Irisin as a muscle-derived hormone stimulating thermogenesis – A critical update

Peptides 54 (2014) 89–100

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Irisin as a muscle-derived hormone stimulating thermogenesis – A critical update Tobias Hofmann a , Ulf Elbelt a,b , Andreas Stengel a,∗ a Charité Center for Internal Medicine and Dermatology, Division of General Internal and Psychosomatic Medicine, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany b Charité Center for Internal Medicine with Gastroenterology and Nephrology, Division of Endocrinology, Diabetes and Nutrition, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany

a r t i c l e

i n f o

Article history: Received 5 December 2013 Received in revised form 18 January 2014 Accepted 18 January 2014 Available online 26 January 2014 Keywords: Brown adipose tissue Exercise FNDC5 Obesity

a b s t r a c t The recently described myokine, irisin is cleaved from fibronectin type III domain containing protein 5 (FNDC5) and has been proposed to be secreted upon exercise to promote the browning of beige fat cells in white adipose tissue that results in enhanced thermogenesis and increased energy expenditure. The initial studies suggested irisin as a treatment option for obesity and associated diseases such as type 2 diabetes mellitus and stimulated further research. However, the results of subsequent studies investigating the regulation of irisin by different types of exercise are partly conflicting and effects were only shown in highly selective patient populations so far. Moreover, other parameters like body weight or fat free mass were shown to influence irisin adding more complexity to the mechanisms regulating this hormone. The present review will describe the discovery of irisin, its potential role in adipose tissuemediated thermogenesis, its regulation by exercise and lastly, discuss current controversies and highlight gaps of knowledge to be filled by future studies. © 2014 Elsevier Inc. All rights reserved.

Contents 1.

2.

3.

4.

Identification of irisin, expression and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of FNDC5 as muscle-derived protein to induce the browning of white adipose tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. 1.2. Structure of FNDC5 and processing to irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conservation of irisin across mammalian species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Expression sites of FNDC5/irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Browning of white adipose tissue and the proposed role of irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Regulation of FNDC5/irisin by exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Alteration of FNDC5/irisin in different diseases and possible implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alteration under conditions of long-term changes in body weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Alteration under conditions of impaired glucose homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Alteration under other conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Gaps to fill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Establishment and validation of antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Evaluation of the translation of human versus mouse FNDC5/irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Identification and regulation of the irisin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Identification of the molecular pathways regulating the processing of FNDC5 to irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Identification of other functions of irisin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: Charité Center for Internal Medicine and Dermatology, Division of General Internal and Psychosomatic Medicine, Charitéplatz 1, 10117 Berlin, Germany. Tel.: +49 30 450 553 002; fax: +49 30 450 553 900. E-mail addresses: [email protected], [email protected] (A. Stengel). 0196-9781/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2014.01.016

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1. Identification of irisin, expression and regulation 1.1. Identification of FNDC5 as muscle-derived protein to induce the browning of white adipose tissue

modification the finally secreted portion of FNDC5, a 112 amino acid peptide, was named irisin in analogy to the Greek messenger goddess, Iris [10]. 1.3. Conservation of irisin across mammalian species

