Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue

Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue

Accepted Manuscript Title: Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue Au...

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Accepted Manuscript Title: Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue Authors: Nazan Uysal, Oguz Yuksel, Servet Kizildag, Zeynep Yuce, Hikmet Gumus, Aslı Karakilic, Guven Guvendi, Basar Koc, Sevim Kandis, Mehmet Ates PII: DOI: Reference:

S0304-3940(18)30286-6 https://doi.org/10.1016/j.neulet.2018.04.023 NSL 33546

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

20-1-2018 9-4-2018 11-4-2018

Please cite this article as: Nazan Uysal, Oguz Yuksel, Servet Kizildag, Zeynep Yuce, Hikmet Gumus, Aslı Karakilic, Guven Guvendi, Basar Koc, Sevim Kandis, Mehmet Ates, Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Regular aerobic exercise correlates with reduced anxiety and incresed levels of irisin in brain and white adipose tissue

Nazan Uysal1*, Oguz Yuksel2, Servet Kizildag3, Zeynep Yuce4, Hikmet Gumus1,5, Aslı Karakilic1, Guven Guvendi1, Basar Koc1, Sevim Kandis1, Mehmet Ates3. of Physiology, Dokuz Eylul University, School of Medicine,

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1 Department

Department of Sports Medicine, Dokuz Eylul University, School of Medicine,

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College of Vocational School of Health Services, Dokuz Eylul University, School of

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Medicine,

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4 Department of Medical Biology and Genetics, Dokuz Eylul University, School of Medicine,

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Izmir, Turkey.

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Dokuz Eylul University, School of Sport Sciences and Technology, Izmir, Turkey.

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Dokuz Eylul University

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Medical Faculty

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Nazan Uysal, MD, Professor

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* Corresponding Author:

Physiology Dept.

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Balcova/Izmir/Turkey

Email: [email protected] Phone: +905304019480/+902324124460

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Highlights    

Irisin production may be depends on exercise intensity. Exercise induced central irisin cause to reduction of anxiety levels. Peripheral irisin maybe affect anxiety levels without bloodstream. Actually, peripheral irisin mostly works on the effects of exercise on peripheral tissues.

Abstract

We have recently shown that regular voluntary aerobic exercised rats have low levels of anxiety. Irisin is an exercise-induced myokine that is produced by many tissues; and the role it plays in anxiolytic behavior is unknown. In this study we aimed to investigate the correlation between anxiety like behavior and irisin levels following regular voluntary aerobic exercise in male mice. We’ve have shown that anxiety levels

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decreased in exercised mice, while irisin levels increased in the brain, brown adipose tissue, white adipose tissue, kidney, and pancreas tissues. No significant difference of

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irisin levels in the liver, muscle and serum were detected in the exercise group, when

compared to controls. In addition, there was a strong positive correlation between brain

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irisin levels and activity in middle area of open field test and in the open arms of elevated

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plus maze test; both which are indicators of low anxiety levels. Our results suggest that

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decrease in anxiolytic behavior due to regular voluntary exercise may be associated with

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locally produced brain irisin.

White adipose tissue irisin levels also correlated very strongly with low anxiety.

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However, no serum irisin increase was detected, ruling out the possibility of increased peripheral irisin levels affecting the brain via the bloodstream. Further research is

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necessary to explain the mechanisms of which peripheral and central irisin effects anxiety

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and the brain region affected. Keywords: Exercise; irisin; anxiety; brain; muscle; brown adipose tissue; white

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adipose tissue; pancreas; kidney; liver. 1. Introduction: Physical activity leads to many health benefits and also prevents and treats various metabolic diseases such as obesity and type 2 diabetes mellitus. Physical activity also

supports the brain and improves cognitive functions [23]. We previously reported the benefits of regular physical activity on cognition and brain biochemistry [9, 41-44]. Irisin is a new hormone/myokine -discovered in 2012- proposed to work as a mediator for the benefits of physical exercise on metobolism [21]. Initial studies reported

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increased irisin secretion from skeletal muscle. Today we know that irisin is also secreted from brown and white adipose tissues, brain, liver, kidney and pancreas; and may be

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related to the beneficial effects of exercise [13, 21]. Exercise exerts its effects via various

molecular pathways and myokines; through autocrine, paracrine, and endocrine mechanisms [2, 3]. Irisin has been suggested to function in the metabolic adaptation

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following regular physical activity. Exercise leads to the reduction of adiposity through

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the usage of fatty acids mobilized from white fat tissue, resulting in its conversion to

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brown adipose tissue [22]. Irisin has been suggested to work as a mediator in the

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conversion of white fat tissue to brown fat tissue; a process that requires energy

