Circulating Levels of Orexin-A,Nesfatin-1,Agouti-Related Peptide,and Neuropeptide Y in Patients with Hyperthyroidism

Abstract

Background:

There is insufficient information about the appetite-related hormones orexin-A, nesfatin-1, agouti-related peptide (AgRP), and neuropeptide Y (NPY) in hyperthyroidism. The aim of the present study was to investigate the effects of hyperthyroidism on the basal metabolic rate (BMR) and energy intake, orexin-A, nesfatin-1, AgRP, NPY, and leptin levels in the circulation, and their relationship with each other and on appetite.

Methods:

In this prospective study, patients were evaluated in hyperthyroid and euthyroid states in comparison with healthy subjects. Twenty-one patients with overt hyperthyroidism and 33 healthy controls were included in the study.

Results:

Daily energy intake in the hyperthyroid state was found to be higher than that in the euthyroid state patient group (p=0.039). BMR was higher in hyperthyroid patients than the control group (p=0.018). Orexin-A was lower and nesfatin-1 was higher in hyperthyroid patients compared to the controls (p<0.001), whereas orexin-A increased and nesfatin-1 decreased after euthyroidism (p=0.003, p<0.001). No differences were found in the AgRP, NPY, and leptin levels between the hyperthyroid and euthyroid states and controls (p>0.05). Orexin-A correlated negatively with nesfatin-1 (p=0.042), BMR (p=0.013), free triiodothyronine (fT3; p<0.001), and free thyroxine (fT4; p<0.001) and positively with thyrotropin (TSH; p<0.001). Nesfatin-1 correlated negatively with orexin-A (p=0.042) and TSH (p<0.001) and positively with fT3 (p=0.005) and fT4 (p=0.001). In the regression analysis, “diagnosis of hyperthyroidism” was the main factor affecting orexin-A (p<0.001).

Conclusions:

Although it seems that no relationship exists among orexin-A, nesfatin-1, and increased appetite in hyperthyroidism, the orexin-A and nesfatin-1 levels are markedly affected by hyperthyroidism.

Introduction

Hyperthyroidism is characterized by increased food intake and appetite, but weight loss is considered to be a result of negative energy balance due to increased energy expenditure (1). It has been suggested that appetite increases with the effect of thyroid hormones on the hypothalamic nuclei, which are likely responsible for the increased energy intake in patients with hyperthyroidism (1). However, the factors and mechanisms involved in increased appetite during hyperthyroidism are not clear.

The effects of neuroendocrine peptides, which are related to appetite in hyperthyroidism, have not yet been fully elucidated. The hypothalamus regulates the complex mechanism of energy balance, which includes eating, digestion, absorption of digested food, metabolism, and storage of energy, and maintains the functionality of these processes. The arcuate nucleus is known as “the center of food intake into the body.” One of the two different neuron groups in this nucleus increases appetite by releasing orexigenic substances named Agouti-related peptide (AgRP) and neuropeptide Y (NPY). Thyroid hormones contribute to energy regulation by controlling the hypothalamus (2). It has been proposed that triiodothyronine (T3) increases the synthesis of AgRP and NPY by acting on the arcuate nucleus (ARC) via the “mammalian target of rapamycin” (mTOR) and uncoupling protein 2 (UCP2) (2). Moreover, Herwig et al. recently reported in a rodent model that T3 may have a direct effect on AgRP mRNA expression in the ARC (3). To the best of the authors' knowledge, there is no study related to the blood levels of AgRP and NPY in hyperthyroidism.

Orexin-A (hypocretin-1) is a peptide synthesized in the lateral hypothalamic area, where feeding behavior and energy balance are regulated (4,5). It has been suggested that orexin-A increases both food intake and energy expenditure to prevent weight gain (5). Many publications have shown that orexin-A is related to sleep-wakefulness, arousal, energy balance, narcolepsy, glucose metabolism, gastric ulcers, and thermogenesis (4,6 –11). The results of animal studies that investigated the relationship of orexin-A with the thyroid axis are contradictory (4,12,13). Mitsuma et al. (13) injected mice with orexin-A and reported that orexin-A suppressed the secretion of TRH from the hypothalamus, whereas release of thyrotropin (TSH) from the anterior hypothalamus was not affected by orexin-A. It was observed that intraparaventricular nuclear orexin-A administration suppressed TSH levels (4). In another study, the levels of prepro-orexin mRNA in the hypothalamus and the adrenal glands of rats were not found to be directly related with thyroid hormones (6). As far as the authors know, there are no studies investigating orexin-A levels in patients with hyperthyroidism.

Nesfatin-1 was recently found to be an important regulator of central energy balance (14,15). Nesfatin-1 reduces food intake, and it is suggested that it acts on other systems in addition to its anorexigenic effect (15,16). Nesfatin-1 has an antihyperglycemic effect that is independent of its anorexigenic effect (15,17). Some studies show that nesfatin-1 is involved in diabetic hyperphagia, mood disorders, epilepsy, stress, sleep, anxiety, depression, reproductive functions, increased insulin secretion, and the regulation of gastrointestinal functions (16 –19). There are conflicting results from two previous studies investigating nesfatin-1 levels in hyperthyroidism. The level of nesfatin-1 was found to be low in children with hyperthyroidism. However, the other study reported that the nesfatin-1 levels in both an overt and subclinical hyperthyroid group were similar to those of the healthy control group (20,21).