Peroxisome proliferator-activated receptor (PPAR)-␥ coactivator-1␣ (PGC-1␣) has been described as a key modulator of biological mechanisms implicated in energy metabolism. Elevated PGC-1␣ mRNA expression was observed in brown adipose tissue and skeletal muscle of mice exposed to cold resulting in an up-regulation of uncoupling protein (UCP) 1 [57]. In addition, PGC-1␣ is induced in skeletal muscle by exercise [55] stimulating mitochondrial biogenesis with beneficial effects on inflammation and several chronic diseases [27]. In line with these findings, transgenic mice overexpressing PGC-1␣ are resistant to age-related weight gain and show an improved insulin sensitivity [78]. These findings suggested a factor derived from skeletal muscle communicating with other organs. Therefore, Boström et al. analyzed adipose tissue of PGC-1␣ overexpressing transgenic mice to identify genes involved in thermogenesis and development of brown fat. Although they did not find any differences in the expression of such genes in interscapular brown or visceral white adipose tissue, they detected elevated levels of UCP1 protein in the inguinal subcutaneous fat layer compared to wild-type controls and a strongly increased UCP1 mRNA expression in response to exercise such as wheel running or swimming giving rise to a PGC-1␣dependent browning of subcutaneous white adipose tissue [10]. Since this effect might be due to direct muscle-to-adipose tissue signaling, white adipocytes were treated with conditioned media from myocytes expressing PGC-1␣ that resulted in an increased mRNA expression of several genes specific for brown fat including UCP1 suggesting a molecule secreted by muscle cells able to induce a thermogenic program [10]. Next, five proteins likely to be targeted by PGC-1␣ and secreted by skeletal muscle were identified by a combination of gene arrays with algorithms to predict secreted proteins. Subsequently, these five proteins, namely interleukin 15 (IL-15), fibronectin type III domain containing protein 5 (FNDC5), vascular endothelial growth factor B (VEGF-␤), leucinerich alpha-2-glycoprotein 1 (LRG1) and metalloproteinase inhibitor 4 (tissue inhibitor of metalloproteinase 4; TIMP4) were shown to be up-regulated on the mRNA level following endurance exercise in mice and humans [10]. However, when applied to subcutaneous white adipocytes, only FNDC5 induced a dose-dependent and robust UCP1 mRNA expression [10]. Conversely, application of an anti-FNDC5 antibody to the conditioned media from muscle cells expressing PGC-1␣ was followed by a marked reduction of UCP1 activity in fat cell cultures, indicating a major physiological action of FNDC5 to induce the browning of white adipose tissue [10]. As shown by immunohistochemistry, FNDC5 also promoted an increase of UCP1 protein expression in adipocytes with multilocular lipid droplets and a higher density of mitochondria as a characteristic feature of brown adipocytes. Lastly, FNDC5 augmented oxygen consumption and energy expenditure predominantly by uncoupling respiration indicating that FNDC5 induces a brown fat-like program in white adipocytes resulting in increased thermogenesis (Fig. 1) [10]. 1.2. Structure of FNDC5 and processing to irisin FNDC5 is composed of a signal peptide, a fibronectin III domain and a hydrophobic C-terminal domain [22,71]. Since this structure is found in type I membrane proteins, proteolytic cleavage and release of FNDC5 was hypothesized. In line with this assumption, while FNDC5 was detected by immunoblot in media containing FNDC5 expressing cells, C-terminally flag-tagged FNDC5 was not detectable indicating the cleavage and release [10]. After further

