Investigation of the relationship between serum irisin level in the idiopathic restless legs syndrome: Could be a marker independent of physical activity?

Abstract Introduction Restless legs syndrome (RLS) is a common but underdiagnosed neurological syndrome. It is characterized by the feeling of discomfort and desire to move, especially in the lower extremities, which often occurs at night, and the cure or relief of symptoms with movement. Irisin is a hormonelike polypeptide that was first identified in 2012, weighs 22 kDa, consists of 163 amino acids, and is mainly synthesized in muscle. Its synthesis increases with exercise. Here in this study, we planned to investigate the relationship among serum irisin level, physical activity, lipid profile, and RLS. Material and methods A total of 35 patients with idiopathic RLS and 35 volunteers were included in the study. Then, venous blood was taken from the participants in the morning after 12 h of night fasting. Results The mean value of serum irisin level was 16.9 ± 14.1 ng/mL in the case group and 5.1 ± 5.9 ng/mL in the control group, which was statistically quite significant (p < .001). A significant efficiency (under the curve area 0.886 [0.804–0.967]) of irisin value was observed in the differentiation of patients in the case and control groups. Discussion Serum irisin level was significantly higher in the case group than in the control group. In conclusion, we suggest that irisin may play a role in the pathophysiology of RLS independently of the intensity and duration of physical activity and anthropometric data, such as body weight, body mass index, and waist/hip ratio.


INTRODUCTION
Restless legs syndrome (RLS), also known as Willis-Ekbom disease, was first described in 1672 and is a common but underdiagnosed neurological syndrome (Gossard et al., 2021). It is characterized by the feeling of discomfort and desire to move, especially in the lower extremities, which often occurs at night, and the cure or relief of symptoms with movement. More rarely, symptoms may occur in other parts of the body and during the day (Klingelhoefer et al., 2016;Manconi et al., 2021). RLS can be seen as idiopathic (primary) or secondary.
There is no etiological factor in idiopathic RLS, but genetic factors could be influential. Secondary RLS occurs in conditions, such as iron deficiency anemia, kidney failure, and pregnancy (Klingelhoefer et al., 2016). Although centuries have passed since the first recognition of RLS, its pathophysiology is still unclear. Brain iron metabolism, dysfunction in dopaminergic pathways, and glutamate and adenosine-related changes are held responsible for the pathophysiology (Gonzalez-Latapi & Malkani, 2019;Manconi et al., 2021).
Irisin is a hormonelike polypeptide that was first identified in 2012, weighs 22 kDa, consists of 163 amino acids, and is mainly synthesized in muscle (Cheng et al., 2021). Its synthesis increases with exercise.
Irisin is formed by the degradation of a transmembrane protein called fibronectin type III domain-containing protein 5 (FNDC5) by enzymatic activity. After irisin is synthesized in the muscle, it passes into the blood and is transported to the adipose tissue. It plays a role in the conversion of subcutaneous white adipose tissue into brown adipose tissue, regulation of insulin metabolism, reduction of insulin resistance, and regulation of energy metabolism (Chen et al., 2016;Huang et al., 2020).
Irisin is also responsible for cholesterol metabolism. With the increase in irisin secretion, total cholesterol synthesis decreases, while hepatobiliary cholesterol secretion and fecal cholesterol excretion increase (Cheng et al., 2021).
Recent studies have shown that the FNDC5/Irisin complex stimulates the release of brain-derived neurotrophic factors (BDNF), especially in the hippocampus, and plays a role in neuroplasticity (Jodeiri Farshbaf & Alviña, 2021;Lourenco et al., 2019). For this reason, recent studies examining the relationship between Alzheimer's disease, cognitive functions, and Irisin are seen in the literature (Lourenco et al., 2019). However, as far as we know, a study has not been examined before to investigate the relationship between the Irisin molecule, released from muscle with exercise, and RLS, a disease in which symptom relief is seen with movement. Here in this study, we planned to investigate the relationship among serum irisin level, physical activity, lipid profile, and RLS. was applied to all participants. IPAQ-SF is a questionnaire consisting of 7 questions in total and evaluating physical activity in the last 7 days according to 4 intensity levels (vigorous-intensity, moderate-intensity, walking, and sitting). The form estimates how many days and how many minutes per day people have been physically active in the last 7 days. The metabolic equivalent task (MET) score is calculated from the total physical active time obtained as a result of the test, according to the formula published by Ainsworth et al. (2000). The MET coefficients of walking, moderate-activity, and vigorous-activity in IPAQ-SF are 3.3, 4, and 8, respectively. A result is obtained by multiplying the number of minutes and days of total physical activity by the MET coefficient. Those who score less than 1500 MET-min/week are defined as physically inactive (sedentary) (Ainsworth et al., 2000;Zhang et al., 2020).