Leptin, which is produced by adipocytes, is accepted as an important regulator of food intake and energy balance (1). The connection between leptin and thyroid metabolism has been investigated for the last two decades (1,22,23), but it remains unclear. Leptin affects specific centers in the brain to increase satiety and reduce food intake (1,22). Leptin centrally regulates orexigenic and anorexigenic neuropeptides. Leptin receptors are located primarily in areas of the brain such as the ARC, in which orexigenic peptides such as AgRP and NPY are produced, and the pituitary gland and paraventricular nucleus, which include TRH secreting neurons (1,22). Leptin decreases AgRP and NPY expression in the ARC to reduce food intake (22). Guo et al. (24) demonstrated that TRH gene expression was increased in the paraventricular nucleus after leptin injection in fasting animals. Additionally, circadian patterns of leptin and TSH have been reported to be similar (25). However, in studies that investigated circulating leptin levels in hyperthyroidism, the results are conflicting (22,23).

As far as the authors know, there is insufficient information about the peripheral blood levels of orexin-A, nesfatin-1, AgRP, and NPY, which are peptides related to appetite, in patients with hyperthyroidism. In this state, a catabolic process is present despite increased appetite. In addition, leptin may participate in the regulation of orexigenic and anorexigenic peptides in hyperthyroidism. However, this issue has yet to be studied. The aim of the present study was to investigate the effects of hyperthyroidism on basal metabolic rate (BMR), food intake, body mass index (BMI), orexin-A, nesfatin-1, AgRP, NPY, and leptin levels and their relationships with each other and in appetite.

Materials and Methods

Patient selection and study design

This study is a prospective longitudinal study in which the patient group was evaluated before treatment in an overt hyperthyroid state and then after 10.2±2.8 months, when the patients were in a euthyroid state, after which they were compared with a healthy control group. The patients, who were diagnosed with overt hyperthyroidism according to their history, physical examination, and laboratory tests in the endocrinology outpatient clinic, were included in the patient group, and healthy individuals were included in the control group. The study protocol was approved by the ethic committee, and informed consent was obtained from all subjects included in the study.

Inclusion criteria for the patient group supporting Graves' disease were overt hyperthyroidism (free T3 [fT3] level >3.71 pg/mL and/or a free thyroxine [fT4] level >1.48 ng/d: and a TSH level <0.35 μIU/mL), increased radioactive iodine (RAI) uptake, positive thyrotropin receptor antibody (TRAb), and/or diffuse involvement in thyroid scintigraphy. Healthy volunteers with normal fT3, fT4, and TSH levels who had no history of thyroid disease and whose age, sex, and BMI were similar to the patient group were invited to participate in the study. The exclusion criteria for the patient and the control group were a history of pregnancy in the last year, lactation, chronic diseases (e.g., renal failure, liver disease, chronic obstructive lung disease), conditions that may affect appetite and endocrine hormones (e.g., malignancy, diabetes mellitus, Cushing's syndrome, acromegaly, gastrointestinal diseases), psychiatric diseases (e.g., anorexia nervosa, major depression, psychosis), or the use of certain drugs that may affect appetite (e.g., corticosteroids, thyroid hormone replacement therapy, antithyroid treatment in the last three months).

Before starting antithyroid treatment in the overt hyperthyroid state, blood samples were obtained from the patients at 08:00–10:00 a.m. after a fasting period of 10 h. Serum and plasma samples were centrifuged and separated. Aprotinin was added to the plasma samples to increase stability, and serum and plasma samples were kept at −80°C. BMR, BMI, and body composition were measured by a dietician. Daily energy and nutrient intake were obtained from three-day food records. Antithyroid treatment (methimazole 15–60 mg) was started to induce euthyroidism after the initial evaluation of the patients. In the follow-up visits, RAI treatment was given only to one patient. The patients in whom euthyroidism could be achieved (serum fT3, fT4, and TSH levels within normal ranges) continued the follow-up. In the follow-up period, the blood levels of orexin-A, nesfatin-1, AgRP, NPY, leptin, BMR, and BMI, and three-day food records were measured again in patients in the euthyroid state. Orexin-A, nesfatin-1, AgRP, NPY, and leptin levels, BMR, BMI, and three-day food records were also measured in healthy subjects in the control group.

Biochemical assessment

TSH (μIU/mL), fT4 (ng/dL), fT3 (pg/mL), anti-Tg (IU/mL), anti-TPO (IU/mL), and TRAb (IU/L) parameters were measured using a chemiluminescence method (Abbott Architecht I200 autoanalyzer). Serum Nesfatin-1 levels were analyzed with the enzyme-linked immunosorbent assay (ELISA) method using an available kit (Phoenix pharmaceuticals Human, Nesfatin-1(1–82) ELISA kit). The assay has a sensitivity of 0.78 ng/mL, an intra-assay variation of <10%, and an inter-assay variation of <15%. Plasma AgRP levels were analyzed with enzyme immunoassay (EIA) using an available kit (Phoenix Pharmaceuticals AgRP (83–132) Amide EIA Kit). The assay has a sensitivity of 0.05 ng/mL, an intra-assay variation of <10%, and an inter-assay variation of <15%. The plasma NPY levels were analyzed with EIA using an available kit (Phoenix pharmaceuticals NPY EIA Kit). The assay has a sensitivity of 0.16 ng/mL, an intra-assay variation of <10%, and an inter-assay variation of <15%. Plasma orexin-A levels were analyzed with EIA using an available kit (Phoenix Pharmaceuticals orexin-A extraction free EIA Kit). The assay as a sensitivity of 0.13 ng/mL, an intra-assay variation of <10%, and an inter-assay variation of <15%. Serum leptin levels were measured using a commercial kit (DIAsource Leptin-EASIA kit, DIAsource Immunoassays S.A.). The assay has a sensitivity of 0.04 ng/mL, an intra-assay variation of <3.5%, and an inter-assay variation of <10.2%.