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expenditure [45]. It has been proposed that this energy expenditure may lead to exerciseinduced weight loss [26]. In addition, studies in both experimental animals and in humans

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have shown that there is a correlation between obesity, metabolic syndrome, insulin

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sensitivity and irisin levels [3, 29]. Recent literature suggests that irisin may also have a role in anxiety. Anxiety is

commonly described as a disorganized, unpleasant, ambiguous prisoner feeling,

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accompanied by autonomic symptoms. Exercise is known to improve one's mood; and it has been shown that voluntary exercise reduces anxiety-like behaviors in rats [31, 43]. However the underlying mechanisms of the anxiolytic effect following exercise is unknown. A recent study reports that patients with distress disorders have lower irisin levels [39]; while another human study, found no association between serum irisin levels

and anxiety [19]. Chiavaroli et al. showed that intracerebroventricular administration of irisin caused an increase in anxiety like behavior in rats [10]. In this study we aimed to investigate the correlation between anxiety like behavior and irisin levels following regular voluntary aerobic exercise in male mice. The voluntary

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exercise group was given free access to a running wheel for 6 weeks following a one-week adaptation period. Anxiety levels were assessed; brain, muscle, pancreas, liver, kidney,

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brown and white fat tissues and blood were collected to determine irisin levels in tissues. 2. Materials and Methods:

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2.1. Subjects

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Eighteen adult male Balb-c mice (Dokuz Eylul University School of Medicine,

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Experimental Animal Laboratory, Izmir, Turkey) were used in this study. All mice were

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housed in individual cages with free access to water and laboratory chow. They were kept in a 12h-light/12h-dark cycle at constant room temperature (22±1°C) and humidity

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(60%). Dokuz Eylul University School of Medicine Animal Care Committee approved all experimental procedures.

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2.2. Experimental design:

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Mice were divided into two groups: (1) control group (n= 8), (2) running wheel exercise group (n= 8). Following an one week adaptation period, the voluntary exercise group had free access to a running wheel (11.5 cm diameter - rotated by animal effort) connected to

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a digital counter, for 6 weeks [9, 42, 43]. At the end of the 6-week exercise period, anxiety was assessed by open field and elevated plus maze tests. Blood samples were obtained under carbon dioxide anesthesia. Whole brain (except cerebellum), brown adipose tissue, white adipose tissue, muscle, liver, kidney, and pancreas tissues were removed.

2.3. Open Field Test: This test is commonly used to assess spontaneous locomotor activity and anxiety. The open field consists of a 1x1m area surrounded with a wall 50cm in height. A video camera was installed 2.5m above the apparatus. Each mouse was placed in the center of the open field and locomotor activity was measured for 5 min in a soundproof observation room,

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illuminated with controlled light (100lx)[8].

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2.4. Elevated Plus Maze:

The elevated plus maze is another commonly used experimental rodent model to assess anxiety. The elevated plus maze apparatus consists of a central platform (5cm×5cm) with

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two open arms (50cm long, 10cm wide and 0.5cm high borders) and two closed arms

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(50cm long, 10cm wide with 40cm high walls), each elevated 50cm above the floor. Mice

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were placed on the platform facing the open arm and were observed for 5 min. The total

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number of entries into the open and closed arms; as well as the entire time spent on the

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open and closed arms were measured [17].

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2.5. Biochemical Analysis:

Blood and all tissue samples were stored at –85°C. Irisin levels were analyzed by mouse

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FNDC5 ELISA kit (catalog no: E-EL-M0392, Elabscience, Wuhan, China - assay sensitivity 37.5 pg/mL and detection range 62.5-4000 pg/mL). Irisin levels were calculated per wet weight. BCA protein Assay kit (Cat No: E- BP-500, Elabscience, Wuhan, China) was used

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for protein analyses, according to the manufacturers’ description. 2.6. Statistical evaluation: All statistical procedures were performed by SPSS software for Windows, Version 11.0 (SPSS, Chicago, IL). Differences between groups were analyzed using Student t-test (two-

sample independent sample unpaired t-test). Correlations among groups were calculated using Pearson correlation analysis. Results are presented as mean ± S.E.M. 3. Results: The exercise group exercised in the running wheel cage; mean running distance is 2.4 

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0.3 km/day, running time is 143  5.18 min/day. In the open field test, the exercise group spent more time in the center of the open field

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arena when compared to the control group (p<0.001)(Fig. 1A). Moreover, movement and walking speed was increased in exercise group (p<0.0001 for both parameters)(Fig. 1B and C).

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compared to control group, (p<0.001)(Fig. 1D).

