Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 5 ) X XX– X XX

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Metabolism www.metabolismjournal.com

Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise Doaa M. Samy a,⁎, Cherine A. Ismail b , Rasha A. Nassra c a b c

Department of Medical Physiology, Faculty of Medicine, Al-Moassat Hospital, University of Alexandria, Alexandria, Egypt Department of Clinical Pharmacology, Faculty of Medicine, Al-Moassat Hospital, University of Alexandria, Alexandria, Egypt Department of Medical Biochemistry, Faculty of Medicine, Al-Moassat Hospital, University of Alexandria, Alexandria, Egypt

A R T I C LE I N FO Article history:

AB S T R A C T Objectives. The speculation that the myokine irisin could regulate whole body energy

Received 1 October 2014

expenditure led to the anticipation that irisin may have therapeutic potential in metabolic

Accepted 4 January 2015

diseases. Regulation of irisin under conditions of metabolic derangements in altered thyroid status, and the changes in irisin response to exercise remain to be investigated.

Keywords:

Methods. Serum irisin concentration was measured in sixty male Wistar rats subjected to

Irisin

either sedentary life or 8-week chronic swimming exercise after induction of hyper- or

Hyperthyroid

hypothyroidism (10 rats/group). The effect of acute exercise on serum irisin was assessed in

Hypothyroid

10 additional rats subjected once to forced swimming against a load (5% of body weight) and

Exercise

compared to sedentary rats.

Oxidative stress

Results. Serum irisin was significantly higher in both sedentary hyper- and hypothyroid rats (by 45%, p < 0.001, and 30%, p < 0.001, respectively) versus euthyroid controls. Serum irisin also increased after acute exercise (p < 0.001 versus sedentary control). Chronic training episodes failed to significantly alter serum irisin in all thyroid hormone profiles. Serum irisin correlated positively with serum creatine kinase (r = 0.45, p < 0.001) and with muscle and liver concentrations of malondialdehyde (r = 0.50 and r = 0.47 respectively, p < 0.001 for both), and negatively with muscle and liver content of reduced glutathione (r = −0.34, p = 0.003 and r = − 0.28, p = 0.018 respectively) in pooled groups. However, significance of these associations was waived when analyzing each group separately. Serum irisin was not associated with skeletal muscle mass, insulin resistance, blood glucose, lipids or TSH. Conclusions. Both hyper- and hypothyroidism are associated with up-regulation of serum irisin in male rats, possibly as a response to oxidative damage and/or myopathy observed in both conditions. Acute exercise, which is also associated with oxidative stress, increases serum irisin. No obvious association was detected linking serum irisin to metabolic abnormalities in thyroid dysfunction. © 2015 Elsevier Inc. All rights reserved.

Abbreviations: PPAR-γ, Peroxisome proliferator-activated receptor-gamma; PGC1α, PPAR-γ coactivator-1 α; FNDC5, Fibronectin type III domain-containing 5 protein; UCP1, Uncoupling protein 1; PTU, 6-n-propyl-2-thiouracil; TSH, Thyroid-stimulating hormone; T3, Triiodothyronine; T4, Thyroxine; IR, Insulin resistance; HOMA-IR, Homeostasis model assessment-insulin resistance; TC, Total cholesterol; TG, Triglycerides; LDL-C, Low density lipoprotein-cholesterol; HDL-C, High density lipoprotein-cholesterol; CK, Creatine kinase; SMM, Skeletal muscle mass; BW, Body weight; PBS, Phosphate buffered saline; GSH, Reduced glutathione; MDA, Malondialdehyde; ATP, Adenosine triphosphate; p38 MAPK, p38 mitogen-activated protein kinase. ⁎ Corresponding author. Tel.: + 20 301002892985. E-mail addresses: [email protected] (D.M. Samy), [email protected] (C.A. Ismail), [email protected] (R.A. Nassra). http://dx.doi.org/10.1016/j.metabol.2015.01.001 0026-0495/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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1.