Sequence analysis indicated 100% identity between murine (mus musculus) and human irisin [10]. Likewise, the conservation rate of irisin for zebrafish (danio rerio) was over 97% and over 80% for Bankiva chicken (gallus gallus) [10] giving rise to the physiological importance of this peptide. However, a very recent study suggested that the human FNDC5 gene differs from that of other species by a mutation in the start codon [58]. Several rodents and other animals exhibit an ATG translation start site, whereas in humans an ATA was found which was shown to result in lower translation efficiency [37]. 1.4. Expression sites of FNDC5/irisin Following the landmark paper of Boström et al. [10], the expression of FNDC5 mRNA in skeletal muscles following exercise has been confirmed by several other groups in rodents [17,59] and humans [35,41]. Moreover, irisin has been detected on the protein level in the plasma of mice [10], rats [65] and humans [10,35,45,69,70,77,83]. An early subsequent study, investigating the distribution of FNDC5 mRNA in different human tissues using quantitative PCR, detected FNDC5 – besides in skeletal muscle – also in other organs, mainly in those containing muscle like heart (including pericardium), tongue and rectum, but also optic nerve and brain and in low levels in other organs such as kidney, liver or lung [35]. In addition, FNDC5 mRNA and irisin peptide has been described in adipose tissue, cardiomyocytes and Purkinje cells of the cerebellum in rodents [4,17,60] and in adipose tissue of humans [35,48], in human cerebrospinal fluid [56] as well as in breast milk of lactating women [3] pointing toward additional functions of this hormone initially thought to be restricted to be a myokine involved in the regulation of energy expenditure. Lastly, irisin has been detected not only in serum but also in saliva [2] which therefore might be a non-invasive alternative to blood withdrawals in future studies. 1.5. Browning of white adipose tissue and the proposed role of irisin In contrast to white adipose tissue which primarily stores energy as triglycerides, brown adipose tissue is known to dissipate energy by uncoupled mitochondrial respiration and therefore play an important role in the regulation of body weight. This is mediated by UCP1 strongly expressed in brown adipose tissue. Despite of earlier studies reporting the detection of brown adipose tissue in adult humans [29,40], for long, the persisting expression and functional relevance of brown adipose tissue was thought to be restricted to small mammals, while in humans it was assumed to disappear after infancy. Recent evidence changed this concept by describing the presence of functional brown adipose tissue in the supraclavicular and neck regions of adult humans in response to cold exposure [50,74]. Moreover, it is known that white adipose tissue contains cells that can express high levels of UCP1 and acquire the multilocular appearance typical for brown fat cells [15]. However, it remained obscure whether this ability was a predisposition of all or most white fat cells, or there was a distinct cell type displaying these properties. The discovery of irisin supported the concept of browning of white subcutaneous adipocytes (Fig. 1) [10]. The results of Boström and colleagues were confirmed by a recent study where recombinant irisin up-regulated PGC-1␣, UCP1 and other brown cell markers in murine adipocytes, an effect likely mediated

T. Hofmann et al. / Peptides 54 (2014) 89–100

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Fig. 1. Proposed pathway of FNDC5/irisin to stimulate energy expenditure. Yet to be established pathways or mechanisms where controversial results have been reported are indicated with question marks. ↑, stimulation; ↓, reduction. Abbreviations: FNDC5, fibronectin type III domain containing protein 5; PGC-1␣, peroxisome proliferator-activated receptor (PPAR)-␥ coactivator-1␣; UCP1, uncoupling protein 1; WAT, white adipose tissue.

by a signaling pathway encompassing p38 mitogen-activated protein kinase (MAPK) and extracellular-signal regulated kinase (ERK) [84]. Since previous studies showed that brown fat cells were derived from a myf-5 muscle-like cell lineage [63], whereas brown-like cells in white adipose tissue were not, a cell type distinct from the classical brown fat cells was suggested and termed beige or brite (brown in white) cells [36,53,63]. In line with this hypothesis, Wu and colleagues described cells within the subcutaneous white adipose tissue that resemble white fat cells and have a low basal expression of UCP1 [81]. However, like brown fat cells, they are able to highly express UCP1 in response to cyclic AMP thereby developing the typical brown fat-like multilocular structure. These cells were shown to express much lower basal levels of UCP1 compared to brown fat cells but reach maximum levels of UCP1 after induction similar to those observed in interscapular brown fat cells [81]. Furthermore, using gene expression analysis, the existence of two genetically diverse cell types in white subcutaneous fat was shown demonstrating that beige cells are a distinct cell type combining the characteristics of both white and brown fat cells. Lastly, the expression pattern in functional human brown fat was much closer to murine beige than to murine brown fat cells suggesting that beige rather than brown adipose tissue is present in human adults [81]. The browning of white adipose tissue is driven by cold exposure [15,43]. In addition to irisin [10] and besides ␤-adrenergic signaling [6,13] likely to predominantly transmit cold-induced browning, there are several peptides known to influence the browning of white adipose tissue. Cold-induced browning is mediated by fibroblast growth factor 21 (FGF 21) [23,34,42] and natriuretic peptides (ANP and BNP) which activate PGC-1␣ and UCP1 expression [9]. In contrast, myostatin (MSTN) has inhibitory effects [64,82] corroborated by findings that myostatin gene knock-out [64] and myostatin