BMR and body composition measurements

BMR was measured using the Fitmate basal metabolism test device (CosMed), which operated via the principle of indirect calorimetry. Using this device, the energy requirement of an individual at absolute rest was determined. The Fitmate facial mask was used for this measurement, and the resting metabolic rate was calculated from the mean oxygen consumption capacity (VO₂).

Segmental fat analysis, skeletal muscle weight, BMI, waist/hip ratio, and the intracellular (ICW) and extracellular water (ECW) levels were determined for five different regions of the body using an INBODY 720 Bioelectrical Impedans Analyzer (Biospace). The degree of adiposity in the abdominal region was determined using the VISCAN (Tanita) device.

Dietary assessments

Food intake of the participants was determined using the three-day food record method. With this method, the types and amounts of the foods consumed throughout the day were detected, and the amounts of energy and nutrients were determined. Daily energy and nutrient intakes were calculated using the computer software Nutrition Information Systems (BeBIS) to prepare Turkish foods. The program includes the definitions and calories of many foods and the menus consumed, and the serving amount may be adjusted.

Statistical evaluation

Statistical analyses were performed using IBM SPSS Statistics for Windows v21.0 (IBM Corp.). The numerical variables are summarized using mean±standard deviation (SD) and median [minimum – maximum] values. The categorical variables are expressed as numbers. A chi-square test was used to investigate differences in terms of categorical variables between the groups. The Shapiro Wilks test was used to examine the normality of numerical variables, and the Levene test was used to examine the homogeneity of the variants. The differences between independent groups in terms of numerical variables were examined using the t-test, if parametric test assumptions were met. If parametric test assumptions were not met, the Mann–Whitney U-test was used to compare the groups. A paired t-test was used to examine whether there was a difference between the patients with hyperthyroidism and euthyroid patients in dependent groups, assuming parametric test assumptions were met. If parametric test assumptions were not met, a Wilcoxon test was used. The relationship between numerical variables were expressed with the Pearson or Spearman correlation coefficient. The factors that affected certain variables were investigated using multiple regression analysis. Transformation methods were used for the variables that did not show a normal distribution in the regression analysis. A p-value of <0.05 was considered significant.

Results

Twenty-one patients with a diagnosis of overt hyperthyroidism and 33 healthy volunteers were included in the study. The two groups were found to be similar in terms of age, sex, and BMI (p>0.05; Table 1). The patients with hyperthyroidism were reevaluated in the euthyroid state for a mean period of 10.2±2.8 months after antithyroid treatment was started. The laboratory values of the patient and control groups are given in Table 1.

Table 1.

Characteristics of Patient and Control Groups

	Patient group (hyperthyroid), n=21	Patient group (euthyroid), n=21	p-Value: hyperthyroid vs. euthyroid in patient group	Control, n=33	p-Value: hyperthyroid patient vs. control	p-Value: euthyroid patient vs. control
Age (years)	39.6±11.6			36.4±14.4	0.39
Sex (F/M)	16/5			25/8	1
BMI (kg/m²)	25±4.4	26.5±4.8	0.001	25±4.3	0.946	0.257
Energy intake (kcal)	1686.5±424.8	1469.6±890.3	0.039	1569.7±262.5	0.267	0.621
BMR (kcal/day)	1668.6±399.7	1554.2±391.9	0.099	1447.7±265.9	0.018	0.239
TSH (μIU/mL)	0.01 (0–0.02)	1.35±0.72	<0.001	1.24 (0.47–2.46)	<0.001	0.998
fT3 (ng/dL)	6.1 (1.6–21.9)	2.86 (2.29–3.50)	<0.001	3.07 (2.5–3.71)	<0.001	0.038
fT4 (pg/mL)	1.9 (0.8–4.64)	1.06±0.063	<0.001	1.16 (0.93–1.45)	<0.001	<0.001
Anti-Tg (IU/mL)	52.2 (1.49–2860.76)	7.08 (0.43–871)	<0.001	1.05 (0.02–162.31)	<0.001	<0.001
Anti-TPO (IU/mL)	249.16 (3.52–29,320.0)	183.5 (0.2–7280.0)	0.001	0.21 (0.01–91)	<0.001	<0.001
TRAb (IU/L)	16.24 (1.35–147)	7.89 (1.62–83.71)	0.004	5.77 (0.36–12)	<0.001	0.034
Orexin-A (ng/mL)	0.83±0.15	1.14±0.27	<0.001	1.23±0.21	<0.001	0.20
Nesfatin-1 (ng/mL)	1.35 (1.04–5.40)	1.09 (0.71–5.54)	0.003	0.97 (0.64–25.55)	<0.001	0.414
AgRP (ng/mL)	0.31 (0.17–0.65)	0.31 (0.12–0.67)	0.768	0.29 (0.14–10.0)	0.887	0.657
NPY (ng/mL)	1.36 (0.35–10.0)	1.63 (1.02–2.43)	0.217	1.36 (0.70–4.36)	0.83	0.29
Leptin (ng/mL)	5 (0.89–18.78)	5.29 (2.08–22.39)	0.614	5.58 (1.78–37.30)	0.409	0.722

The values that showed a normal distribution were expressed as the mean±standard deviation, and the values that did not show a normal distribution were expressed as median [minimum – maximum].

