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Metabolism www.metabolismjournal.com

Influence of thyroid dysfunction on serum levels of angiopoietin-like protein 6 Jung Ah. Lim a, b , Hyo Jeong Kim c , Hwa Young Ahn a, d , Kyoung Un Park e , Ka Hee Yi a , Do Joon Park a , Hak Chul Jang a , Young Joo Park a,⁎ a

Department of Internal Medicine, Seoul National University College of Medicine, Seoul Department of Internal Medicine, National Medical Center, Seoul c Department of Internal Medicine, Eulji University School of Medicine, Eulji Medical Center, Seoul d Department of Internal Medicine, Chung-Ang University Hospital, Seoul e Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea b

A R T I C LE I N FO

AB S T R A C T

Article history:

Angiopoietin-like protein 6 (ANGPTL6) is a novel metabolic regulator that modulates energy

Received 10 February 2015

expenditure as well as glucose and lipid metabolism. Thyroid hormone can induce

Accepted 19 June 2015

metabolic changes that are similar to those induced by ANGPTL6. Herein, we investigated whether circulating ANGPTL6 levels change according to thyroid hormone status in

Keywords:

humans. We measured the serum levels of ANGPTL6 and metabolic parameters in 150

Thyroid

drug-naïve subjects with overt hyperthyroid, subclinical hyperthyroid, euthyroid,

Angiopoietin-like 6 protein

subclinical hypothyroid, or overt hypothyroid status (n = 30 in each group). Serum

Human

ANGPTL6 levels were significantly higher in patients with overt hypothyroidism than in

Adipokines

the other subjects. Women had significantly higher serum levels of ANGPTL6 than men. ANGPTL6 levels correlated positively with thyroid stimulating hormone (TSH), total cholesterol, aspartate aminotransferase, and alanine aminotransferase (ALT) and negatively with serum free thyroxine (T4) level. Multiple stepwise linear regression analysis revealed that sex, TSH, free T4, and ALT were independent predictors of serum ANGPTL6 levels. In summary, serum ANGPTL6 levels increased in patients with a hypothyroid status, and both TSH and free T4 levels are associated with ANGPLT6 levels, suggesting a possible association between thyroid function and ANGPTL6 levels. Whether the upregulated ANGPTL6 level in the hypothyroid status is primarily owing to a direct association or a compensatory mechanism remains to be determined © 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Several adipocyte- and hepatocyte-derived factors, the socalled adiopokines and hepatokines, are a critical link between obesity and metabolic disease [1,2]. A novel

hepatokine, angiopoietin-like protein 6 (ANGPTL6) has been introduced as a member of the ANGPTL family, whose members are circulating orphan peptides secreted by the liver [3,4]. It is also known as angiopoietin growth factor, which induces angiogenesis and skin cell proliferation, and

Abbreviations: ANGPTL6, angiopoietin-like protein 6; TSH, thyroid stimulating hormone; AST, aspartate aminotransferase; ALT, alanine aminotransferase; T4, thyroxine; BMI, body mass index; LDL, low-density lipoprotein. ⁎ Corresponding author at: Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu,Seoul 110-744, Korea. Tel.:+82 2 2072 4183; fax:+82 2 762 2292. E-mail address: [email protected] (Y.J. Park). http://dx.doi.org/10.1016/j.metabol.2015.06.015 0026-0495/© 2015 Elsevier Inc. All rights reserved.

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thereby wound healing [3,5]. It was recently shown that ANGPTL6 is involved in the development of obesity and its related insulin resistance, and ANGPTL6 deficiency led to reduced energy expenditure in a mouse model [6]. In accordance with these findings, the mice with transgenic ANGPTL6 overexpression were lean and insulin sensitive and they showed increased energy expenditure and resistance to high fat diet-induced obesity, insulin resistance, and nonadipose tissue steatosis [6]. However, in contrast to the rodent data, human data showed elevated ANGPLT6 levels in patients with metabolic syndrome, preeclampsia, polycystic ovary syndrome, or type 2 diabetes mellitus [7–12]. This paradoxical elevation has been understood as ANGPTL6 resistance or a compensatory mechanism [9,10,12]. Thyroid hormone has an important role in different physiological processes such as development, growth, and metabolic control [13]. It has been recognized for more than 100 years that thyroid hormone increases obligatory thermogenesis [14], although the mechanisms involved are poorly understood. In fact, hypothyroid animals and patients typically experience a 20%–30% reduction in the resting metabolic rate and are more likely to develop hypothermia, whereas treatment with thyroid hormone increases the oxygen consumption in most tissues [13,15]. It has been suggested that adipokines and hepatokines as well as thyroid hormones share some physiological actions such regulating energy expenditure or lipid and glucose metabolism [16,17]. Because both thyroid hormones and ANGPTL6 are important regulators of energy metabolism, it could be hypothesized that the thyroid hormone status influences ANGPTL6 regulation. However, there are no human studies about the correlation between serum ANGPTL6 levels and thyroid hormones to date. Thus, here we evaluated the influence of the thyroid hormone status on serum ANGPTL6 levels in humans.

