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Short-Term Hyperthyroidism Has No Effect on Leptin Levels in Man¹

Department of Medicine, Divisions of Endocrinology and Metabolism, Gerontology, and Bone and Mineral Metabolism, Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory of the Beth Israel Hospital, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Alan C Moses, Clinical Research Center, GZ 800, Beth Israel Hospital, Harvard Medical School, 99 Brookline Avenue, Boston, Massachusetts 02215. E-mail moses{at}sprcore bih.harvard.edu.

	Abstract

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Leptin, a 16-kDa adipocyte-derived protein whose circulating levels reflect energy stores, increases the resting metabolic rate and thermogenesis in rodents. Thyroid hormones also increase the basal metabolic rate, but nothing is known about possible interactions between leptin and thyroid hormone. Activation of ß-adrenergic receptors decreases leptin levels in rodents. To test the hypothesis that thyroid hormones, by causing a "functional hyperadrenergic" state, result in decreased leptin concentrations in humans, we studied 22 normal healthy men before and after the administration of T₃ for 1 week to induce moderate hyperthyroidism. Short term thyroid hormone excess does not alter circulating leptin concentrations despite a demonstrated effect on heart rate, systolic blood pressure, cholesterol levels, and metabolic indexes of bone turnover. Elucidation of the apparently separate pathways by which thyroid hormones, ß-agonists, and leptin regulate energy expenditure and food intake may have important implications for our understanding of the mechanisms for regulating energy homeostasis in health and disease.

	Introduction

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THE REGULATION of body weight requires a balance between energy intake and energy expenditure. A major advance in our understanding of the physiological system that is responsible for the long term regulation of energy balance and body weight (1, 2) was the recent discovery of leptin (2). Leptin is produced by adipocytes (3, 4, 5), and its circulating levels reflect energy stores in adipose cells (4, 5). Genetic defects that either impair the production of this molecule (2, 6) or produce resistance to its actions (7, 8) cause severe obesity in rodents (2, 7, 8). Administration of leptin decreases food intake and increases the resting metabolic rate and thermogenesis (9, 10, 11, 12), providing evidence that leptin plays a key role in a feedback loop maintaining energy balance.

Thyroid hormones directly increase the basal metabolic rate in man and have a permissive effect on adaptive thermogenesis in small animals (13, 14). The potential mechanisms responsible for thyroid hormone-controlled energy expenditure, including uncoupled oxidative phosphorylation, are complex and not yet fully elucidated (13, 14). Moreover, the potential interaction of thyroid hormones with the leptin system remains to be explored.

Recent studies indicate that stimulation of ß-adrenergic receptors decreases leptin expression in rodent adipocytes (12, 15). The current study was conducted to test the hypothesis that thyroid hormones would decrease leptin concentrations by causing a "functional hyperadrenergic" state, by decreasing energy stores as fat, or through other mechanisms.

	Subjects and Methods

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Subjects

Twenty-two healthy male volunteers between the ages of 18–35 yr were recruited. Subjects were excluded if they had either a medical condition that predisposed them to adverse effects from treatment with excess thyroid hormone or any condition that could alter the metabolic end points of the study. The study protocol was approved by the Beth Israel Hospital committee on clinical investigations, and written informed consent was obtained. The part of this study relating to the effect of moderate hyperthyroidism on bone metabolism has been described previously (16).

Protocol

Subjects were admitted to Boston’s Beth Israel Hospital General Clinical Research Center (GCRC), and after an overnight fast they were instructed to void and then drink 0.5 L water at 0600 h. At 0800 h on day 1, a fasting blood sample and a 2-h fasting urine sample were obtained. The hydroxyproline to creatinine ratio was measured in the urine. Leptin, osteocalcin, total cholesterol, T₃, T₄, T₄-binding globulin (TBG), TSH, complete blood count (CBC), and differential and serum chemistry profile were determined in the blood.