BMI, body mass index; fT3, free triiodothyronine; fT4, free thyroxine; TSH, thyrotropin; BMR, basal metabolic rate; Anti-Tg, antithyroglobulin antibody; Anti-TPO, thyroid peroxidase antibody; TRAb, TSH receptor antibody; NPY, neuropeptide Y; AgRP, agouti-related peptide.

All body composition measurements except for body fat percentage and weight were increased in the euthyroid state compared with those in the hyperthyroid patient group (p<0.05; Table 2). BMI was not different between hyperthyroid patients and the control group (p=0.946; Table 1). However, BMI was increased in the euthyroid state compared with the hyperthyroid state in the patient group (p=0.001). In this study, only BMI was used in the correlation analyses.

Table 2.

Body Composition Measurements in the Patient and Control Groups

	Patient group (hyperthyroid), n=21	Patient group (euthyroid), n=21	p-Value: hyperthyroid vs. euthyroid in patient group	Control, n=33	p-Value: hyperthyroid patient vs. control	p-Value: euthyroid patient vs. control
Abdominal fat (%)	35.9 (19.7–50.6)	39.5 (20.3–50.8)	0.003	31.9 (15.6–56)	0.729	0.152
WC (cm)	94 (73–117)	101 (79–121)	0.001	93 (73–138)	0.790	0.234
Waist/hip ratio	0.89 (0.77–1)	0.92 (0.79–1.03)	<0.001	0.9 (0.79–1.01)	1	0.180
Intracellular water (L)	18.7 (17–27.7)	20.75 (17.8–29.4)	<0.001	20.2 (16.4–14.9)	0.07	0.690
Extracellular water (L)	11.8 (10.7–17)	12.6 (11.1–18)	0.003	12.4 (10.2–20.4)	0.356	0.638
Total body water (L)	30.4 (27.7–44.6)	33.3 (28.9–47.1)	<0.001	32.6 (26.7–55.3)	0.166	0.638
Fat mass (kg)	21 (9.2–39.2)	24.2 (11.1–43.3)	0.031	18.3 (7.7–41.7)	0.472	0.198
Fat free mass (kg)	41.5 (37.7–60.6)	46.3 (39.2–64)	<0.001	44.1 (36.5–75.5)	0.163	0.316
Skeletal muscle weight (kg)	22.4 (20.2–34.1)	25.05 (21–36.4)	<0.001	24.4 (19.4–43.5)	0.084	0.743
Body fat percentage (%)	33.6 (19.6–49)	33.05 (19.8–49.6)	0.641	29.6 (13.4–46)	0.435	0.260

WC, waist circumference; ICW, intracellular water; ECW, extracellular water.

No significant difference was observed in the amount of energy intake between hyperthyroid patients and the control group (p=0.267). However, the amount of energy intake in the hyperthyroid state was higher compared with that of euthyroid patients (p=0.039). BMR was found to be higher in the hyperthyroid patient group than in the control group (p=0.018). The BMR after euthyroidism in the patient group was found to be lower compared with the hyperthyroid state, but the difference was not statistically significant (p=0.09).

The orexin-A level was lower in the patient group compared with the control group (p<0.001). The orexin-A level was increased in the euthyroid state compared with the hyperthyroid state in the patient group (p<0.001). The orexin-A level was found to be similar between euthyroid patients and the control group (p=0.2). The nesfatin-1 level was higher in the hyperthyroid patient group than in the control group (p<0.001). The nesfatin-1 level in the patient group was higher than that of the euthyroid state (p=0.003), although it was similar in euthyroid patients and the control group (p=0.414; Table 1). No significant differences were found in the AgRP, NPY, and leptin levels between the hyperthyroid and control group (p=0.887, p=0.838, and p=0.409, respectively). Additionally, no significant differences in these hormones were found between the hyperthyroid and euthyroid states of the patient group (p=0.768, p=0.217, and p=0.614, respectively; Table 1).

In the whole study group (the patient group in the hyperthyroid state and the control group taken together; n=54), a strong positive correlation was found between orexin-A and TSH (r=0.613, p<0.001), and negative correlations were found between orexin-A and fT3, and fT4 (r=−0.564 p<0.001; r=−0.507, p<0.001), BMR (r=−0.336, p=0.013), and nesfatin-1 (r=−0.278, p=0.042; Table 3). By multiple regression analysis, the model was conducted with the plasma orexin-A level as a dependent variable, and diagnosis of hyperthyroidism, BMR, nesfatin-1, and NPY as independent variables. Based on the model, diagnosis of hyperthyroidism was the only significant predictor for orexin-A levels (β=−0.402, std-β=−0.712 [CI −0.512 to −0.292], R ²=0.507, p<0.001).