2.

Material and Methods

2.1.

Study Population

The medical records of consecutive subjects without previous history of thyroid disease who underwent a self-referred health checkup at the Seoul National University Bundang Hospital (SNUBH) Health Promotion Center between January 2008 and December 2009 were reviewed. We excluded subjects who presented with any known chronic disease requiring treatment, such as diabetes mellitus or chronic liver disease. We ultimately included 150 subjects (52 men, 98 women; mean age, 43.6 ± 10.0 years). The patients were then divided into overt hyperthyroid, subclinical hyperthyroid, euthyroid, subclinical hypothyroid, and overt hypothyroid groups (n = 30 each). The study protocol was approved by the Institutional Review Board of SNUBH (IRB No. B-0801-053-015).

2.2.

Anthropometric and Biochemical Measurements

The height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively, and the body mass index (BMI) was calculated as weight divided by height squared (kg/m2) at the

time of blood sampling. A venous blood sample was taken after a minimum 12-h fast. Serum thyroid-stimulating hormone (TSH) and free thyroxine (T4) were measured by using immunoradiometry with commercial kits (TSH, CIS Bio International, Gif-sur-Yvette, France; free T4, DiaSorin, Saluggia, Italy). Euthyroidism was defined as normal levels of TSH (range, 0.4–4.5 mIU/L) and free T4 (range, 0.7–1.5 ng/dL). Overt hyperthyroidism was defined as a TSH level < 0.4 mIU/L and a free T4 level > 1.5 ng/dL; subclinical hyperthyroidism was defined as a TSH level < 0.4 mIU/L and a free T4 level within the normal range. Overt hypothyroidism was defined as a TSH level > 4.5 mIU/L and a free T4 level < 0.7 ng/dL; subclinical hypothyroidism was defined as a TSH level > 4.5 mIU/L and a free T4 level within the normal range. The levels of glucose, total cholesterol, triglycerides, lowdensity lipoprotein (LDL) cholesterol, high-density lipoprotein cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using the Toshiba 200FR Neo Chemistry autoanalyzer (Toshiba Medical Systems, Tokyo, Japan). Serum ANGPTL6 levels were measured by using a Luminex® Multiplex assay using MILLIPLEX MAP Human Liver Protein Magnetic Bead Panel (Millipore, MO, USA). The system had an inter-assay coefficient of variation (CV) of 7.5%–18.4% and an intra-assay CV of 1.5%–3.3%.

2.3.

Statistical Analysis

All normally distributed continuous variables are expressed as means ± SD, and variables with a skewed distribution are expressed as median and range. Variables with skewed distribution were log-transformed for the statistical analysis. Baseline characteristics were compared between groups using analysis of variance (ANOVA). To evaluate the differences in the levels of ANGPTL6 in various states of thyroid function, ANOVA with Bonferroni’s post hoc analysis was applied. Pearson’s correlation analysis identified significant correlations between ANGPTL6 and various metabolic parameters. Stepwise linear regression analysis was used to identify independent predictors of ANGPTL6 levels. Statistical analyses were performed using SPSS for Windows version 16.0. (SPSS, Chicago, IL, USA). Values of P < 0.05 were considered significant.

3.

Results

3.1. Subjects’ Clinical Characteristics and Serum ANGPTL6 Levels The subjects’ clinical characteristics are summarized in Table 1. The mean age did not differ among groups. The female to male ratio was higher in the overt hypothyroid subjects than in the others (P = 0.003). The mean BMI was comparable in all five groups. However, the lipid profiles and serum level of AST and ALT differed significantly among the groups: serum levels of total cholesterol, LDL-cholesterol, triglycerides, and AST were significantly higher in the overt hypothyroid subjects than in the other subjects (Table 1). The significance remained similar after the adjustment for sex (data not shown).

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Table 1 – Clinical characteristics of the subjects.

Age (years) Sex M/F (female %) Weight (kg) BMI (kg/m2) TSH (mIU/L) Free T4 (ng/dL) Glucose (mg/dL) Triglyceride (mg/dL) Total cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) AST (IU/L) ALT (IU/L)

Overt hyperthyroidism

Subclinical hyperthyroidism

Euthyroid

Subclinical hypothyroidism

Overt hypothyroidism

45.0 ± 12.2 8/22 (73.3)

45.1 ± 11.3 12/18 (60.0)

42.9 ± 7.4 12/18 (60.0)

39.7 ± 10.4 17/13 (43.3)

45.1 ± 8.7 a-d 3/27 (90.0)

60.7 ± 10.8 23.4 ± 4.2 0.02 ± 0.07 1.95 (1.59–3.80) 87.9 ± 9.5 84.4 ± 44.7 162.4 ± 30.4 51.1 ± 10.9 84.1 ± 25.1 24.4 ± 11.1 30.8 ± 21.8

60.8 ± 10.0 22.5 ± 3.2 0.19 ± 0.15 1.28 (0.94–1.92) 82.4 ± 11.6 84.6 ± 38.7 185.8 ± 30.9 57.8 ± 12.4 102.0 ± 25.1 19.4 ± 5.4 18.0 ± 8.3 a