peptide inhibition [82] increase the browning process. Also FNDC5 is influenced by myostatin signaling as shown by the up-regulation of FNDC5 in myostatin knock-out mice resulting in the browning of white adipose tissue, an effect abolished by an FNDC5 antibody [64]. 1.6. Regulation of FNDC5/irisin by exercise Boström et al. initially reported irisin to be expressed dependent on endurance exercise. Irisin levels were elevated after a 3-week free wheel running period in mice, a finding that also held true in eight healthy human adults after a 10 weeks period of endurance training (Fig. 1) [10]. A subsequent study investigated FNDC5 and PGC-1␣ mRNA expression in 24 male systolic heart failure patients with a mean ejection fraction of 29.5%. According to their aerobic performance as measured by oxygen consumption (VO2 ) and ventilatory efficiency (VE/VCO2 ), patients were divided into two groups: high versus low aerobic performance. The group with high peak VO2 and better VE/VCO2 displayed higher FNDC5 and PGC-1␣ mRNA expression in muscle (M. vastus lateralis) compared to the low peak VO2 and worse VE/VCO2 group [41]. A recent study concordantly observed a significant increase of FNDC5 mRNA in skeletal muscle following a 12-week combined strength and endurance training with significantly higher elevation in a group with pre-diabetes and overweight compared to normoglycemic and normal weight male subjects (Table 1) [51]. However, in contrast to the initial report, Timmons and colleagues reported a lack of differences in muscle FNDC5 mRNA expression between either sedentary compared to endurance exercise-trained younger subjects and sedentary versus resistancetrained subjects (Table 1) [72]. Interestingly, when separately analyzing a subgroup of older endurance-trained subjects compared to untrained and younger trained or untrained subjects, an

92

Table 1 Studies in humans investigating the influence of exercise on FNDC5/irisin. Group size

Sex

Age

Study design

Intervention/Grouping

Analyzed material

Key findings

Influence of exercise on FNDC5/irisin

obese (BMI 33) non-diabetic adults

n=8

Male

∼52 years

Interventional

10 weeks of supervised endurance exercise training

Plasma, muscle biopsy

Two-fold increase of circulating plasma irisin; increase of FNDC5 mRNA expression in muscle

+

Timmons et al. [72]

Younger and older subjects

n = 10 per group

Male and female

Younger (18–28) and older (65–80) subjects

Interventional

Resistance exercise training twice weekly on non-consecutive days for 26 weeks

Muscle biopsy

30% greater FNDC5 mRNA expression in muscle of older trained compared to older sedentary subjects

+

Lecker et al. [41]

Patients with heart failure (left ventricular ejection fraction ≤40%)

n = 24

Male

Mean age 67.2 ± 9.2 years, age range 50–86)

Cross-sectional

High versus low aerobic performance: based on peak VO2 (>14 versus ≤14 ml O2 kg−1 min−1 [n = 15 and 9, respectively] and VE/VcO2 <34 versus ≥34 [n = 14 and 10, respectively])

Muscle biopsy

FNDC5 mRNA expression in muscle higher in high aerobic performance group

+

Huh et al. [35]

Healthy normal weight adults

n = 15

Male

20.5 ± 1.5 years

Interventional

2 or 3 sets of 2 m × 80 m sprints

Serum

Induction of serum irisin 30 min after exercise (sprints)

+

Kraemer et al. [38]

Healthy volunteers

n = 12

Male (n = 7) and female (n = 5)

18–35 years

Interventional

90 min treadmill aerobic exercise

Plasma

Transient elevation of circulating irisin concentration after 1 h of treadmill exercise, no sex differences

+

Aydin et al. [2]

Sedentary volunteers; obese (34.8 ± 4.0 kg/m2 , n = 7) and normal weight subjects (26.6 ± 6.0 kg/m2 , n = 7)

n = 14

Male

40.5 ± 6.5 years

Interventional

45 min running (5.5 km)

Saliva

Elevation of irisin levels in saliva

+

Norheim et al. [51]

Healthy and physically inactive men

Normoglycaemic and normal weight (n = 13); slightly hyperglycaemic and over-weight (n = 13)