Introduction

Physical exercise is one of the key non-pharmacological approaches for control of commonly encountered metabolic disorders [1–3]. Mechanisms by which exercise exerts its protective effects remain to be fully elucidated. Recently, it was suggested that during muscular exercise, peroxisome proliferator-activated receptor-gamma (PPAR-γ) coactivator-1α (PGC1α) expression is increased in muscle [3]. This induces the expression of fibronectin type-III domain-containing 5 (FNDC5), a membrane protein that is proteolytically cleaved to form the hormone, irisin [4]. In a landmark study, Boström et al. [4] showed that irisin, a newly identified myokine, induces browning of subcutaneous white adipocytes by increasing uncoupling protein-1 (UCP1), with subsequent stimulation of oxygen consumption and thermogenesis. This effect improved metabolic profile and increased wholebody energy expenditure [4,5]. Recently, Huh et al. [6] suggested that irisin could regulate muscle growth and improve adipocyte metabolism. Since irisin was found to be altered in obesity, diabetes and metabolic syndrome [5,7–9], it was postulated that irisin may have therapeutic potential in metabolic and muscle disorders. Although the initial data from irisin in mice were promising, little knowledge is available about other animal species. In humans, recent studies raised many questions about regulation and function of irisin, and its relation to exercise [10–12]. Thyroid hormones play a critical role in carbohydrate, lipid and protein metabolism; and regulate basal metabolic rate and thermogenesis [13]. Thyroid dysfunction, one of the most common endocrine diseases, is associated with metabolic imbalance, abnormal energy homeostasis, oxidative stress and muscle disorders [14–17]. Given that both irisin and thyroid hormones are linked to body energy expenditure, metabolism, and muscle physiology, we, therefore, hypothesized that serum irisin may be altered in states of thyroid dysfunction. Moreover, since irisin is assumed to be derived mainly from skeletal muscle, it is plausible that irisin could be one mechanism underlying the beneficial effects of exercise on metabolism, oxidative stress and myopathy [2,3]. In the present study, we investigated the alterations of irisin hormone in rat models of hypo-and hyperthyroidism and compared the impact of acute and chronic exercise on serum irisin.

2.

Materials and Methods

2.1.

Experimental Animals

2.2.1.

Protocol I

This experiment was designed to investigate changes in serum irisin, and metabolic and oxidative stress parameters, in hypo and hyperthyroid rats with or without chronic swimming exercise. Sixty rats were randomly divided into six groups (N = 10 each) (1) sedentary euthyroid controls, (2) an euthyroid group subjected to chronic swimming exercise, (3) a sedentary hypothyroid group, (4) hypothyroid rats subjected to chronic swimming exercise, (5) a sedentary hyperthyroid group, (6) hyperthyroid group subjected to chronic swimming exercise (Fig. 1A).

2.2.1.1. Thyroid Dysfunction. Hyperthyroidism and hypothyroidism were induced by adding 0.012% L-thyroxine and 0.05% of 6-n-propyl-2-thiouracil (PTU) to drinking water, respectively [18,19]. Both drugs were purchased from SigmaAldrich, St. Louis, MO. Three weeks later, thyroid dysfunction was confirmed by measuring serum thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4). Treatment was continued until 48 hrs before animal euthanization. After establishment of thyroid dysfunction, exercise groups were subjected to a chronic swimming exercise protocol, while sedentary groups were kept in their cages without intervention. 2.2.1.2. Chronic Swimming Exercise.

Animals were made to swim in a plastic tank (80 cm diameter/100 cm height/40 cm water depth) filled with water maintained at 35 ± 1 °C. Rats were habituated to the swimming exercise during the first week. Initially, rats swam for 15 min, with increments of additional 15 min daily, until a swimming period of one hr was attained. Subsequently, a daily swimming period of one hr, 5 times/week, was maintained for 8 weeks [20]. At the end of each exercise session, animals were dried and kept in a warm environment. All rats completed the training period except one hyperthyroid and one hypothyroid rat that were exhausted early and failed to continue the swimming program; those rats were replaced. Rats were sacrificed 48 hrs after last exercise session to minimize acute effects of exercise.

2.2.2.

Protocol II

To evaluate the effects of acute exercise on serum irisin, a forced swim test was performed once to a single group of 10 rats and results were compared with those of the sedentary euthyroid group in protocol I.

2.2.2.1. Acute Swimming Exercise.

Adult male Wistar rats (200–220 g) were supplied by the Animal Experiment Centre of Alexandria University. Animals received care in compliance with national animal care guidelines approved by the Faculty of Medicine, Alexandria University Ethics Committee. Animals were maintained in a 12 hr light/dark cycle with free access to standard rat chow and water.

Before performing forced swim test, rats were acclimated to swimming for 10 min/day for 3 days, followed by 2-day rest, to wash out any preconditioning training effects. On the test day, rats were fasted for 12 hrs, then forced to swim against a load (5% of body weight) attached to the tail for 100 min, in the same tank conditions described above for chronic exercise [21]. These rats were immediately sacrificed at the end of the exercise period (Fig. 1B).

