enzyme chemiluminescence immunoassay (Immulite 2000 DPC). Assay sensitivity was 2 mIU/L. The intra- and inter-assay CV were below 5.5% and 7.3%, respectively. Insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR) and calculated from fasting plasma insulin and fasting plasma glucose concentrations as follows: HOMA-IR index=fasting insulin (mIU/L)×fasting glucose (mmol/L)/22.5.

Data were first tested for normal distribution using the Kolmogorov–Smirnov test to apply the adequate analysis. Data were expressed as the mean (standard deviation) and/or median (2.5 and 97.5 percentiles). Groups were compared by Student's t-test or the ANOVA test. Qualitative clinical data were compared using the chi-square test. Correlations between variables were tested using univariate analyses (Pearson's or Spearman's correlations where appropriate). Multiple regression analyses by enter methodology was performed to identify the factors affecting TSH concentrations by entering variables with a significant univariate relationship into the equation. Parameters that did not follow a normal distribution were natural logarithm (ln)–transformed before analysis. All the statistical analyses were performed with the statistical software package SPSS, version 12.0 (SPSS, Chicago, IL). Multivariate analyses were performed using SAS statistical software version 9.2 (SAS Institute Inc., Cary, NC). All the tests were two-tailed and a p-value <0.05 was considered statistically significant.

Results

Descriptive data

The characteristics of the 894 subjects studied are summarized in Table 1. Men were older than women (46.3±14.8 vs. 43.8±15.1 years, p<0.01). Mean BMI for the total group was 26.2±4.6 kg/m², being slightly higher for men than for women (26.6±3.8 vs. 25.8±5.0 kg/m², p<0.001) and abdominal circumference was abnormally high, according to US National Cholesterol Education Program Adult Treatment Panel III criteria, in 15.5% of men and 29.5% of women (p<0.001). The median leptin concentration was 5.53 (1.24–20.95) μg/L in men and 15.57 (3.7–46) μg/L in women. Positive thyroid antibodies were detected in 49 of 812 (6.03%) individuals, 7 men and 42 women. All the subjects were euthyroid, and there were no differences in thyroid hormone levels between men and women. The median urine iodine concentration was 150.0 μg/L.

Table 1.

Characteristics of the 894 Subjects Studied

Number	894
Men (N/%)	390/43.6
Age (years)	44.9±15
Smokers (%)	32.8
BMI (kg/m²)	26.2±4.6
Leptin (μg/L), median (percentiles 2.5–97.5)
Men	5.53 (1.24–20.95)
Women	15.57 (3.7–46)
HOMA-IR, median (percentiles 2.5–97.5)	2.26 (0.99–8.27)
Anti-TPO and/or anti-Tg antibody positivity (%) (TA)	6.03%
TSH (mIU/L), median (percentiles 2.5–97.5)
Men	1.42 (0.50–3.29)
Women	1.41 (0.46–3.33)
FT₄ (pmol/L)
Men	18.1±2.44
Women	17.37±2.19

BMI, body mass index; HOMA-IR, homeostasis model assessment of insulin resistance; TPO, thyroid peroxidase; Tg, thyroglobulin; TA, thyroid autoimmunity; TSH, thyrotropin; FT₄, free thyroxine.

As shown in Table 2, the proportion of smokers was significantly greater in men than in women. Smokers were younger and had lower BMI, leptin, HOMA-IR, and TSH and higher FT₄ than nonsmoker individuals, and all these differences were statistically significant.

Table 2.

Differences Between Smoker and Nonsmoker Individuals

	Smoker	Nonsmoker	p
Men (%)	38.7	61.3	0.001
Women (%)	28.2	71.8
Age (years)	39.3±13.3	47.6±15.1	<0.001
BMI (kg/m²)	25.2±4.2	26.7±4.7	0.001
Leptin (μg/L)
Men	4.4 (1.2–17.6)	6.4 (1.3–24)	<0.001
Women	13.1 (3–39.9)	16.3 (4.8–48.7)	0.002
HOMA-IR	2.09 (0.84–7.83)	2.39 (1.03–8.4)	<0.001
TSH (mIU/L)	1.35 (0.45–3.07)	1.45 (0.50–3.4)	0.012
FT₄ (pmol/L)	18.01±2.44	17.50±2.31	0.008

Univariate correlations between TSH and study variables

Univariate analyses (Table 3) showed that, in the overall group, TSH values correlated positively with BMI, leptin, and HOMA-IR (p=0.039, p<0.001, and p=0.010, respectively). In men, TSH correlated directly with leptin (p=0.004), and in women, directly with leptin (p=0.002) and HOMA-IR (p=0.031), and inversely with FT₄ (p=0.024). When analyses were performed according to smoker status, a positive correlation between TSH and leptin (p=0.010) and HOMA-IR (p=0.024) was present in smoker men, but was absent in nonsmokers. Also, in smoker women, TSH correlated directly with leptin (p=0.004), but these correlations were not found in nonsmoker women, which showed only a negative correlation with FT₄ (p=0.047).