60.5 ± 10.2 22.4 ± 2.7 1.66 ± 0.76 1.11 (0.97–1.35) 85.1 ± 7.7 79.0 ± 33.9 185.2 ± 23.7 61.4 ± 14.6 91.9 ± 18.5 21.4 ± 5.2 20.3 ± 7.5 a

65.8 ± 12.3 23.3 ± 3.1 7.38 ± 2.25 1.00 (0.75–1.33) 89.4 ± 6.9 b 121.5 ± 75.1 194.7 ± 31.8 a 54.7 ± 12.7 107.6 ± 25.1 a 19.4 ± 3.5 a 20.3 ± 7.0 a

61.8 ± 8.4 23.7 ± 2.8 86.7 ± 19.4 0.23 (0.10–0.47) 86.7 ± 6.2 156.2 ± 120.5 a-c 236.5 ± 29.9ad 58.3 ± 16.3 140.9 ± 28.3 a-d 27.4 ± 5.1 b-d 28.1 ± 7.4 b

Except for the sex, the p values are from ANOVA with Bonferroni’s post hoc analysis. M, male; F, female; BMI, body mass index; TSH, thyroid stimulating hormone; HDL. high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine transaminase. a p < 0.05 compared with overt hyperthyroidism. b p < 0.05 compared with subclinical hyperthyroidism. c p < 0.05 compared with normal thyroid. d p < 0.05 compared with subclinical hypothyroidism.

The mean serum ANGPTL6 levels were 50.8 ng/mL, 44.1 ng/mL, 57.6 ng/mL, 37.8 ng/mL, and 79.5 ng/mL in the overt hyperthyroid, subclinical hyperthyroid, euthyroid, subclinical hypothyroid, and overt hypothyroid subjects, respectively. ANOVA with post hoc analysis revealed that ANGPTL6 levels in subjects with overt hypothyroidism were significantly higher than in those in other groups (P < 0.001; Fig. 1). The serum ANGPTL6 levels were significantly higher in

women than in men (median 54.5 [11.8–189.0] ng/mL vs. 43.9 [18.9–89.9] ng/mL, P = 0.002).

3.2. Correlation Between ANGPTL6 Levels and the Thyroid Function Status We next investigated whether serum ANGPLT6 levels were correlated with biochemical parameters or thyroid hormone levels (Table 2). In all subjects, serum ANGPTL6 was positively associated with serum TSH (r = 0.25, P = 0.002), total cholesterol (r = 0.20, P = 0.01), AST (r = 0.27, P = 0.001), and ALT level (r = 0.18, P = 0.03). The ANGPTL6 level correlated inversely with the free T4 level (r = − 0.28, P < 0.001). We then performed a stratified analysis to exclude the effects of sex. In women, the serum TSH (r = 0.366, P < 0.001) and AST levels (r = 0.266, P = 0.009) showed similar correlations with ANGPTL6 levels. Age (r = −0.221, P = 0.031) and serum free T4 level (r = − 0.395, P < 0.001) were also negatively associated with ANGPTL6 levels. However, no correlation between serum ANGPTL6 and thyroid hormones was observed in men (Table 2).

3.3.

Fig. 1 – Serum ANGPTL6 level according to functional thyroid status.ANGPTL6, angiopoietin-like protein 6. * p < 0.05 compared with overthypothyroidism, † p < 0.05 compared with overthypothyroidism, ‡ p < 0.05 compared with overthypothyroidism, || p < 0.05 compared with overthypothyroidism.

Factors Affecting Serum ANGPTL6 Levels

To determine the factors affecting serum ANGPTL6 levels, multivariate stepwise linear correlation analysis was performed. Serum TSH and free T4 were separately analyzed using different models because they can influence each other. Since there were fewer men than women, we did not stratify the subjects by sex; rather, we used sex as a covariant. Serum TSH level remained positively and free T4 level remained negatively associated with circulating ANGPTL6 levels after adjustment for sex (Table 3, Model 1 and 2). Sex and serum TSH and ALT levels were analyzed as statistically significant factors affecting circulating ANGPTL6 levels independent of other components, including age, BMI, serum total cholesterol, and AST levels (Table 3, Model 3). In addition, serum free

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Table 2 – Correlations between serum ANGPTL6 levels and various metabolic parameters. Total

Age BMI TSH a Free T4 Glucose Triglyceride a Total cholesterol HDL-cholesterol LDL-cholesterol AST ALT

Men

Women

r

P value

r

P value

r

P value

−0.11 −0.11 0.25 −0.28 −0.06 0.04 0.20 0.13 0.13 0.27 0.18

0.19 0.23 0.002

Influence of thyroid dysfunction on serum levels of angiopoietin-like protein 6.

Angiopoietin-like protein 6 (ANGPTL6) is a novel metabolic regulator that modulates energy expenditure as well as glucose and lipid metabolism. Thyroi...
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