Circulating and/or cutaneous irisin resistance: A novel link among androgenetic alopecia, comorbid metabolic syndrome and cardiovascular risks

Androgenetic alopecia (AGA) is a common cause of hair loss in both genders that may be associated with disturbed systemic metabolism. Irisin is a hormone‐like myokine that greatly influences systemic metabolism and is linked to cardiovascular diseases.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is the most common cause of hair loss in both genders. It is often considered a cosmetic problem, but evidence shows potential comorbid conditions. Over the past few decades, authors have reported significant associations between AGA and metabolic syndrome (MetS) 1 and its components with an increased risk of coronary heart disease, hypertension, hyperinsulinemia, insulin resistance, and dyslipidemia. Also, AGA and cardiovascular disease (CVD) are linked to each other. 2 Some may even regard AGA as a simple marker of subclinical atherosclerosis. 3 Irisin is a novel myokine that is derived from fibronectin type III domain-containing protein 5 (FNDC5) and acts by changing white adipose tissue to brown adipose tissue to enhance systemic metabolism and insulin sensitivity. 4 It is synthesized mainly in skeletal muscle in response to physical exercise, and is also found in cardiac muscle, adipose tissue, liver, brain, bones, sebaceous glands, pancreas, kidneys, and others. 5 However, in 2012, Timmons and colleagues' study doubts muscle FNDC5 induction and argues that it happens only in a minority of subjects (highly active elderly people) in whom FNDC5 expression was unrelated to metabolic status. 6 Although the exact physiology of irisin in humans is still under investigation, some studies show an association between irisin levels and abnormal metabolic conditions, such as obesity, type 2 diabetes mellitus and MetS. 7 Moreover, some studies have demonstrated irisin levels as a strong positive predictor of 10-year CVD risk in general population. 8 Many debates about irisin molecule pathophysiology, detection methods, and receptors are not yet fully elucidated. 9 In 2015, Albrecht and colleagues raised a concern regarding the lack of specificity in anti-irisin antibodies. 10 Also, there were contradictory observations about the existence of irisin by experimental evidence, but many sensitive approaches-including ELISA assays and quantitative mass spectrometry-have been later implemented successfully to prove irisin's identity and to quantify humans' circulating irisin. 11 Irisin receptor, as well, is not yet fully identified. Although, αV/ β5 integrins have been discovered recently as receptors for irisin in some tissues such as bone and fat, 12 there are still ongoing trials to discover other receptors both within and outside of the integrin family. 11 Since both irisin and AGA are linked to metabolic dysregulations, the present study aimed to assess the serum level of irisin hormone, in addition to scalp immunohistochemical expression of irisin in AGA patients as well as healthy controls. It also correlates them with the prevalence of MetS and the associated cardiovascular risk in a trial to shed the light on irisin as a predictor for such comorbidities.

| Study place and population
This study was implemented on 44 AGA patients (

| Exclusion criteria
Patients with chronic dermatologic diseases, smokers, patients using drugs that are known to cause hyperglycemia, hyperlipidemia, hypertension, or hormonal therapy in the previous 3 months, and patients on systemic or topical treatment for AGA within the last 3 months were excluded from the study.

| All subjects underwent
Complete history: Early onset AGA was diagnosed if started before 36 years old. 13 General examination: Anthropometric measures were taken.
Body mass index (BMI = kg/m2) was calculated, 14 and blood pressure was measured twice in a resting state and the average was considered. Blood pressure was considered high if it is greater than 130/85. 15 Dermatologic examination: to diagnose AGA and assess its grading and severity using modified Norwood-Hamilton classification in males and Ludwig classification in females. Grade I-III in males and I-II in females were considered mild to moderate AGA, while male grade VI and higher and female grade III were considered as severe AGA. 16

| Blood sampling
Two (5 mL) venous blood samples were withdrawn from each subject following 12 h fasting and centrifuged for 10 min at 4000 r.p.m.
The serum obtained was kept frozen at −20°C till analysis. The first sample was used to assess fasting blood sugar (FBS) and lipid profile. The second one was used for serum irisin measurement using the SUN RED (ST) ELISA Kit, from Shanghai. The kit uses a doubleantibody sandwich enzyme-linked immunosorbant assay. Two antibodies specific for irisin were used, the first one has been pre-coated onto a microplate. The second one is, a biotin-conjugated antibody K E Y W O R D S androgenetic alopecia, carotid intima media thickness, immunohistochemistry, irisin, metabolic syndrome specific for irisin which was added to the wells. This ELISA is specific for human irisin, and quality controls were included in all ELISA measurements with the results falling within the expected range. All samples were analyzed in duplicate. No significant cross-reactivity or interference between human irisin and analogues were observed (as stated by the manufacturer).