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In the elevated plus maze test, the exercise group spent more time in the open arms when

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Brain, brown adipose tissue, white adipose tissue, kidney, and pancreas irisin levels were

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higher in exercise group when compared to controls (p<0.005 in brain and pancreas,

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p<0.0001 in kidney, brown and white adipose tissues). No significant change was detected in the muscle, liver and serum irisin levels following exercise (p>0.05).

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A very strong positive correlation was found between irisin levels of the brain and activity in the middle area of the open field test and activity in the open arms of elevated plus

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maze test (r = 0.756, p<0.0001 and r= 0.818, p<0.0001) (Fig. 3A). In addition, a very strong positive correlation was found between irisin levels of white adipose tissue and activity

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in the middle area of open field test and activity in the open arms of elevated plus maze test (r = 0.878, p<0.0001 and r= 0.858, p<0.0001)(Fig. 3B). Another very strong correlation was found between brown and white adipose tissue irisin levels (r= 0.928, p=0.0001). The irisin levels of the kidney strongly correlated with both type of adipose

tissues (r= 0.810, p=0.0001 for brown adipose tissue; r= 0.824, p=0.0001 for white adipose tissue). 4. Discussion: In this study, we were able to demonstrate that regular voluntary exercise leads to decreased anxiety correlated with brain and white adipose tissue irisin levels. We also

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showed that physical activity induced irisin expression in the brain, kidney, pancreas, white and brown adipose tissues. Regular voluntary exercise did not change serum and

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muscle irisin levels.

Exercise is suggested to increase resistance to stress-induced situations such as

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anxiety disorders. In literature there are some conflicting reports on the relationship

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between chronic voluntary exercise and anxiety. There are a couple of studies that report

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chronic voluntary exercises leads to a decrease in anxiety-like behaviors [5, 12, 36].

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Whereas other studies report that anxiety levels are not affected or increase following exercise [7, 14, 34]. We also demonstrated that chronic voluntary exercise decreased

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anxiety levels in a previous study [43]. Our exercised mice had higher ambulation in

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center cells of open field arena, and in the open arms of elevated plus maze test; indicators associated with less anxiety.

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We showed that irisin levels increased in brain, pancreas, kidney, brown and white adipose tissues after regular physical activity. The relationship between irisin and physical activity has been reported in human and experimental animal studies. Physical

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activity affects tissues differently, which may result in different tissue irisin levels. During exercise, muscles release myokines (such as irisin), which are important molecules in the regulation of energy homeostasis. Exercise induced irisin is involved in mitochondrial biogenesis and muscle fiber type changes in skeletal muscle [38]. The irisin is produced in the tissue and then released into the blood. It is estimated that about %72 of the

circulating irisin is released by skeletal muscle [6]. There are many conflicting reports on the effect of exercise and plasma and muscle irisin levels. While some studies report no change in plasma and muscle irisin levels following exercise; other have reported an increase in irisin levels [2, 24, 35, 40]. In a recently published study, only high intensity exercise was shown to increase irisin levels in the muscle [2, 35]. These inconsistencies

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may be due to differences in the amount of energy required for the physical activity tested. One theory is that irisin only increases when excessive energy is consumed. Without

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excessive physical activity, the necessity of ATP in the muscles rise enough for irisin to be expressed and secreted [15]. In our study, voluntary regular exercise did not change the

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irisin levels in the muscle. This result may be due to the fact that the exercise intensity of

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the mice was not a high intensity exercise. In addition, no significant changes in serum

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irisin levels were detected in the exercised mice.

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White adipose tissue is the biggest energy store as well as the largest endocrine organ of the body. It releases hormones such as leptin, which regulates metabolic

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homeostasis, appetite, angiogenesis, immune and cardiovascular systems and a variety of physiological functions [1, 11]. Brown adipose tissue functions in adaptive thermogenesis

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that maintains homeostasis in response to thermal stimuli. It is paramount to activate

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these adipose tissues, to struggle obesity and related cardio metabolic diseases because of their important functions in metabolism [28]. Irisin has been reported to improve adipose tissue metabolism. Irisin induces browning (convergence of white adipose tissue

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to brown adipose tissue) of adipose tissue, which is very important in glucose homeostasis and treatment of obesity [15]; and increases glucose tolerance and insulin sensitivity [30]. Recently, it has been shown that irisin is secreted from the pancreas and is expressed in both Langerhans and serous secretory cells [4, 16]. In humans, irisin is positively associated with the circulation of insulin and the homeostasis model

assessment (HOMA) and was suggested to be involved in the regulation of β-cell function [33, 47]. However, there is no information on how regular physical activity affects pancreas irisin levels. In this current study we show that irisin levels increase in the pancreas, and in both brown and white fat tissues of exercised mice. Irisin is thought to be part of the molecular link between muscle and brain [20]. It

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is required for proper neural differentiation of embryonic stem cells in mice; and it plays

a role in hippocampal neurogenesis in a dose-dependent manner [18]. Another study also

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demonstrated its positive effects on the hippocampus [46]. Irisin has been shown to reduce ischemia induced neuronal damage; therefore, it is thought to be a

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neuroprotective agent [25]. In this current study found that regular exercise increased

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brain irisin levels. Recent studies indicate that irisin crosses the blood brain barrier [48].