Table 3.

The Correlations Between Nesfatin-1, Orexin-A, AgRP, NPY, BMR, TSH Levels, and Other Parameters in the Whole Study Group

	Nesfatin-1		Orexin-A		AgRP		NPY		Leptin		BMR		TSH
	r	p	r	p	r	p	r	p	r	p	r	P	r	P
Age (years)	0.086	0.537	0.1	0.473	0.129	0.353	0.378	0.005	−0.017	0.902	−0.031	0.824	−0.057	0.680
BMI (kg/m²)	0.008	0.955	−0.068	0.624	0.041	0.766	−0.011	0.935	0.423	<0.001	0.072	0.607	−0.055	0.692
Energy intake	0.111	0.425	−0.144	0.298	−0.042	0.761	−0.91	0.514	−0.146	0.292	0.079	0.572	−0.047	0.736
BMR	0.042	0.761	−0.336	0.013	0.036	0.794	−0.058	0.680	−0.076	0.583	—	—	−0.235	0.088
Nesfatin-1	—	—	−0.278	0.042	−0.167	0.228	−0.155	0.263	−0.104	0.455	0.042	0.761	−0.506	<0.001
Orexin-A	−0.278	0.042	—	—	0.077	0.578	0.251	0.067	0.064	0.644	−0.336	0.013	0.613	<0.001
AgRP	−0.167	0.228	0.077	0.578	—	—	0.480	<0.001	−0.116	0.404	0.036	0.794	0.026	0.853
NPY	−0155	0.263	0.251	0.067	0.480	<0.001	—	—	0.004	0.976	−0.058	0.680	0.060	0.664
Leptin	0.104	0.455	0.064	0.644	−0.116	0.404	0.04	0.976	—	—	−0.076	0.583	0.120	0.388
TSH (μIU/mL)	−0.506	<0.001	0.613	<0.001	0.026	0.853	0.060	0.664	0.120	0.388	−0.235	0.088	—	—
fT3 (ng/dL)	0.378	0.005	−0.564	<0.001	0.020	0.887	−0.039	0.782	−0.171	0.216	0.435	0.001	−0.673	<0.001
fT4 (pg/mL)	0.423	0.001	−0.507	<0.001	0.029	0.833	−0.024	0.861	−0.232	0.091	0.467	<0.001	−0.688	<0.001

The whole group (n=54) consisted of hyperthyroid patients (n=21) and the control group (n=33) together.

In the whole study group, negative correlations were found between nesfatin-1 and orexin-A, and TSH (r=−0.278, p=0.042; r=−0.506, p<0.001, respectively), and positive correlations were found between nesfatin-1 and fT3, and fT4 (r=0.378, p=0.005; r=0.423, p=0.001, respectively; Table 3). To avoid the problem of multicolinearity, thyroid hormones were not entered together into the model. Because the number of variables that would be entered into the model was low, multiple linear regression analysis was not performed. No statistically significant correlations were found between AgRP and NPY and the other parameters in the whole study group. A positive correlation was found between AgRP and NPY (r=0.480, p<0.001). A positive correlation was found between BMR and fT3 and fT4 (r=0.435, p=0.001; r=0.467, p<0.001, respectively). As shown in Table 3, the leptin level significantly correlated only with BMI in the whole group (r=0.423, p<0.001).

Discussion

In the present study, the orexin-A levels were decreased and the anorexigenic hormone nesfatin-1 was increased, whereas energy intake was increased in patients with overt hyperthyroidism. When the patients with hyperthyroidism became euthyroid, the orexin-A levels increased, the nesfatin-1 levels decreased, and both were similar with those in the control group.

Body composition analysis and food intake were investigated in this study because a catabolic state is expected during hyperthyroidism. BMI and the majority of the body composition measurements were found to be lower in the hyperthyroid state compared with the euthyroid state. The studies in the literature also support these findings (1,26,27). It was observed that energy intake in the hyperthyroid state was higher than that in the euthyroid state. It is known that energy intake is increased in hyperthyroidism (1,28). In 1998, Lönn et al. (29) reported that food intake was decreased in patients with hyperthyroidism who were being treated. The reason for increased appetite in hyperthyroidism is not known. However, the blood levels of the peptides related to appetite investigated in this study do not explain this increase in appetite, as discussed below. More specifically, the present results for nesfatin-1 and orexin-A are not compatible with the increased caloric intake in patients with hyperthyroidism in this study, but it is not possible to explain the underlying mechanism. In this study, the amounts of dietary energy intake were calculated as an indicator of energy intake, and BMR was measured as an indicator of energy expenditure because BMR is the largest component of energy expenditure (30). BMR was increased in patients with hyperthyroidism compared with healthy controls, and BMR increased when fT3 and fT4 were increased in this study. Some studies have investigated the mechanism of increased BMR in hyperthyroidism (31,32). The increase in BMR, which was the result of increased glucose utilization and increased protein and lipid catabolism, has been suggested as an explanatory mechanism (33).