Table 3.

Correlations Between Serum Thyrotropin Levels and Study Variables: Univariate Analyses

	Age	BMI	FT₄	Leptin	HOMA-IR
Men and women	r=0.008	r=0.069	r=−0.063	r=0.136	r=0.086
	p=0.804	p =0.039	p=0.062	p <0.001	p =0.010
Men	r=−0.19	r=0.088	r=−0.001	r=0.145	r=0.075
	p=0.714	p=0.083	p=0.977	p =0.004	p=0.140
Women	r=0.038	r=0.068	r=−0.101	r=0.136	r=0.097
	p=0.399	p=0.130	p =0.024	p =0.002	p =0.031

There was no correlation between iodine urinary concentrations and TSH values, neither in the whole group nor when sex or smoker status was considered.

Multiple regression analysis

The multiple regression analysis is shown in Table 4. BMI and abdominal circumference has been mean-centered before inclusion in the model to minimize collinearity. The interaction between ln-leptin and smoking status was significant and has also been included as an independent variable. The analysis after dichotomy by sex showed no significant differences in the mean of the ln-TSH levels between men and women. Age (β=−0.00310; p=0.0265), smoking status (β=−0.24085; p=0.0202), and thyroid autoimmunity (β=0.20652; p=0.0075) were independent predictors of TSH variations. Leptin was a significant independent predictor of TSH variations only in smokers (β=−0.16451; p=0.047). After adjustment for the other variables, for every 10 years of age, TSH decreased by 3.0%, smoking status decreased TSH by 21.4%, and the categorical positivity of thyroid autoimmunity increased TSH by 22.9%. In smokers, when leptin increases by 20%, TSH increases by 3%.

Table 4.

Correlations Between Natural Logarithm–Transformed Serum Thyrotropin Levels and Study Variables: Multiple Regression Analysis Enter Methodology

Variable	β	p	95% CI β
Intercept	0.57760	0.0014	0.22400 to 0.93120
Age	−0.00310	0.0265	−0.00583 to −0.00036
Female	−0.06651	0.3148	−0.19631 to 0.06330
Smoker	−0.24085	0.0202	−0.44399 to −0.03772
BMI^a	−0.00745	0.3280	−0.02240 to 0.00749
Waist circumference^a	−0.00416	0.1468	−0.00146 to 0.00979
Ln-leptin	−0.07480	0.0979	−0.01381 to 0.16340
Ln-leptin/smoking^b	−0.08971	0.0475	0.00098 to 0.17844
Ln-HOMA-IR	0.01273	0.7448	−0.06402 to 0.08948
FT₄	−0.17089	0.0846	−0.36517 to 0.02340
TA	0.20652	0.0075	0.05536 to 0.35769

Dependent variable: ln-TSH (mIU/L). R ²=0.0539.

BMI and abdominal circumference has been mean centered before included in the model.

Interaction between ln-leptin and smoking status.

95% CI, 95% confidence interval; Ln, natural logarithm–transformed.

Discussion

In our study of euthyroid iodine-sufficient adults, TSH showed positive correlations with indirect adiposity parameters such as BMI, leptin, and the HOMA-IR index, in agreement with some of the clinical and population-based studies recently published (5,7,8,33 –37). Multiple regression analysis showed that age, smoking status, and the presence of thyroid autoimmunity were independent predictors of TSH variations in the whole group. Leptin was a significant independent predictor only in smokers. The issue of the relationship between thyroid function and adiposity is a controversial one, and other recent reports have not found such a relationship (38 –41). In fact, the magnitude of the relationships found in the present study is low, as shown by the correlation coefficients obtained in univariate analysis (values near 0.1 and the coefficient of determination [R ²] of the multiple regression model of 0.0539), indicating that this model explains 5.39% of TSH variation. These limited results are in agreement with others (7) and clearly indicate that thyroid function, and TSH in particular, is influenced by an important number of different factors besides adipose tissue. However, some of the last published studies have shown univariate correlation coefficients in the range of 0.2–0.3 and even higher at high BMI categories (36). A majority of published data support a positive association between adiposity and TSH, and most importantly, two longitudinal studies seem to confirm such a relationship (5,42). The direction of the relationship remains to be established, and a bidirectional influence is not excluded. TSH has direct effects on adipocyte differentiation (23), and conversely, TSH values decrease after amelioration of obesity by hypocaloric diet or bariatric surgery (10 –13). Expression of the TSHR gene in subcutaneous and visceral fat is reduced in obesity, and this expression increases with weight loss when decreasing serum TSH concentrations. This suggests a role for adipocytes in TSH regulation (43). The former mechanism has also been explored in vitro models showing that TSH is able to directly stimulate leptin secretion in human omental adipocyte cultures (21), which would explain the coordinated pulsatile plasma profile of both hormones (44,45). In fact, hyperleptinemia has been proposed as one of the effectors that modulate different pituitary axes in obese subjects. In particular, leptin exerts a stimulatory effect on TSH secretion by a direct action on the hypothalamic–pituitary–thyroid axis (17,20).