MATERIAL AND METHODS
In this study, serum irisin level, complete blood count, ferritin, and serum iron levels were studied in all participants. For Irisin study, venous blood was taken from the participants in the morning after 12 h of night fasting. In addition, it was instructed to the participants that they should not do physical activity before the blood sampling.
Relevant blood samples were centrifuged at 4000 rpm for 10 min; then their serums were separated and stored at −80 • C until the study day. All samples were thawed at the same time on the study day. Serum irisin levels were studied according to the manufacturer's instructions and standards using the enzyme-linked immunosorbent assay method with a commercially available kit. Within-assay and between-test confidence intervals were <10% and <12%, respectively.
In the descriptive statistics of the data, mean, standard deviation,

RESULTS
The ages of 70 participants included in the study ranged from 27 to 61, and the mean age was 44. group ranged from 10 to 37, and the mean score was found to be 25 ± 7.
The participants' total physical activity time calculated according to IPAQ-SF ranged from 271 to 4111 min, and the mean physical activity duration was 1291 ± 872 min. When the serum irisin levels of the participants were examined, they ranged from 3.1 to 54.5 ng/mL, and the mean serum irisin level was found to be 11 ± 12.2 ng/mL (Table 1).
When the control group and case group were compared in terms of demographic data, height, body weight, BMI, waist circumference, hip circumference, waist/hip ratio, and metabolic complication risk by waist circumferences and waist/hip ratio, there was no statistically significant difference between groups ( Table 2). The mean IPAQ-SF (min) in the control group was 1208 ± 792, whereas it was 1374 ± 950 in the case group. In the IPAQ-SF (min) scores and physical activity intensities, no significant difference was observed between the control and case groups in light, moderate, and high-intensity activity ( Table 3).
The mean value of serum irisin level was 16.9 ± 14.1 ng/mL in the case group and 5.1 ± 5.9 ng/mL in the control group, which was statistically quite significant (p < .001) (Table 3, Figure 1). When the serum ferritin, iron, triglyceride, total cholesterol, and LDL values in the case and control groups were examined, no significant difference was observed, but the mean HDL value in the case group was 43.9 ± 9.8 mg/dL, whereas it was 51 ± 10.6 mg/dL in the control group, which is statistically quite significant (p = .007) was detected (Table 3).

F I G U R E 1
Irisin level was statistically significantly higher in the case group than in the control group.
F I G U R E 2 ROC (receiver operating characteristic) curve analysis assesses serum irisin's feasibility as a diagnostic indicator of primary restless legs syndrome (RLS  Figure 2). ULAŞ ET AL.

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TA B L E 1 Sociodemographic and anthropometric data of the participants, duration of restless legs syndrome (RLS) diagnosis, severity of RLS, physical activity scale scores, and serum irisin and lipid profile values.