Orexin-A, which is an orexigenic peptide, was found to be low in patients with hyperthyroidism, but it was increased after euthyroidism in these patients and became similar to the levels found in healthy controls. Supporting this finding, a negative correlation was found between orexin-A and fT3 and fT4, and a strong positive correlation was found between orexin-A and TSH. In this study, it was shown that a diagnosis of hyperthyroidism was the strongest predictive factor for orexin-A levels. This is the first study in this area. The mechanism of these alterations cannot be explained by current scientific knowledge. In the literature, the levels of ghrelin and obestatin, which are among the orexigenic peptides associated with appetite, were investigated in patients with hyperthyroidism. Similar to the present findings, the ghrelin and obestatin levels were also found to be low in patients with hyperthyroidism (34,35). However, it has not yet been explained how ghrelin secretion is inhibited in hyperthyroidism (35). Serum ghrelin levels were not responsible for the increased appetite observed in hyperthyroidism, although they were reported to be associated with glucose in hyperthyroidism (34).

The present study found that the serum levels of nesfatin-1, an anorexic hormone, were increased in hyperthyroidism and decreased after euthyroidism occurred, ultimately becoming similar to those of the control group. There are two recently published studies that were contrary to these results. In a study from Poland, published in Polish, the nesfatin-1 levels were reported to be lower in children with hyperthyroidism (20). However, in a cross-sectional study, the nesfatin-1 levels were investigated in overt hyperthyroid patients, subclinical hyperthyroid patients, and healthy subjects in a larger study group compared to those in the present study (21). These authors found no significant differences in nesfatin-1 levels among the groups after adjusting for age and BMI (21). It is not clear whether patients with other causes of hyperthyroidism, such as subacute thyroiditis, were included in that study (21). However, in the present study with a prospective design, the patient group consisted only of overt hyperthyroid patients due to Graves' disease. Consequently, the cause of the discrepancy among different studies may be the different design, analytical and statistical methods, the etiology of hyperthyroidism, disease duration, and inclusion criteria. Moreover, possible various systemic effects of nesfatin, as discussed above (15 –19), may also lead to different results.

In the present study, the nesfatin-1 levels also increased as fT3 and fT4 increased; a negative correlation was found between TSH and nesfatin-1 levels. These findings support the result that nesfatin-1 levels increase during hyperthyroidism. Interestingly, Gungunes et al. observed a statistically significant correlation between nesfatin-1 and fT3, which is similar to the present result (21). Moreover, a negative correlation was reported between plasma nesfatin-1 levels and TSH in patients with type 2 diabetes in a recent study by Liu et al. (36).

There are studies suggesting that nesfatin-1 and orexin-A may be related to many disorders in addition to appetite (10,11,16,37). Moreover, orexin-A has pleiotropic effects, including increasing body temperature, heart rate, and blood pressure by stimulating the sympathetic nervous system (9,38). It has been shown that after a central orexin-A injection, body temperature increases and UCP-1 synthesis, which is thought to be a biomarker of peripheral thermogenesis, remains unaffected in mice (38). In another study conducted with mice, it has been shown that a central injection of orexin-A increased BMR rather than food intake (39). However, there was a negative correlation between orexin-A and BMR in the present study. The fact that the orexin-A level was found to be low in the present study might be a compensatory mechanism for BMR, which was increased in hyperthyroidism. Further studies are needed to confirm the current results and investigate the mechanism of orexin-A and nesfatin-1 alterations in hyperthyroidism.

No significant differences were observed in the levels of AgRP and NPY between the hyperthyroid and control groups. Similarly, the levels of these hormones did not change with treatment. AgRP and NPY are among the strongest orexigenic substances (40). However, peripheral circulating levels of AgRP and NPY may not reflect their central effects in hyperthyroidism.

No relationship was found with serum leptin and other measured peptides. Similar to AgRP and NPY, the serum leptin levels were not different between hyperthyroid patients, euthyroid patients, and the control group in the present study. Although some previous studies have reported increased or decreased circulating leptin levels in hyperthyroidism, the majority of the studies showed that the leptin levels were unchanged, suggesting that circulating leptin levels may not be affected by hyperthyroidism (22,23).

As far as the authors know, this is the first study to analyze plasma orexin-A, NPY, and AgRP in hyperthyroid patients and the first prospective study of serum levels of nesfatin-1 in these patients. Only the peripheral levels of the investigated peptides were examined because of the in vivo nature of the study. The biological effects of these peptides occur mostly via their effect on specific areas of the central nervous system. Additionally, peripheral receptors might contribute to this very complex and poorly understood mechanism. For example, the expression of NPY receptors in adipose tissue was shown to be correlated with the serum NPY levels (41). The physiological significance of the levels of the investigated peptides in the peripheral circulation has not been well described. However, nesfatin-1 level in plasma has been shown to be correlated with nesfatin-1 in cerebrospinal fluid in a previous human study (42). Additionally, Strawn et al. showed that the levels of orexin-A were strongly correlated with their measurements in plasma and cerebrospinal fluid (43). Further research is still required to clarify this issue. Moreover, the relatively small number of subjects is a limitation of the present study. Studies with larger numbers of patients with hyperthyroidism investigating orexin-A and nesfatin-1 levels may support the importance of these results.