On day 7, subjects returned to the GCRC at 1700 h for a repeat of the testing performed on days 0–1. Subjects began taking T₃ (Cytomel, Smith-Kline and French, Philadelphia, PA: 25-µg tablets, two tablets twice daily, i.e. 100 µg daily) on day 8 and continued until day 15 inclusive. Compliance was verified by daily telephone calls and pill counts on day 15. On days 15 and 29, subjects returned to the GCRC at 1700 h for a repeat of the protocol performed on days 7–8.

Measurements

All serum specimens, with the exception of CBC and chemistry profiles, were frozen at -70 C after collection and assayed at the end of the study at once to avoid interassay variability.

Serum

TSH, T₃, T₄, and TBG were measured by a commercially available fluoroimmunometric assay using a Delfia kit (Wallac, Gaithersburg, MD). The free T₄ index was calculated as the product of total T₄ x 20/TBG. Serum osteocalcin levels were determined by RIA as described previously (17). CBC and automated serum chemistry profiles were performed by Bioran (Cambridge, MA).

Serum leptin was measured by RIA (Linco Research, Indianapolis, IN). The limit of detection was 0.5 ng/mL, the within-assay coefficient of variation was 4.39% for low levels (2.9 ng/mL) and 5.66% for high levels (14.1 ng/mL), and the between-assay variations were 6.9% and 9%, respectively. All assays were performed in duplicate.

Urine

Measurement of urinary creatinine was based on the modified Jaffe method (19). Urinary hydroxyproline was determined using the Hypernosticon kit (Organon-Teknika, Boxtel, Holland) (16).

Statistical analysis

Values are reported as the mean ± SEM. Repeated measures ANOVA followed by multiple comparison testing were performed using the SAS statistical program (SAS Institute, Cary, NC).

	Results

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Baseline characteristics

The mean (± SEM) age of participants in this study was 23.36 ± 1.08 yr (range, 18–29 yr), and their mean (±SEM) height was 176.19 ± 2.6 cm (range, 160–197). Their weight was 75.91 ± 3.79 kg (range, 60.4–104.7) and remained stable for the duration of the study. Baseline serum leptin and thyroid hormone indexes were normal (Table 1).

After 7 days of treatment with T₃ (study day 16), mean serum T₃ values tripled (P < 0.01 vs. study day 1; Table 1), and mean serum TSH decreased to 15% of the baseline value (P < 0.01 vs. study day 1; Table 1). By study day 30 (2 weeks after the discontinuation of T₃), thyroid hormone indexes returned to baseline levels (Table 1). Additionally, the free T₄ index was not significantly different from baseline.

Effect of T₃ treatment on heart rate and blood pressure levels

After treatment with T₃, heart rate increased by 15% compared to the baseline level (P < 0.01 vs. baseline; Table 1), systolic blood pressure increased by 5% (P < 0.05 vs. baseline; Table 1), and diastolic blood pressure did not change (Table 1).

Effect of T₃ treatment on serum cholesterol and indexes of bone metabolism

After treatment with T₃, serum cholesterol concentrations fell to 70% of the baseline level (P < 0.05 vs. baseline; Table 1), but were not different from the baseline on study day 30 (Table 1). After treatment with T₃, serum osteocalcin and the urinary hydroxyproline to creatinine ratio rose significantly on day 16 (P < 0.05 vs. baseline), as described previously (16). However, bone turnover indexes were not different from the baseline on study day 30 (16).

Effect of T₃ treatment on serum leptin concentrations

Serum leptin did not change significantly during thyroid hormone administration (Table 1).

Side-effects of treatment

Two of the 22 subjects reported mild insomnia while taking thyroid hormone. No symptoms consistent with overt hyperthyroidism were reported.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This study investigated the possible interrelationships between two molecules that are involved in the physiological regulation of energy homeostasis, leptin, and thyroid hormone. Leptin is a circulating adipocyte-derived molecule whose levels reflect the magnitude of fat stores (2, 3, 4, 5). The demonstrated actions of leptin in rodents include inhibition of food intake and stimulation of energy expenditure (3, 5, 9, 10, 11). Leptin gene expression is regulated by direct actions on the adipocyte by glucocorticoids, insulin, and, recently, activation of ß-adrenergic receptors (12, 15). Thyroid hormone increases the resting metabolic rate in man and has a permissive effect on adaptive thermogenesis in small animals (13, 14). The potential mechanisms responsible for thyroid hormone-controlled energy expenditure are complex and have not been fully elucidated (13, 14).