| Skin biopsy
A four-millimeter punch biopsy was taken from the scalp vertex of AGA patients and matched site of controls. The specimens were fixed in 10% formalin solution and submitted for routine tissue processing and embedding in paraffin blocks. Two sections of 4 μm thick were cut. One section was for hematoxylin and eosin (H&E) staining for histopathological examination and the other was for staining immunohistochemically by polyclonal antibody-against FNDC5 (Irisin).

| Histopathological examination
It included examination of H&E sections for: total number of the hair follicles, number of telogen hairs, number of anagen hairs, anagen/telogen ratio, presence or absence of perifollicular lymphohistiocytic infiltration, and presence or absence of perifollicular collagen.

| Steps of immunostaining
The sections were deparaffinized in xylene and then rehydrated by immersion in three changes of alcohols and then washed in phosphate-buffered saline (PBS). Excess liquid was dried by absorbent paper, avoiding drying the section. The slides were transferred to a humidity chamber where they were incubated in hydrogen peroxide (3% H 2 O 2 in absolute methyl alcohol) as a blocking solution for 7-10 min. After washing, the slides were subjected to an antigen retrieval step using citrate buffer (pH 6.0) in the microwave. In the humidity chamber, Ultra v block was applied and the sections were then incubated with a bovine serum to block nonspecific background staining. The primary antibody was a polyclonal antibody against FNDC5 (Irisin). It was received in concentrated form from Chongqing Biospes Co., Ltd (Chongqing, China; www.Biosp es.com) and was diluted 1:100 prior to application to sections.
In each run, a negative control slide was prepared by omitting the primary antibody from the staining procedure and a positive control slide of human skeletal muscle was included. After washing with PBS, sections were incubated with the secondary antibody and then with preformed streptavidin peroxides. The DAB chromogen substrate was used to elicit the reaction with Mayer's hematoxylin (Bio Genex, cat. no. 94583) as a counter stain.

| Interpretation and scoring of immunostaining of FNDC5 (Irisin)
The interfollicular and follicular epidermis were assessed for:  20 ; H score = 1x% of mildly stained cells +2x% of moderately stained cells +3x % of strongly stained cells.
Furthermore, the median value of the score was used as a reference to compare low versus high levels of expression.

| Radiological examination
Carotid Doppler ultrasonography (US) was performed on patients with a 7.5 MHz linear array imaging probe. To maximize the lumen, transducer was placed in longitudinal plane 1 cm proximal to the carotid bifurcation while the patient was lying supine. The carotid intima media thickness (CIMT) of the far wall was evaluated as the distance between the lumen-intima interface and the media-adventitia interface. Measurements were obtained from five contiguous sites at 1 mm intervals bilaterally, and the average was registered. CIMT values of more than 1 mm were accepted as abnormal. 21

| Statistical analysis
Data were tabulated using SPSS statistical package version 23 (SPSS Inc. Released 2015. IBM SPSS statistics for windows, version 23.0, Armnok, NY: IBM Corp.). Groups were compared qualitatively using chi-square or quantitatively using either the t-test (normally distributed) or the Mann-Whitney and Kruskal-Wallis test (non-normally distributed). Spearman's correlations were used to draw linear relation between quantitative variables. Regression analysis was used to study the relationship between a dependent variable and one or more independent variables (or 'predictors'). Roc-curve was used to assess for different possible cut-off values of the diagnostic marker.
Significance was considered if p value ˂0.05. Table 1 Patients and controls were age and sex matched. Among studied subjects, BMI (p ˂ 0.001), blood pressure (p ˂ 0.001 for diastolic BP, p = 0.002 for systolic BP) and FBS (p ˂ 0.001) were significantly higher in AGA patients compared to controls. Dyslipidemia was detected in 33 (75%) patients versus none of the controls (p ˂ 0.001) with higher mean cholesterol, LDL-C, and TGs (p ˂ 0.001 for all) and

| H and E staining of studied controls and AGA patients
There was a significant lower total number of hair follicles, anagen hair follicles, and anagen/telogen ratio in AGA patients compared to controls (p < 0.001 for all). However, number of telogen hair follicles was significantly lower in control subjects compared to AGA patients (p = 0.01). Presence of fibrous tracts/perifollicular collagen and presence of peri-infundibular lymphocytic cell infiltrate were significantly associated with AGA patients (p < 0.001) (data not shown).  Figures 3B and 4E).
There was a tendency toward positive epidermal irisin expression in male compared to female patients (p = 0.06). Also, epidermal nucleocytoplasmic expression was significantly associated with male gender (p = 0.002) ( Table 4).