It was conclusively found that the levels of orexin-A, which are known to increase appetite, were low and that the level of anorexigenic nesfatin-1 was high in the circulation. However, energy intake was high and BMR was low in patients with overt hyperthyroidism. Moreover, the orexin-A and nesfatin-1 levels were similar to those of the control group after euthyroidism was provided in patients with hyperthyroidism. The levels of AgRP, NPY, and leptin did not change in hyperthyroidism. The investigated peptide levels in this study do not explain the reason of increased appetite during hyperthyroidism. The levels of orexin-A and nesfatin-1 were markedly affected by hyperthyroidism, but the mechanism of these alterations in orexin-A and nesfatin-1 is not yet known.

Footnotes

Acknowledgments

We gratefully acknowledge Sevilay Karahan for her help in statistical analysis. This project was supported by Gazi University Research Foundation (BAP -01/2012-42).

Author Disclosure Statement

The authors have nothing to disclose.

References

Santini

, Marzullo

, Rotondi

, Ceccarini

, Pagano

, Ippolito

, Chiovato

, Biondi

. 2014. Mechanisms in endocrinology: the crosstalk between thyroid gland and adipose tissue: signal integration in health and disease. Eur J Endocrinol, 171:R137–R1522.

López

, Alvarez

, Nogueiras

, Diéguez

. 2013. Energy balance regulation by thyroid hormones at central level. Trends Mol Med, 19:418–427.

Herwig

, Campbell

, Mayer

, Boelen

, Anderson

, Ross

, Mercer

, Barrett

. 2014. A thyroid hormone challenge in hypothyroid rats identifies T3 regulated genes in the hypothalamus and in models with altered energy balance and glucose homeostasis. Thyroid, 24:1575–1593.

Russell

, Small

, Sunter

, Morgan

, Dakin

, Cohen

, Bloom

. 2002. Chronic intraparaventricular nuclear administration of orexin A in male rats does not alter thyroid axis or uncoupling protein-1 in brown adipose tissue. Regul Pept, 104:61–68.

Sellayah

, Sikder

. 2013. Feeding the heat on brown fat. Ann N Y Acad Sci, 1302:11–23.

López

, Seoane

, Señarís

, Diéguez

. 2001. Prepro-orexin mRNA levels in the rat hypothalamus, and orexin receptors mRNA levels in the rat hypothalamus and adrenal gland are not influenced by the thyroid status. Neurosci Lett, 300:171–175.

Fronczek

, Overeem

, Reijntjes

, Lammers

, van Dijk

, Pijl

. 2008. Increased heart rate variability but normal resting metabolic rate in hypocretin/orexin-deficient human narcolepsy. J Clin Sleep Med, 4:248–254.

Szlachcic

, Majka

, Strzalka

, Szmyd

, Pajdo

, Ptak-Belowska

, Kwiecien

, Brzozowski

. 2013. Experimental healing of preexisting gastric ulcers induced by hormones controlling food intake ghrelin, orexin-A and nesfatin-1 is impaired under diabetic conditions. A key to understanding the diabetic gastropathy?. J Physiol Pharmacol, 64:625–637.

Girault

, Yi

, Fliers

, Kalsbeek

. 2012. Orexins, feeding, and energy balance. Prog Brain Res, 198:47–64.

10.

Tsujino

, Sakurai

. 2013. Role of orexin in modulating arousal, feeding, and motivation. Front Behav Neurosci, 7:28.

11.

Giardino

, de Lecea

. 2014. Hypocretin (orexin) neuromodulation of stress and reward pathways. Curr Opin Neurobiol, 29:103–108.

12.

Ishii

, Kamegai

, Tamura

, Shimizu

, Sugihara

, Oikawa

. 2003. Hypothalamic neuropeptide Y/Y1 receptor pathway activated by a reduction in circulating leptin, but not by an increase in circulating ghrelin, contributes to hyperphagia associated with triiodothyronine-induced thyrotoxicosis. Neuroendocrinology, 78:321–330.

13.

Mitsuma

, Hirooka

, Mori

, Kayama

, Adachi

, Rhue

, Ping

, Nogimori

. 1999. Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm Metab Res, 31:606–609.

14.

García-Galiano

, Navarro

, Gaytan

, Tena-Sempere

. 2010. Expanding roles of NUCB2/nesfatin-1 in neuroendocrine regulation. J Mol Endocrinol, 45:281–290.

15.

, Zhang

, Tang

, Bi

, Liu

. 2010. The novel function of nesfatin-1: antihyperglycemia. Biochem Biophys Res Commun, 391:1039–1042.

16.

Emmerzaal

, Kozicz

. 2013. Nesfatin-1; implication in stress and stress-associated anxiety and depression. Curr Pharm Des, 19:6941–6948.

17.

, Wang

, Chen

, Guan

, Jiang

. 2010. Fasting plasma levels of nesfatin-1 in patients with type 1 and type 2 diabetes mellitus and the nutrient related fluctuation of nesfatin-1 level in normal humans. Regul Pept, 159:72–77.

18.

Aydin

. 2013. Multi-functional peptide hormone NUCB2/nesfatin-1. Endocrine, 44:312–325.

19.

Shimizu

, Mori

. 2013. Nesfatin-1: its role in the diagnosis and treatment of obesity and some psychiatric disorders. Methods Mol Biol, 963:327–338.

20.

Sawicka

, Bossowski

. 2013. Analysis of serum levels of nesfatin-1 in children and adolescents with autoimmune thyroid diseases. Pediatr Endocrinol Diabetes Metab, 19:5–10.