Thyroid hormones produce a hyperresponsiveness of peripheral tissues to adrenergic hormones. Given our previous findings in mice, supporting an acute effect of a ß₃-adrenergic agonist to increase energy expenditure and decrease serum leptin concentrations (12), we hypothesized that thyroid hormones might produce a similar effect. Alternatively, it is possible that the effect of thyroid hormones to increase energy expenditure could be mediated in part by a thyroid-induced increase in leptin concentrations. We, therefore, examined the potential effects of a short term increase in the level of T₃ on the serum leptin concentration in humans.

Exogenously administered T₃ produced a hyperthyroid state, as assessed by T₃ levels, suppressed TSH, and several metabolic indexes, including decreased cholesterol concentrations and increased indexes of bone formation and resorption. Hyperthyroidism was also assessed by a functional hyperadrenergic state, as indicated by the increases in heart rate and systolic blood pressure. Despite these changes, thyroid hormone excess did not change serum leptin concentrations in this group of young men.

We previously have shown that stimulation of ß₃-adrenergic receptors in mice, which causes increased energy expenditure, acutely suppresses the expression and circulating levels of leptin (12). Other work shows that this is a cAMP-dependent process in white adipose tissue (15). A decrease of leptin concentrations might, therefore, have been expected in response to the functional hyperadrenergic state produced by T₃ in this study, assuming that a hyperadrenergic state exists at the level of the adipocyte. A previous study has shown that leptin messenger ribonucleic acid expression is significantly decreased after T₄ administration to Zucker rats (20). However, the rats had lost a significant amount of weight (20), and the reduced leptin expression in this case most likely reflects significantly decreased adipose stores.

What are the implications of the fact that no changes in leptin concentrations were observed in the present study? First, it is likely that the ability of thyroid hormones to regulate energy expenditure does not operate through increases in leptin levels in humans. We might have expected thyroid hormone-induced hypermetabolism to cause a fall in leptin levels. However, as no weight change was seen with the short term hyperthyroidism, no alteration in leptin levels would be expected on the basis of changes in fat mass. It might be argued that the dose of thyroid hormone and/or the duration of treatment were insufficient for an effect on leptin concentrations to be observed. However, definite biological effects of thyroid hormone treatment were documented in this study. It remains to be determined whether higher doses of T₃ or a longer duration of treatment would produce changes in leptin concentrations.

In summary, a short term thyroid hormone excess of sufficient magnitude to affect heart rate, systolic blood pressure, serum cholesterol concentrations, and biochemical indexes of bone turnover does not alter circulating early morning leptin concentrations in young men.

	Acknowledgments

 
We thank Janet Hurwitz for performing the RIA analyses of thyroid hormones and TSH. Without the efforts of the nursing staff of the GCRC, this study would not have been possible.

	Footnotes

 
¹ This work was supported by NIH Grant DK-28082 (to J.S.F.), USPHS Bureau of Health Professions Faculty Training Project in Geriatric Medicine (to H.N.R.), Grant D31-PE-91000, Boots Pharmaceuticals, the Endocrine Fellows Foundation, Smith Kline Beecham Pharmaceuticals, Ciba-Geigy Pharmaceuticals, and Beth Israel Hospital General Clinical Research Center Grant M01-RR-01032.

² Member of the Clinical Investigator Training Program, Beth Israel Hospital, Harvard-Massachusetts Institute of Technology Health Sciences and Technology, in collaboration with Pfizer, Inc.

³ Recipient of the Clinical Associate Physician Award through the General Clinical Research Center.

Received July 11, 1996.

Revised September 10, 1996.

Accepted September 26, 1996.

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