DISCUSSION
The superior result of our study is that serum irisin level was significantly higher in the RLS case group than in the control group. RLS is a common sensorimotor disease affecting sleep quality and quality of life (Harrison et al., 2018). To date, many studies on its pathophysiology have been carried out. Dysfunction of dopaminergic neurons in the spinal cord, genetic factors, and brain iron deficiency is thought to play a role in the pathophysiology (Ainsworth et al., 2000;Allen et al., 2014;Chen et al., 2016;Cheng et al., 2021;Gonzalez-Latapi & Malkani, 2019;Harrison et al., 2018;Huang et al., 2020;Jodeiri Farshbaf & Alviña, 2021;Lourenco et al., 2019;Manconi et al., 2021;Phillips et al., 2014;Trenkwalder et al., 2018;World Health Organisation (WHO), 2008, Zhang et al., 2020. In a 2021 study, the involvement of the hippocampus and mesolimbic dopaminergic pathway was also pointed up in RLS patients (Mogavero et al., 2021). Relief of symptoms with movement in the clinical diagnosis of RLS suggests that exercise-related mediators may also play a role in the pathophysiology. Irisin is a polypeptide that release is increased by exercise. Recent studies have shown that irisin is not only found in serum but also crosses the blood-brain barrier Since irisin is secreted from the muscle and then goes to the adipose tissue, it has been shown in previous studies that serum irisin levels can be affected by the ratio of muscle and adipose tissue. It has been previously reported that there is a positive correlation between serum irisin levels and body weight, BMI, but no correlation between age and waist/hip ratio and serum irisin levels (Stengel et al., 2013). In another study, it was specified that there was a positive correlation between serum irisin levels and the amount of muscle (Huang et al., 2020). In our study, we evaluated the age, gender, height, body weight, BMI, waist circumference, hip circumference, and waist/hip ratio and we did not find a significant difference between the case and control groups.
Another main factor affecting serum irisin levels is physical activity. In a study, RLS symptom severity and physical activity level were evaluated using the IPAQ and were not found to be significant, but it was still stated that in cases with RLS, those who were more physically active had milder symptoms, less pain, and were less physically restricted (Daniele et al., 2013). In our study, we applied IPAQ-SF to all participants to question physical activity, and we did not find a ULAŞ ET AL.

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TA B L E 3 The International Physical Activity Questionnaire-Short Form (IPAQ-SF) (min), physical activity intensity, and serum irisin, lipid profile levels of control and case groups. Irisin is also a mediator involved in cholesterol metabolism. In a study, an inverse relationship was reported between serum irisin levels and triglyceride, total cholesterol, and LDL cholesterol levels (Oelmann et al., 2016). Similarly, a recent study stated that as serum irisin levels increased, total cholesterol levels decreased (Cheng et al., 2021). Previous studies have shown that serum HDL levels increase with physical activity (Palazón-Bru et al., 2021;Sanllorente et al., 2021). However, IPAQ-SF is a limited and subjective test for evaluating physical activity. The low serum HDL levels in the case group in our study can also be considered evidence that the physical activity of the case group was not higher than the control group. Therefore, the fact that serum irisin levels were significantly higher in the case group than in the control group is not related to physical activity and muscle-adipose tissue ratios, sug-gesting that irisin may directly play a role in the pathophysiology of RLS.

Control group
In conclusion, we suggest that Irisin may play a role in the pathophysiology of RLS independently of the intensity and duration of physical activity and anthropometric data. The wide and diverse range of duties of irisin suggests that it may also play a role in central and peripheral nervous system-related diseases. We believe that irisin, which is secreted from the muscle and has effects on the central nervous system with BDNF, may be associated with the pathogenesis of RLS, a disease in which symptoms are relieved by movement. Our results are also promising in this regard. However, the limited number of patients, the use of only IPAQ-SF to evaluate physical activity, and the clinical diagnosis of RLS without polysomnography are the limitations of our study. It will be more beneficial to assess the relationship between RLS and irisin with a larger number of multicenter cases and use more objective tests to evaluate physical activity. Nevertheless, we believe that the fact that it is the first study in the literature to investigate the relationship between RLS and irisin and that serum irisin levels are significantly higher in the case group will guide future studies.

DATA AVAILABILITY STATEMENT
The data are not publicly available due to privacy or ethical restrictions; research data are not shared.