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Serum irisin levels did not change in our study; reinforcing the assumption that locally

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produced irisin were measured in the brain. We found a very strong positive correlation between exercise-induced brain irisin levels and low levels of anxiety like behavior. There

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are only four publications reporting a possible relationship between anxiety and irisin expression. One study reported that serum irisin levels were are positively correlated

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with distress disorder in bronchial asthma patients [39]. Whereas another study reported

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that there was no association between plasma irisin and anxiety levels in exercised obese women [19]. Our results support the latter report; we also did not find any association between serum irisin levels and the degree of anxiety in exercised mice. In a recent report,

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Siteneski et al. showed that intracerebroventricular irisin administration decreased anxiety levels [37]. In a conflicting report Chiavaroli et al. demonstrated increased anxiety, after intracerebroventricular administration of irisin [10]. The concentrated of irisin administrated is one of the main differences between the two experimental designs. Siteneski et al. administrated a 0.5-1 ng dose of irisin whereas Chiavaroli et al.

administrated 0.4-0.6 mcg doses. When comparing both studies we see that Chiavaroli et al.’s administration of high doses of intracerebroventricular irisin, increased anxiety; while Siteneski et al.’s administration of a low dose resulted in decreased anxiety; implying an dose dependent dual function. Low levels of irisin may lead to a reduction in anxiety; whereas high doses may have the opposite effect by increasing anxiolytic

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behavior. In this current study irisin levels in the brain following physical activity are

lower than previously reported literature, nevertheless we demonstrate a strong

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correlation with reduced anxiety. Furthermore, we found a very strong positive

correlation between exercise-induced white adipose tissue irisin levels and low levels of

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anxiety like behavior. We did not find significant differences in blood irisin levels;

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suggesting that increased peripheral –white adipose tissue- irisin production cannot

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affect the brain through the bloodstream.

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In addition, we found that kidney irisin levels increased in exercised mice. Clinical reports show that exercise can reduce the risk of developing progressive chronic kidney

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disease and limit the degree of damage to kidney cells. Recombinant irisin attenuated kidney damage and and improved renal function in mice. Irisin stimulated mitochondrial

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functions in renal tubule cells [32]. Aydin et al. showed that acute swimming exercise

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increased renal irisin levels [3]. Positive effects of physical activity on the kidney may be modulated through irisin. Strenuous exercise can cause damage to the kidney [27], so

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again the positive effects associated with irisin may appear to be dose-dependent. This study mainly aimed to investigate the relationship between anxiety and tissue

irisin levels in exercising mice. According to our knowledge, this is the first experimental study of its kind. Reduced anxiety due to regular voluntary exercise correlates with increased levels of locally produced brain irisin. Exercise also led to the production of irisin in the pancreas, liver, kidney, brown and white adipose tissues; suggesting different

parallel outcomes of regular physical activity, other than the known beneficial health effects. White adipose tissue irisin levels correlated very strongly with low anxiety levels. However, levels of serum irisin did not change after exercise, implying that the increase in peripheral irisin does not directly affect the brain. In our previous studies we showed that anxiety levels decreased in regular voluntary exercised rats. The observed decrease

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in anxiety was associated with prefrontal cortex IGF1 and leptin levels [41, 43]. Further research is needed to investigate the mechanisms of which peripheral and central irisin

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effects anxiety, the role of leptin and IGF1, and the brain region affected; in addition to dose-dependent dual functions of irisin.

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The authors have no conflict of interests to disclose.

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A

Figure legends:

Figure 1: Behavioral test results. (A) Open field test result, time spends in middle cells of open field arena. (B) Open field test result, moving duration in the open field arena. (C) Open field test result, moving speed in the open field arena. (D) Elevated plus maze test result, time spends in the open arms of T-maze. * Compared to control group, p<0.001.

Figure 2: Biochemical investigation results. (A) Irisin levels of brain tissue, (B) Irisin levels of brown adipose tissue, (C) Irisin levels of white adipose tissue, (D) Irisin levels of kidney, (E) Irisin levels of pancreas. * Compared to control group, p<0.05. Figure 3: Correlation analysis results. (A) Correlation scatterplot between brain irisin levels and time spent open arms of elevated plus maze. (B) Correlation scatterplot

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