Pituitary response to thyrotropin releasing hormone in children with overweight and obesity

Thyroid stimulating hormone (TSH) concentrations in the high normal range are common in children with overweight and obesity, and associated with increased cardiovascular disease risk. Prior studies aiming at unravelling the mechanisms underlying these high TSH concentrations mainly focused on factors promoting thyrotropin releasing hormone (TRH) production as a cause for high TSH concentrations. However, it is unknown whether TSH release of the pituitary in response to TRH is affected in children with overweight and obesity. Here we describe TSH release of the pituitary in response to exogenous TRH in 73 euthyroid children (39% males) with overweight or (morbid) obesity. Baseline TSH concentrations (0.9–5.5 mU/L) were not associated with BMI z score, whereas these concentrations were positively associated with TSH concentrations 20 minutes after TRH administration (r2 = 0.484, p < 0.001) and the TSH incremental area under the curve during the TRH stimulation test (r2 = 0.307, p < 0.001). These results suggest that pituitary TSH release in response to TRH stimulation might be an important factor contributing to high normal serum TSH concentrations, which is a regular finding in children with overweight and obesity. The clinical significance and the intermediate factors contributing to pituitary TSH release need to be elucidated in future studies.

in the children with obesity, and 2.5 (0.9-5.5) mU/L in the children with morbid obesity. FT4 concentrations were within normal range in all children (13.3 ± 2.0 pmol/L). All participant characteristics are presented in Table 1.
Baseline serum TSH concentrations were stratified into quartiles to evaluate TSH response of the pituitary in response to exogenous TRH stimulation for children in the higher and lower normal ranges of baseline serum TSH concentration (quartile 1 = < 2.05 mU/L; quartile 2 = 2.05-2.99 mU/L; quartile 3 = 3.00-3.69 mU/L; quartile 4 = > 3.69 mU/L). This is shown in Fig. 1. The TSH iAUC during the TRH test was significantly different between children in the different quartiles (p < 0.001). Post-hoc analysis showed a significant difference between quartile 1 and quartile 3 (p = 0.002), and between quartile 1 and quartile 4 (p < 0.001).  . The TSH iAUC during the TRH test was significantly different between the baseline serum TSH concentration quartiles (p < 0.001). Post-hoc analysis showed a significant difference between quartile 1 and quartile 3 (p = 0.002), and between quartile 1 and quartile 4 (p < 0.001). Baseline serum TSH concentrations were within the normal range in all children based on age specific references ranges 31 . TSH = thyroid stimulating hormone; TRH = thyrotropin releasing hormone; Q1 = quartile 1; Q2 = quartile 2; Q3 = quartile 3; Q4 = quartile 4.
Scientific RepoRts | 6:31032 | DOI: 10.1038/srep31032 Baseline serum TSH concentrations were positively associated with both, serum TSH concentrations twenty minutes after TRH administration (t 20 ) (r 2 = 0.484, p < 0.001) and the TSH incremental area under the curve (iAUC) during the TRH stimulation test (r 2 = 0.307, p < 0.001) ( Fig. 2A,B). Furthermore, the serum TSH concentration at t 20 showed an inverse association with age (r 2 = 0.056; p = 0.044), while no associations were found between age and the TSH iAUC during the TRH stimulation test. There were no gender differences regarding baseline serum TSH concentrations, serum TSH concentrations at t 20, and TSH iAUC during the TRH stimulation test.
BMI z-score and waist circumference z-score showed no significant associations with baseline serum TSH concentrations, serum TSH concentrations at t 20 , or the TSH iAUC during the TRH stimulation test. Significant inverse associations between serum c-reactive protein (CRP) concentrations and serum TSH concentrations at t 20 (r 2 = 0.142, p = 0.01), and the TSH iAUC during the TRH stimulation test (r 2 = 0.124, p = 0.007) were demonstrated. Plasma interleukin 6 (IL-6) concentrations were also significantly negative associated with serum TSH concentrations at t 20 (r 2 = 0.118, p = 0.026, respectively) and with the TSH iAUC during the TRH stimulation test (r 2 = 0.116, p = 0.009).