2.2.

2.3.

Experimental Protocols

After 1 week of acclimatization, rats were randomized to one of 2 protocols.

Blood Sampling and Biochemical Analysis

At the end of experimental period, sedentary and chronic exercise rats were fasted for 12 hrs, and then subjected to

Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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habituation

A

3

Swim 1h/d, 5d/wk

L-thyroxin, PTU or vehicle treatment Weeks

0

3

4

12

Check thyroid status

Sacrifice 48h after cessation of exercise and therapies

B Swim 10min/d

Days

0

1

2

rest

3

4

5 Swimming for 100 min then sacrifice

Fig. 1 – Experimental design of the study; L-thyroxine and 6-n-propyl-2-thiouracil (PTU) were supplemented to induce rat models of thyroid dysfunction. Three weeks later, thyroid function tests were carried out to confirm thyroid hormone conditions. Then, rats were kept sedentary or subjected to swimming training (1 hr/day, 5 days/week) for 8 weeks after one week of gradual habituation to exercise (n = 10/group). After 48 hrs, rats were sacrificed for biochemical tests (A). Ten euthyroid rats were acclimated to swimming for 10 min/day for 3 days, followed by 2 days rest. Then, animals were forced to swim against a load (5% of body weight) attached to the tail for 100 min. The rats in this group were immediately sacrificed following exercise (B). terminal anesthesia using thiopental (40 mg/kg). Blood was collected by cardiac puncture and immediately centrifuged at 3000 rpm for 10 min. Fasting serum glucose was measured by an enzymatic colorimetric method, and the remaining serum aliquots were stored at −20 °C until further biochemical analysis.

2.3.1.

Measurement of Serum Irisin Concentrations

Serum irisin was determined using a commercial enzymelinked immunosorbent assay kit following the manufacturer's instructions (Irisin EIA kit EK-067–16; Phoenix Pharmaceuticals, Burlingame, CA), on a spectrophotometric reader at a wavelength of 450 nm. This test provided a range of detection of 0.1–1000 ng/mL and had a coefficient of variation of 6–10% inter- and intra-assay.

the formation of NADPH in a rate directly proportionate to CK activity [24] and measured bi-chromatically at 340 and 540 nm; with an analytical measurement range of 7–1000 U/L.

2.4.

Immediately after animals were sacrificed, the gastrocnemius, soleus and extensor digitorum longus muscles were carefully dissected out and weighed using a digital scale. The sum of their wet weights was calculated as a measure for skeletal muscle mass (SMM). These muscles were chosen since they are heavily involved in swimming and are affected by thyroid states in rats [25]. All weights were expressed in mg/g body weight (BW).

2.5. 2.3.2.

Lipid Profile

Serum total cholesterol (TC) and triglycerides (TG) were assayed using enzymatic colorimetric methods. HDL-cholesterol (HDL-C) was analyzed using NS Biotec HDL-precipitating reagent. LDL-cholesterol (LDL-C) was calculated using Friedewald formula [23]: LDL-C (mg/dL) = TC – HDL-C – (TG/5)

2.3.4.

Oxidative Stress in Liver and Skeletal Muscle

Serum Insulin and Calculation of Insulin Resistance

Fasting serum insulin was quantified using Rat Insulin ELISA Kit following the manufacturer's instructions (WKEA MED Supplies, NY, USA). Insulin resistance (IR) of individual rats was evaluated using the homeostasis model assessment (HOMA-IR) index [22], calculated as: [HOMA-IR] = fasting serum glucose (mg/dL) × fasting serum insulin (μU/mL)/405.

2.3.3.

Skeletal Muscle Mass

Creatine Kinase

As a marker for muscle damage, serum total creatine kinase (CK) was assayed via a coupled enzyme reaction resulting in

Liver and gastrocnemius muscle tissues were immediately frozen in liquid nitrogen and kept at −80 °C until homogenization. Tissue samples were weighed and a 10% homogenate was made with phosphate buffered saline (PBS) solution, pH 7.4. The tissue homogenates were centrifuged at 4000 rpm for 15 min. Supernatants were analyzed for reduced glutathione (GSH) and the oxidative damage marker, malondialdehyde (MDA), using commercial colorimetric kits (Biodiagnostic, Cairo, Egypt), according to the manufacturer’s instructions.

2.6.

Statistical analysis

Values are presented as mean ± S.E.M. All biochemical concentrations were normally distributed (according to the Shapiro– Wilk test). In protocol 1, groups were compared by one way ANOVA. When significant, ANOVA was followed by post-hoc pairwise comparisons using the least significant difference test.

Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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In protocol 2, unpaired t-test was used. Additionally, a general linear model was used to estimate mean serum irisin after adjustment for CK or oxidative stress parameters. Pearson correlations were used to test for associations of irisin with the different variables. Statistical analysis was performed using the Statistical Package for Social Sciences 20.0 for Windows (SPSS, Chicago. IL). P < 0.05 was considered statistically significant.

Chronic exercise had no effect on serum irisin in either thyroid status. In contrast, a single episode of acute exercise significantly increased serum irisin by 47% versus sedentary controls (p < 0.001) (Table 1). Irisin adjustment for oxidative stress parameters, but not for CK, reversed the effects of acute and chronic exercise on serum irisin (Supplementary Table 1).

3.1.3.

3.

Results

3.1.

Blood Biochemical Findings

3.1.1.

Serum TSH

Sedentary hyperthyroid rats showed significantly lower TSH, while hypothyroid rats had significantly higher TSH compared to euthyroid controls. Chronic exercise significantly increased TSH only in euthyroid and hypothyroid rats (Fig. 2A). Similarly, acute exercise increased serum TSH (Table 1).

Serum Irisin

TSH (mIU/L)

12

A

SEDENTARY ### EXERCISED ***

9

6

3

#

Euthyroid

CK (U/L)

3.2.

Skeletal Muscle Mass (SMM)

There was a significant reduction in SMM in sedentary hyperthyroid rats (p < 0.001), but not hypothyroid rats, compared to

800

*** **

400

200

0

Hyperthyroid

Euthyroid

Hypothyroid

D 10

Hyperthyroid

##

Hypothyroid

#

***

***

300

B

600

C

400

Serum Creatine Kinase (CK)

Both hyper- and hypothyroidism, as well as acute exercise caused muscle damage as shown by elevated CK. Chronic swimming significantly reduced CK in hyper- and hypothyroid rats (Table 1, Fig. 2C).

*

0

500

3.1.4.

Irisin (ng/mL)

As shown in Fig. 2B, sedentary hyperthyroid and hypothyroid rats had higher serum irisin (by 45%, p < 0.001 and 30%, p < 0.001, respectively) compared to sedentary euthyroid control. Yet, serum irisin levels were not significantly different between the two groups of thyroid dysfunction (P = 0.08). After adjusting for CK levels, the significant elevation in irisin disappeared in the hypothyroid but not the hyperthyroid groups. Adjustment for oxidative stress parameters in a separate model abolished the differences in irisin in both, hyper- and hypothyroid rats versus controls (Supplementary Table 1).

#

###

200 100 0

SMM (mg/g BW)

3.1.2.

Metabolic Parameters

Fig. 3 reveals that sedentary hyperthyroid rats showed higher plasma glucose (p = 0.019), and insulin (p < 0.001), with subsequently higher insulin resistance (HOMA-IR) (p < 0.001) versus euthyroid rats. Meanwhile, sedentary hypothyroid group exhibited higher values of serum TC (p < 0.001), TG (p < 0.001), and LDL-C (p < 0.001) versus euthyroid control rats. The 8-week swim training significantly reduced the increased HOMA-IR observed in hyperthyroid rats and plasma TC, TG and LDL-C in all thyroid states. Acute exercise reduced insulin resistance marker (p < 0.001) but had no impact on lipid profile (Table 1).

8

##

***

6 4 2 0

Euthyroid

Hyperthyroid Hypothyroid

Euthyroid

Hyperthyroid

Hypothyroid

Fig. 2 – Differences in serum TSH (A), serum irisin (B), creatine kinase (C) and skeletal muscle mass (SMM) (D) between sedentary and exercised groups of induced thyroid dysfunction. Values are means ± SEM (n = 10/group). *P < 0.05, **P < 0.01, ***P ≤ 0.001 versus sedentary euthyroid group; # P < 0.05, ## P < 0.01, ### P ≤0.001 versus corresponding sedentary group. Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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Table 1 – Effect of acute exercise on serum irisin, metabolic parameters, oxidative stress, TSH, CK and SMM in rats.