21.

Gungunes

, Ozbek

, Ginis

, Sahin

, Demirci

, Cakir Ozkaya

, Karbek

, Sayki Arslan

. 2014. Serum nesfatin-1 levels in overt and subclinical hyperthyroidism. Minerva Endocrinol, 39:209–214.

22.

Iglesias

, Díez

. 2007. Influence of thyroid dysfunction on serum concentrations of adipocytokines. Cytokine, 40:61–70.

23.

Feldt-Rasmussen

. 2007. Thyroid and leptin. Thyroid, 17:413–419.

24.

Guo

, Bakal

, Minokoshi

, Hollenberg

. 2004. Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo . Endocrinology, 145:2221–2227.

25.

Mantzoros

, Ozata

, Negrao

, Suchard

, Ziotopoulou

, Caglayan

, Elashoff

, Cogswell

, Negro

, Liberty

, Wong

, Veldhuis

, Ozdemir

, Gold

, Flier

, Licinio

. 2001. Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans. J Clin Endocrinol Metab, 86:3284–3291.

26.

Moretto

, Pedro

, Leite

, Romaldini

. 2012. Evaluation of body weight in patients with Graves' disease during the treatment with methimazole. Arq Bras Endocrinol Metabol, 56:364–369.

27.

De la Rosa

, Hennessey

, Tucci

. 1997. A longitudinal study of changes in body mass index and total body composition after radioiodine treatment for thyrotoxicosis. Thyroid, 7:401–405.

28.

Ebert

. 2010. The thyroid and the gut. J Clin Gastroenterol, 44:402–406.

29.

Lönn

, Stenlöf

, Ottosson

, Lindroos

, Nyström

, Sjöström

. 1998. Body weight and body composition changes after treatment of hyperthyroidism. J Clin Endocrinol Metab, 83:4269–4273.

30.

Goran

. 2000. Energy metabolism and obesity. Med Clin North Am, 84:347–362.

31.

Levine

, Nygren

, Short

, Nair

. 2003. Effect of hyperthyroidism on spontaneous physical activity and energy expenditure in rats. J Appl Physiol (1985), 94:165–170.

32.

Lahesmaa

, Orava

, Schalin-Jäntti

, Soinio

, Hannukainen

, Noponen

, Kirjavainen

, Iida

, Kudomi

, Enerbäck

, Virtanen

, Nuutila

. 2014. Hyperthyroidism increases brown fat metabolism in humans. J Clin Endocrinol Metab, 99:E28–35.

33.

Tauveron

, Grizard

, Thieblot

, Bonin

. 1992. Metabolic adaptation in hyperthyroidism. Implication of insulin. Diabete Metab, 18:131–136.

34.

Altinova

, Törüner

, Aktürk

, Elbeğ

, Yetkin

, Cakir

, Arslan

. 2006. Reduced serum acylated ghrelin levels in patients with hyperthyroidism. Horm Res, 65:295–299.

35.

Kosowicz

, Baumann-Antczak

, Ruchała

, Gryczyñska

, Gurgul

, Sowiñski

. 2011. Thyroid hormones affect plasma ghrelin and obestatin levels. Horm Metab Res, 43:121–125.

36.

Liu

, Yang

, Gao

, Liu

, Chen

. 2014. Decreased plasma nesfatin-1 level is related to the thyroid dysfunction in patients with type 2 diabetes mellitus. J Diabetes Res. 128014.

37.

Celik

, Gurger

, Can

, Balin

, Gul

, Kobat

, Gumusay

, Sahan

, Bursal

, Celiker

, Aydin

. 2013. The effect of nesfatin-1 levels on paroxysmal supraventricular tachycardia. J Investig Med, 61:852–855.

38.

Yoshimichi

, Yoshimatsu

, Masaki

, Sakata

. 2001. Orexin-A regulates body temperature in coordination with arousal status. Exp Biol Med (Maywood), 226:468–476.

39.

Lubkin

, Stricker-Krongrad

. 1998. Independent feeding and metabolic actions of orexins in mice. Biochem Biophys Res Commun, 253:241–245.

40.

Williams

, Harrold

, Cutler

. 2000. The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box. Proc Nutr Soc, 59:385–396.

41.

Sitticharoon

, Chatree

, Churintaraphan

. 2013. Expressions of neuropeptide Y and Y1 receptor in subcutaneous and visceral fat tissues in normal weight and obese humans and their correlations with clinical parameters and peripheral metabolic factors. Regul Pept, 185:65–72.

42.

Tan

, Hallschmid

, Kern

, Lehnert

, Randeva

. 2011. Decreased cerebrospinal fluid/plasma ratio of the novel satiety molecule, nesfatin-1/NUCB-2, in obese humans: evidence of nesfatin-1/NUCB-2 resistance and implications for obesity treatment. J Clin Endocrinol Metab, 96:E669–673.

43.

Strawn

, Pyne-Geithman

, Ekhator

, Horn

, Uhde

, Shutter

, Baker

, Geracioti

Jr . 2010. Low cerebrospinal fluid and plasma orexin-A (hypocretin-1) concentrations in combat-related posttraumatic stress disorder. Psychoneuroendocrinology, 35:1001–1007.