DISCUSSION
This is the first study investigating pituitary TSH release in response to exogenous TRH stimulation in a large group of euthyroid children with overweight and obesity. A positive association between baseline serum TSH concentrations and TSH release of the pituitary in response to exogenous TRH stimulation was demonstrated. This suggests that TSH release of the pituitary in response to TRH stimulation might be an important factor contributing to the frequently found high normal baseline serum TSH concentrations in children with overweight and obesity (Fig. 3), which is associated with several obesity related complications [1][2][3][4][5][6][7] .
Studies investigating TSH release of the pituitary in response to exogenous TRH stimulation in children with overweight and obesity are limited to small study populations [15][16][17] . In line with our findings in children, studies in adults with obesity demonstrated a higher TSH release in response to exogenous TRH stimulation as compared to lean adults [10][11][12] . Besides these HPT axis alterations, hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis has been described in adults with obesity when adrenocorticotropic hormone (ACTH) and cortisol concentrations were studied [18][19][20] . Since previous studies have shown that the HPA-axis can be influenced by pro-inflammatory cytokines 21,22 and the fact that obesity is characterized by a chronic state of low-grade inflammation 23 , it is tempting to suggest that presence of pro-inflammatory mediators might also play a role in the alterations in the other hypothalamic axes. However, results of this study showed that serum CRP concentrations and plasma IL-6 concentrations were negatively associated with serum TSH concentrations at t 20 and with the TSH iAUC during the TRH stimulation test, ruling out inflammatory stimulation as a contributing factor to high pituitary TSH release. Interestingly, this study showed that TSH release of the pituitary in response to exogenous TRH stimulation and high normal baseline serum TSH concentrations are not simply the consequence of excess body weight, since BMI z-score and waist circumference z-score were not associated with baseline serum TSH concentrations, serum TSH concentrations at t 20   Thus, the results of the current study showed that neither pro-inflammatory cytokines nor the amount of excess body weight is associated with high TSH release of the pituitary in response to exogenous TRH stimulation. Considering the evidence that in mice leptin contributes to regulation of HPT-axis activity 24 , there might also be a role for adipokines influencing the HPT-axis and pituitary TSH response to TRH in humans. Since the objective of this study was to evaluate the direct effect of TRH on pituitary TSH release, adipokines concentrations were not determined and cannot be evaluated as intermediate factors to high pituitary TSH release in this study. Furthermore, a recent review suggested that the HPT-axis activity is influenced by nutritional status and stressful situations including physical activity 25 . Oppert et al. also demonstrated an increased pituitary TSH release in response to exogenous TRH stimulation in young adults during long-term overfeeding as compared to the preoverfeeding TSH release 26 . Feeding status was not assessed in our study, but it is tempting to suggest that children with overweight and obesity are often exposed to overfeeding. Future studies are necessary to determine which factors might also affect pituitary TSH release in children with overweight and obesity.
In conclusion, baseline serum TSH concentrations are associated with TSH release of the pituitary in response to exogenous TRH stimulation in euthyroid children with overweight and obesity. The clinical significance and the intermediate factors contributing to pituitary TSH release need to be elucidated in future studies.

Methods
Study participants. This cross-sectional study was designed and conducted within the setting of the Centre for Overweight Adolescent and Children's Healthcare (COACH) at the Maastricht University Medical Centre (Maastricht UMC+ ). Within COACH, the health status of children with overweight, obesity, and morbid obesity is evaluated, and they are monitored and guided as described previously 27 . All children received a TRH stimulation test at the beginning of their participation in the COACH program. Children without a complete TRH stimulation test were excluded in this retrospective study. Further, children with baseline serum TSH concentrations above the normal range and children with thyroid diseases were excluded. Finally, 73 children were eligible for inclusion. Disease-related causes for overweight were ruled out in all children. The study was conducted in concordance with the guidelines laid down in the Declaration of Helsinki and approved by the medical ethical committee of the Maastricht UMC+ . Informed consent was obtained from all subjects or their parent or legal guardian.
Participant characteristics. Anthropometric data were obtained while children were barefoot and wearing only underwear. Body weight was determined using a digital scale (Seca) and body length was measured using a digital stadiometer. BMI was calculated and BMI z-scores were obtained using a growth analyser (Growth Analyser VE). Based on the International Obesity Task Force criteria children were classified as overweight, obese, or morbidly obese 28 . Waist circumference was measured with a non-elastic tape at the end of a natural breath at midpoint between the top of the iliac crest and the lower margin of the last palpable rib. Hip circumference was measured at the widest portion of the buttocks. Waist-and hip circumference z-scores were determined 29 , waist-to-hip (WHR) ratio was calculated, and ethnicity was defined 30   Scientific RepoRts | 6:31032 | DOI: 10.1038/srep31032 fluoroimmunoassay system (PerkinElmer). Serum TSH concentrations were considered within the normal range or above the normal range based on age specific references ranges 31 . Serum fT4 concentrations were considered normal between the range of 8-18 pmol/L. TRH stimulation test. At the start of the TRH stimulation test non-fasting serum TSH concentrations (t 0 ) were determined. A bolus of 200 μ g TRH was given intravenously, subsequently venous blood samples were obtained to determine serum TSH concentrations at 20 minutes (t 20 ), 40 minutes (t 40 ), 60 minutes (t 60 ), and 90 minutes (t 90 ) after the TRH administration.
Statistical analysis. All statistical analyses were performed using SPSS 23.0 for Windows (SPSS Inc).
Shapiro-Wilk test was performed to test for normality. Serum baseline TSH concentrations were stratified into quartiles. The TSH iAUC was calculated using the trapezoidal method. A one-way analysis of variance (ANOVA) with Bonferroni as post hoc analysis was used to evaluate differences in iAUC between serum baseline TSH concentration quartiles. Associations between variables were determined by linear regressions models. Since TSH concentrations are age dependent 31 associations were adjusted for age. A p-value below 0.05 was considered statistically significant. Data are presented as mean with standard deviation or as median with the minimum and maximum.

Clinical trial registration.
Clinical trial registration at ClinicalTrial.gov; Registration Number: NCT02091544.