Serum irisin (ng/mL) Fasting glucose (mg/dL) Fasting insulin (IU/mL) HOMA-IR a Total-C b (mg/dL) Triglycerides (TG) (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Muscle MDA c (nmol/g tissue) l=Liver MDA (nmol/g tissue) Muscle GSH d (nmol/g tissue) Liver GSH (nmol/g tissue) TSH e (mIU/L) CK f (U/L) SMM (mg/g BW) g

Sedentary Control

Acute Exercise

P (T-Test)

409.48 101.7 7.61 1.91 94.4 67.3 42.6 38.3 54.0 44.8 30.9 84.4 0.850 184.5 7.16

601.68 78.3 5.79 1.13 93.0 61.9 42.5 38.1 79.8 76.0 19.8 55.2 1.216 250.8 6.79

<0.001 0.003 0.007 <0.001 0.839 0.190 0.976 0.976 0.001 <0.001 0.015 <0.001 0.011 0.017 0.311

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

21.54 5.9 0.43 0.142 5.2 2.7 2.4 6.6 4.8 4.7 3.0 4.2 0.134 13.0 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

27.40 3.6 0.42 0.109 4.3 2.9 2.2 2.8 4.9 5.3 2.8 5.7 0.097 21.5 0.19

Data represent mean ± SEM and analyzed by unpaired t-test; n = 10 per group. HOMA-IR, homeostatic model assessment of insulin resistance index; b C, cholesterol; c MDA, malondialdehyde; d GSH, reduced glutathione; e TSH, thyroid stimulating hormone; f CK, creatine kinase; g SMM, skeletal muscle mass; BW, body weight. a

30 0

D

4

#

9 6

90 60

##

##

3

1

0

0

E

***

80 60 40

30

20

0

0

60

# #

##

***

SEDENTARY ##

EXERCISED

2

3

## 120

C

***

12

100

***

B

40

20

0

F

80

LDL-C (mg/dL)

60

Muscle and Liver Oxidative Stress

Both hyper and hypothyroid rats had increased lipid peroxidation, assessed by muscle and liver MDA levels, although

HOMA-IR

90

15

3.3.

HDL-C (mg/dL)

*

120

150

Total-C (mg/dL)

A

Fasting Insulin (IU/mL)

150

TG (mg/dL)

Fasting glucose (mg/dL)

euthyroid controls. After an 8-week swimming protocol, SMM was significantly increased in all thyroid states versus sedentary counterparts (Fig. 2C). On the other hand, acute exercise did not significantly affect SMM (p = 0.484) (Table 1).

G

*** #

60 40 20

# #

0

Fig. 3 – Metabolic changes in sedentary and exercised groups of induced thyroid dysfunction; fasting glucose (A), fasting insulin (B), HOMA-IR (C) and lipid profile (D–G). Values are means ± SEM (n = 10/group). *P < 0.05, **P < 0.01, ***P ≤ 0.001 versus sedentary euthyroid group; #P < 0.05, ##P < 0.01, ###P ≤0.001 versus corresponding sedentary group. Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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A

100

***

80

##

*

60

## #

40 20

Liver MDA (nmol/g tissue)

Muscle MDA (nmol/g tissue)

100

0

C

Hyperthyroid

EXERCISED

80

## 60

# #

40 20

Hypothyroid

Euthyroid

D

###

120

50

##

##

40 30

**

20

*

10

Liver GSH (nmol/g tissue)

Muscle GSH (nmol/g tissue)

SEDENTARY

***

0 Euthyroid

60

B

Hyperthyroid

Hypothyroid

##

100

###

###

80

***

60 40 20 0

0 Euthyroid

Hyperthyroid

Hypothyroid

Euthyroid

Hyperthyroid

Hypothyroid

Fig. 4 – Oxidative stress in sedentary and exercised groups of induced thyroid dysfunction; muscle and liver malodialdehyde MDA (A and B) and reduced glutathione (GSH) content (C and D). Values are means ± SEM (n = 10/group). *P < 0.05, **P < 0.01, ***P ≤ 0.001 versus sedentary euthyroid group; #P < 0.05, ##P < 0.01, ###P ≤ 0.001 versus sedentary corresponding group.

the increase in liver MDA was not significant (p = 0.145). The muscle and liver contents of the antioxidant GSH were significantly reduced in sedentary hyper- and hypothyroid rats, versus euthyroid controls (Fig. 4). Chronic exercise

significantly reduced oxidative stress in all trained groups versus sedentary counterparts (Fig. 4). In contrast, acute forced exercise significantly increased oxidative stress parameters (Table 1).

Fig. 5 – Scatter diagram showing associations between serum irisin and oxidative stress parameters/serum creatine kinase (CK) in pooled groups (n = 70). Serum irisin is positively correlated with muscle/liver content of malodialdehyde (MDA) and CK (up); and negatively correlated with muscle and liver reduced glutathione (GSH) content (down). Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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3.4.