| DISCUSS ION
Although the present study investigated a relatively small sample of AGA patients (44 patients) of both genders, its significant inference warrants attention and further research. Over the past two decades, there has been increasing evidence showing the association between AGA and MetS. 15,22,23 This goes with the current results, In the present study, CIMT was abnormal in 65.9% of AGA patients with a significantly higher measurement than controls. Both Dogramaci et al., 26 and Erkoç et al., 21 found a relationship between severe alopecia and greater CIMT. However, Agac et al., 27 reported a non-significant correlation between AGA score and CIMT.
Although CV risk in AGA patients is well established, the pathomechanisms involving this risk have not yet been finally elucidated.
The current study showed a significantly higher serum irisin level in AGA patients compared to controls, especially in severe alopecia grades irrespective of patients' age or disease duration. This was in contrast to Ebrahim et al.'s study, 28 who reported lower serum irisin levels in AGA than controls and showed progressive reduction with increasing disease duration. This debate may be related to their study population's criteria, as they excluded patients with any condition that may predispose to metabolic dysregulation, and their BMI was matched with controls.
The higher serum irisin level present in our studied AGA patients, which was significantly associated and positively correlated with BMI, can be explained by the positive correlation between AGA and BMI as the high serum irisin level may be either an increased baseline secretion of irisin by the increased adipose/ muscle tissue in obesity and/or a compensatory increase of irisin to combat obesity, MetS, and/or decreased sensitivity to irisin's effects (irisin resistance). 29 Many studies similarly agreed upon the positive correlation between serum irisin and BMI 7,30-32 and suggested a possible mechanism of resistance to irisin in situations of elevated BMI.
The elevated concentration of circulating irisin in overweight and obese subjects suggests that they have a great amount of brown   These conflicting results may be attributable to differences in the pathogenesis of the studied diseases or the assays used. Also, the supposed irisin resistance in our patients could explain CIMT abnormality despite high serum irisin levels, which also does not ameliorate the beneficial role of irisin in preventing atherosclerosis.
BMI and serum irisin levels were discovered to be independent predictors of CIMT abnormality in AGA patients. This may increase awareness in patients with AGA to assess serum irisin levels as a predictor for MetS and CV risk.
When comparing AGA cases and controls regarding irisin immunohistochemical expression, positive epidermal expression was significantly associated with control skin. This may indicate a physiological role of irisin in the normal scalp.
The upregulated epidermal irisin expression in severe AGA and the positive correlation seen between follicular H score and disease duration could be explained by the recent discovery of irisin as a novel ligand for multiple integrins, including αLβ2 and α4β7, facilitating lymphocyte adhesion and migration in inflammation. 44 This was evident in the studied AGA scalp biopsies in which peri-infundibular lymphocytic infiltrate and perifollicular fibrous tract/collagen tended to be present in cases showing high epidermal irisin H-score, signifying a possible role of cutaneous irisin in AGA pathogenesis and its severity. However, it may also support the concept of compensatory increase in irisin secretion to overcome resistance and decreased sensitivity to irisin effect in severe and long standing cases.
Furthermore, obesity is considered a state of chronic inflammation of adipose tissue. Despite its low-grade nature, it negatively affects distant organ function enhancing complications of obesity including metabolic ones. 45 Hence, being linked to inflammation, upregulated irisin in AGA patients might act as a promoting factor of adipose tissue inflammation and in turn metabolic complications.
Similar to serum irisin, epidermal irisin upregulation was associated with each of MetS, dyslipidemia, and CIMT abnormality.
This more or less proves higher irisin expression in AGA patients with metabolic alterations and associated cardiovascular risks.
The relatively recent discovery of irisin in humans and the lack of previous research studying its expression in healthy and diseased skin made it difficult to discuss our results in comparison with other studies. Moreover, the small sample size that was investigated in the present study limits its results and makes it difficult to be generalized to all settings except after more extensive research on larger cohort of patients and healthy controls.
However, the reported data will surely shed light on this novel focus and will provide a base for future research that is warranted to clarify the exact role of irisin in normal scalp physiology and also in AGA pathogenesis and its associated comorbidities.

| CON CLUS ION
High serum irisin and upregulated scalp irisin expression are associated with the incidence of MetS, dyslipidemia, and cardiovascular risk among AGA patients. In addition, serum irisin levels could be used to predict MetS and cardiovascular risk. This may indicate increased muscle/adipose tissue/cutaneous secretion as a compensatory mechanism to overcome resistance to irisin, which hinders its favorable cardiometabolic actions. However, further studies on larger samples are warranted to investigate the concept of irisin resistance in AGA patients, which was uniquely discussed in the present study.

FU N D I N G I N FO R M ATI O N
None.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Most of data generated or analyzed during this study are included in this article. Enquiries can be directed to the corresponding author.