As in other studies (46 –48), we found marked differences in leptin concentrations between men and women by an order of magnitude of threefold. Although men had higher BMI than women, double the number of women had a pathologic waist circumference; however, when normal BMI subjects were analyzed, sex differences in leptin concentrations were still evident (data not shown). In this regard, leptin has been proposed to be involved in the development of autoimmune diseases and in particular in thyroid autoimmunity, which is remarkably more prevalent in women (2,14). We also found a higher proportion of thyroid autoantibody positivity in women in parallel to the already mentioned higher leptin values in women.

An interesting finding of our study is the possible influence of smoking habits in the relationship between TSH and indirect adiposity parameters, as we found that the positive correlation between TSH and leptin was present only in smokers, in both men and women, but not in nonsmoker individuals. As far as we know, only three other studies have evaluated the smoking status in relation to the TSH–adiposity relationship with some controversial results (5,28,29). In a Brazilian study by de Moura and Sichieri (29), including only women, a strong relationship between TSH and BMI was found only in smokers, whereas in the study by Nyrnes et al. (5), studying both sexes, the relationship was found only in nonsmoker individuals. In a study performed in Norway with more than 27,000 subjects, the relationship was present in both smokers and nonsmokers, although smoker men showed the strongest association (28). Our results are in agreement with those of de Moura and Sichieri (29), as the relationship between TSH and BMI was present only in smokers, in both men and women. Smoking is known to affect thyroid function and indirect adiposity parameters; in our study, it was an independent predictor of TSH variations.

The influence of tobacco consumption on thyroid function is complex and could even be bidirectional. The studies aiming to elucidate whether tobacco exerts certain actions in one direction or another have found inconclusive results. Smoking appears to be negatively associated with hypothyroidism and positively with TSH decreases in the National Health and Nutrition Examinations Survey III (49). Our group previously reported that in a representative sample of noninstitutionalized Catalan adult subjects, hyperthyrotropinemia was more frequent in nonsmoker than in smoker individuals (50), a finding that is in agreement with the data recently published by Mehran et al. (51). A potential negative influence of tobacco consumption on thyroid function has been observed in iodine-deprived populations, in which lower TSH values have been found in smokers than in nonsmokers (52,53), and components of tobacco such as nicotine, thiocyanate, and benzopyrene may be responsible for these effects. In our study, iodine status appears to be optimal given that the median urinary iodine concentration was 150.0 μg/L, but TSH was lower in smokers. Hypothalamic AMP-activated protein kinase, a crucial enzyme inducing feeding-independent weight loss and an increased expression of thermogenic markers in brown adipose tissue (54), is inhibited by nicotine-inducing weight loss by a decreased orexigenic signaling in the hypothalamus (55). The PI3K-AKT, mTOR-p70S6 kinase, and AMP-activated protein kinase pathways play distinct and critical roles in metabolic regulation, and each of these pathways is necessary for leptin's anorexigenic actions in the hypothalamus. The effect of smoking on leptin regulation is also controversial. Smoking may induce low-grade inflammation at the adipose tissue, and in a recent study in subjects with BMI over 25 kg/m², smokers exhibited a significantly lower serum leptin level and leptin gene expression was markedly suppressed in adipocytes. Furthermore, nicotine suppressed leptin gene expression (56). Thus, our finding that leptin was lower in smoker men and women may also be due to a primary effect of nicotine and not just related to the fact that smokers had lower BMI than nonsmokers. Therefore, decreased feeding behavior and the effect of tobacco components may act in concert to explain both a lower TSH, as well as a lower BMI and decreased circulating leptin levels in smokers, as found in the present study.

Aging is associated with changes in the pituitary–thyroid axis function, as well as with an increased prevalence of autoimmune and nodular thyroid disease (57,58). Previous studies suggested that, in the absence of thyroid disease, aging was associated with reduced TSH secretion (56,59). However, data from the National Health and Nutrition Examinations Survey III (60) showed that serum TSH concentrations increase with age in people with no clinical or biochemical evidence of thyroid disease in conditions of iodine sufficiency. Our study was carried out in a representative sample of euthyroid adult individuals of Catalonia, and subjects with previous thyroid disorders and pregnant women were excluded. Under these conditions of subject selection, aging was also associated with decreasing serum TSH concentrations.

The strengths of our study are its population-based design covering all inhabitants of Catalonia and that the number of individuals included has sufficient statistical power to draw consistent correlations between the studied variables. The weaknesses include a nonquantification of tobacco consumption and the lack of monitoring physical activity and food intake.

In summary, leptin is an independent predictor of TSH concentration variations only in euthyroid smoker subjects of both sexes with different BMIs living in Catalonia, Spain. Age, smoking status, and thyroid autoimmunity positivity also influenced TSH variability.

Footnotes

Acknowledgments

The authors gratefully acknowledge the assistance of Mr. Gary Shivel (Barcelona, Spain) for manuscript review.

Author Disclosure Statement

No competing financial interests exist.

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