Correlations Between Serum Irisin and Other Variables

As shown in Fig. 5, a significant positive correlation was observed between serum irisin and CK (r = 0.45, p < 0.001) in the pooled groups. Serum irisin also correlated positively with muscle (r = 0.50, p < 0.001) and liver (r = 0.47, p < 0.001) MDA contents, and negatively with muscle and liver GSH contents (r = − 0.34, p = 0.003 and r = − 0.28, p = 0.018, respectively). Although these associations within each group were less consistent, we observed significant/borderline correlations between irisin and oxidative stress markers/creatine kinase in some groups, in line with the pooled data (Supplementary Table 2). Notably, serum irisin did not correlate with SMM, plasma glucose, insulin, HOMA-IR, lipids or TSH (Table 2).

4.

Discussion and Conclusion

Various aspects of irisin biology have been investigated yet its regulation and specific role are not completely understood [26]. We investigated the changes in serum irisin in response to altered thyroid function and physical exercise in adult rats. Both hyper- and hypothyroidism increased serum irisin, with a non-significant trend toward higher serum irisin in hyperthyroid versus hypothyroid rats. Chronic swimming exercise did not affect serum irisin in all thyroid states, while an acute exercise episode in euthyroid rats increased serum irisin.

Irisin concentrations were positively associated with oxidative stress in muscle and liver and with serum CK, in the pooled data and some but not all groups. However, serum irisin did not correlate with TSH, metabolic parameters, or SMM. Since oxidative stress and myopathy have been linked to hyperthyroidism, hypothyroidism and acute exercise [16,17,21], we postulate that irisin may increase in these settings as an adaptive response to oxidative stress and/or myopathy. In this study, we tracked the potential links between irisin and prominent pathophysiological insults common to both thyroid dysfunction models, namely myopathy, oxidative stress and metabolic disorders. Our data are partly consistent with a recent cross sectional human study comparing serum irisin in 20 hyper- and hypothyroid patients [27]. Ruchala et al. [27] reported a borderline elevation of serum irisin in hyperthyroid versus hypothyroid patients. They reported a negative correlation between serum irisin and CK, linking their finding to the muscle damage-induced by hypothyroidism, hence raising CK and decreasing serum irisin. Nevertheless, both hypo- and hyperthyroidism are known to adversely affect muscle metabolism [28,29]. In the present study, CK was significantly elevated in both thyroid disorders and coincided with an increase in serum irisin. Our results suggest a positive association between serum irisin and muscle damage-induced by thyroid hormone deficiency. However, since adjustment of irisin means for CK canceled the significant irisin increase only in the hypothyroid rats,

Table 2 – Correlation matrix of the studied variables.

SMM (mg/g r BW) P FBG (mg/dL) r p Insulin (IU/mL) r p HOMA-IR r p Total-C (mg/ r dL) p TG (mg/dL) r p HDL-C (mg/dL) r p LDL-C (mg/dL) r p Muscle MDA r (nmol/g tissue) p Liver MDA r (nmol/g tissue) p Muscle GSH r (nmol/g tissue) p Liver GSH r (nmol/g tissue) p Serum CK (U/L) r p TSH (mIU/L) r p

Irisin SMM

FBG

−0.16 0.188 −0.08 0.522 0.21 0.080 0.12 0.342 −0.05 0.707 −0.10 0.423 −0.01 0.915 −0.03 0.777 0.50 <0.001 0.47 <0.001 −0.34 0.004 −0.28 0.018 0.45 <0.001 −0.01 0.916

0.46 <0.001 0.78 <0.001 0.04 0.741 0.21 0.077 0.12 0.340 −0.03 0.817 0.07 0.544 0.01 0.923 −0.12 0.318 −0.28 0.019 0.08 0.502 0.02 0.865

−0.25 0.035 −0.22 −0.066 −0.30 0.011 −0.11 0.358 −0.19 0.108 −0.05 0.655 −0.08 0.499 −0.42 <0.001 −0.49 <0.001 0.48 <0.001 0.54 <0.001 −0.26 0.030 0.27 0.024

Insulin HOMA- Total- TG IR C

0.91 <0.001 −0.11 0.345 0.02 0.881 −0.20 0.100 −0.06 0.598 0.18 0.138 0.20 0.097 −0.10 0.411 −0.11 0.370 0.19 0.115 −0.08 0.523

−0.05 0.656 0.12 0.321 −0.08 0.502 −0.05 0.670 0.19 0.116 0.19 0.121 −0.15 0.215 −0.24 0.048 0.18 0.136 −0.06 0.605

0.58 <0.001 0.45 <0.001 0.93 <0.001 0.07 0.583 0.00 0.985 −0.29 0.014 −0.22 0.069 0.36 0.002 0.65 <0.001

HDLC

0.36 0.002 0.40 0.10 0.001 0.414 0.17 0.09 0.148 0.478 −0.02 0.06 0.872 0.608 −0.30 −0.08 0.010 0.504 −0.29 0.03 0.016 0.831 0.26 0.11 0.030 0.345 0.50 0.26 <0.001 0.030

LDLC

Muscle Liver MDA MDA

Muscle Liver GSH GSH

0.02 0.869 −0.02 0.850 −0.27 0.025 −0.22 0.062 0.35 0.003 0.59 <0.001

0.58 <0.001 −0.38 0.001 −0.44 <0.001 0.49 <0.001 −0.22 0.064

0.68 <0.001 −0.53 <0.001 −0.04 0.771

−0.39 0.001 −0.27 0.026 0.51 <0.001 −0.30 0.011

Serum CK

−0.51 <0.001 0.02 0.16 0.899 0.188

SMM, skeletal muscle mass; FBG, fasting blood glucose; HOMA-IR, homeostatic model assessment of insulin resistance index; C, cholesterol; TG, triglycerides; MDA, malondialdehyde; GSH, reduced glutathione; TSH, thyroid stimulating hormone; CK, creatine kinase; r, Pearson’s correlation coefficient for pooled data (n = 70).

Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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serum irisin could hence be a marker for muscle damage in hypothyroidism. This is consistent with Pothineni et al. [30], who suggested that increased serum irisin with simvastatin administration could be an early sensitive predictor for statininduced myopathy even before rise in CK. Anastasilakis et al. [31] argue that it is unlikely that irisin leaks from damaged muscle cells due to the different day-night rhythm patterns of irisin and CK; however, their findings are derived from healthy rather than metabolically challenged individuals. Previous reports that T3 increases expression of the irisin precursor PGC-1α in muscle [32,33] could support our findings and explain why irisin up-regulation in hyperthyroidism in the present study appeared myopathy-independent. However, assessment for myopathy using more specific markers than CK is required to confirm this conclusion. To date, the relationship between thyroid hormone and irisin levels is not fully understood. In the present study, the correlation between irisin and TSH levels was not significant. In line, Stengel et al. [34] and Ellefsen et al. [35] reported lack of associations between levels of irisin, TSH and/or thyroid hormones in euthyroid individuals. However, Ruchala et al. [27] observed a negative correlation between irisin and TSH. Further studies are warranted to explore the interplay between irisin and thyroid axis. We observed that chronic swimming exercise failed to alter serum irisin regardless of thyroid status. In contrast, acute forced swimming increased serum irisin in euthyroid rats. In line with these findings, several animal and human studies reported that the stimulatory effect of chronic exercise on irisin production or muscle FNDC5 expression is limited [10–12,31,35–40]. However, the effect of different acute exercise programs on serum irisin is controversial, with some studies reporting an increase in serum irisin [10,11,31,40–42], while others reporting no effect [12,38,39]. Huh et al. [10] proposed that up-regulation of irisin after acute exercise may be related to ATP depletion in these untrained muscles, while with chronic exercise ATP reserve is conserved. Recently, it is suggested that induced irisin during exercise facilitates glucose and lipid metabolism in human muscle through AMP kinase phosphorylation [41]. In contrast to our data, increases in serum irisin, FNDC5 or PGC1-α after chronic exercise have been reported in mice and humans [4,43,44]. In addition to the species differences, these discrepancies may be related to physiological and technical differences between studies. The different types (sprint, cycling or swimming; endurance or resistance) and intensities of exercise would have diverse influence on muscle metabolism and disruption, and in turn, on irisin concentrations [45,46]. Another factor is the timing of irisin measurement after exercise; since studies have tested irisin in different time points, the possibility exists that irisin increases for a short period post exercise, after which it returns to baseline concentrations [41,46]. There may also be a mechanism for irisin uptake or clearance from circulation, which has not been assessed yet [41]. Beyond differences in energy expenditure and muscle damage, oxidative stress may be another factor that differs between acute and chronic exercise. Acute strenuous exercise is accompanied by a remarkable increase in oxygen consumption with concomitant production of free-radicals and

subsequent oxidative stress [21,47]. On the other hand, chronic exercise improves muscle oxidative balance by upregulation of endogenous antioxidant defense systems and improvement of mitochondrial respiratory capacities [47,48]. We observed elevation of MDA and decrease of GSH in muscle only after acute exercise, together with elevated irisin concentrations. The observed associations of irisin with oxidative stress may explain the rise of serum irisin after acute exercise, and not after chronic one, especially since adjustment for oxidative stress parameters increased irisin after chronic exercise and abolished the up-regulation of irisin after acute exercise. Oxidative stress stimuli are known to stimulate p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinase (ERK) that activate the transcriptional factor PGC-1α, the major regulator of mitochondrial biogenesis, leading to cleavage of the irisin precursor FNDC5 into circulating irisin [49]. Therefore, serum irisin may be up-regulated in both thyroid disorders as an adaptive compensatory response to oxidative stress; possibility exists that irisin may serve as a myokine with antioxidant action. Gouni-Berthold et al. [50] attributed the in vivo and in vitro increase in serum irisin after simvastatin administration, to a protective mechanism against simvastatin-induced cellular stress. In contrast, Hou et al. [51] reported a negative correlation between serum irisin and MDA in lean and obese subjects. Interestingly, the incidence of oxidative stress, in the two groups of thyroid dysfunction was coincided, in our work, with increased irisin, which was canceled after adjustment of irisin levels for oxidative stress parameters. Notably, serum irisin was elevated in both hyperthyroidism and hypothyroidism, which have distinct metabolic profiles, characterized chiefly by insulin resistance in hyperthyroidism and dyslipidemia in hypothyroidism. However, serum irisin did not correlate with markers of insulin resistance or lipidemia. Chronic exercise ameliorated insulin resistance in hyperthyroidism and dyslipidemia in hypothyroidism, but did not affect serum irisin in either condition. However, chronic exercise also failed to alter fasting glucose and HDL-C, suggesting that our rats were not strongly metabolically challenged. A potential link between exercise, energy metabolism and irisin therefore cannot be completely excluded, and may be elicited using a more severe model of metabolic disturbance. Recently, a role of irisin in amelioration of insulin resistance was proposed, by promoting the expression of betatrophin hormone that stimulates β-cell regeneration via the p38MAPK/PGC-1α axis [49]. However, in literature, the effect of irisin hormone on metabolic parameters is debatable [5,10,31,36,50,52,53]; thus, further studies on different patterns of metabolic challenges are warranted. Interestingly, some authors suggested an irisin resistance phenomenon in the same pattern of insulin [5,9,45]; whether this is related to thyroid dysfunction remains to be elucidated. There are several limitations to the present study. The study lacked mechanistic data on muscle expression of the irisin precursors, PGC1-α and FNDC5, which would have helped clarify the mechanism and implication of irisin elevation in thyroid disorders and its relation to oxidative stress and/or myopathy. However, evidence in literature linking this novel hormone to thyroid function is scarce, and our study benefits from investigating multiple thyroid models

Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001

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in sedentary and exercise conditions. To confirm our interpretation that irisin increases in thyroid disorders due to oxidative stress, study of the effect of antioxidants in these conditions is warranted. Lack of baseline measurements, to enable pre- and post-intervention comparisons within groups, is another limitation Finally, we investigated adult male rats and our results may not be necessarily generalizable to female rats or to humans, since irisin seems to be regulated differently in animals compared to humans and a gender dimorphism was recently reported [31,53]. In summary, circulating irisin is up-regulated in rat models of hyper- and hypothyroidism. Acute, rather than chronic exercise was associated with significant increase in serum irisin. Overall, our findings point to connections between irisin, oxidative stress and myopathy, but do not support a beneficial role for irisin on metabolic regulation in thyroid disorders. Future studies are needed to determine the molecular mechanisms of irisin release and explore its therapeutic potential. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.metabol.2015.01.001.

Author contributions DMS designed the study; DMS, CAI and RAN contributed to acquisition, analysis and interpretation of data; DMS and CAI wrote the manuscript; DMS and RAN critically revised the manuscript. All authors approved the final version of the manuscript.

Funding This research was supported by Alexandria University Research Award.

Disclosure statement The authors do not have any conflict of interest related to this manuscript.

Acknowledgements Authors would like to thank Dr. Amany Elshorbagy for review of the study, Mrs. Salwa Mohamed for her contribution in the measurement of biochemical parameters.

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Please cite this article as: Samy DM, et al, Circulating Irisin Concentrations in Rat Models of Thyroid Dysfunction — Effect of Exercise, (2015), http://dx.doi.org/10.1016/j.metabol.2015.01.001