Male

40–65 years

Interventional

Combined strength (2×/week) and endurance (2×/week) training for 12 weeks; 45 min and 70% of VO2max ergometer cycling before and after training period

Muscle biopsy, subcutaneous adipose tissue

Chronic training enhanced muscle FNDC5 mRNA expression; higher FNDC5 expression in pre-diabetes group compared to controls after chronic training

+

Sedentary subjects

Total: n = 24; high responder group (n = 8) and low responder group (n = 8)

Male

23 ± 1 (low res-ponders) and 24 ± 1 (high respon-ders)

Interventional

6 weeks intense endurance cycling

Muscle biopsy

No change of FNDC5 mRNA expression in muscle

−

Positive effect on irisin Boström et al. [10]

No effect on irisin Timmons et al. [72,73]

T. Hofmann et al. / Peptides 54 (2014) 89–100

Population

Reference

Healthy non-trained individuals

n = 43

Male and female

20–80 years

Interventional

20 weeks of supervised resistance training

Muscle biopsy

No change of FNDC5 mRNA expression in muscle

−

Huh et al. [35]

Healthy normal weight adults

n = 15

Male

20.5 ± 1.5 years

Interventional

8 weeks of intermittent sprint running 3 times per week (2 or 3 sets of 2 × 80 m sprints)

Serum

No effect after 8 weeks of training on serum irisin

−

Besse-Patin et al. [7]

Obese (BMI 32.6 ± 2.3 kg/m2 ) non-diabetic volunteers

n = 11

Male

35.4 ± 1.5 years

Interventional

8 weeks endurance training

Muscle biopsy

No change of FNDC5 mRNA expression in muscle

−

Pekkala et al. [52]

Normal weight and overweight untrained healthy volunteers

n = 56

Male

(1) 50 ± 4 years (n = 17); (2) 27 ± 3 years (n = 10); 62 ± 5 years (n = 11); (3) 57 ± 7 years (n = 9); 62 ± 5 years (n = 9); (4) 34 ± 7 years (n = 14)

Interventional

(1) Acute low intensity exercise training (bicycle ergometry for 1 h) (2) Single resistance exercise training (5 × 10 repetitions of leg press) (3) Heavy intensity endurance exercise combined with resistance exercise training 4 times/week for 21 weeks (4) Heavy resistance exercise training (5 × 10 knee bilateral extensions)

Muscle biopsy; serum

No changes in muscle FNDC5 mRNA or serum irisin

−

Kraemer et al. [38]

Healthy volunteers

n = 12

Male (n = 7) and female (n = 5)

18–35 years

Interventional

90 min treadmill aerobic exercise

Plasma

No elevation of circulating irisin concentration immediately at the end of exercise and 20 min after exercise, no sex differences

−

Aydin et al. [2]

Obese (n = 7) and normal weight (n = 7)

n = 14

Male

40.5 ± 6.5 years

Interventional

45 min running (5.5 km)

Serum

No changes of irisin levels in serum

−

T. Hofmann et al. / Peptides 54 (2014) 89–100

Timmons et al. [72], Phillips et al. [54]

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Table 1 (Continued) Population

Group size

Sex

Age

Study design

Intervention/Grouping

Analyzed material

Key findings

Influence of exercise on FNDC5/irisin

Hecksteden et al. [30]

Untrained normal weight healthy volunteers

(1) n = 62 (control group n = 39, and intervention group n = 23) (2) n = 79 (control group n = 39, and intervention group n = 40)

(1) 26 females/13 males in control group; 15 females/8 males in intervention group (2) 26 females/13 males in control group; 23 females/7 males in intervention group

50 ± 7 years in control group and 49 ± 7 years in intervention group

Interventional

(1) Aerobic endurance training, 45 min running, three times a week for 6 months (2) Strength endurance training, machine-based exercises, three times a week for 6 months

Serum

No training-induced changes in irisin serum concentrations

−

Norheim et al. [51]

Healthy and physically inactive men

Normoglycaemic and normal weight (n = 13); slightly hyperglycaemic and over-weight (n = 13)

Male

40–65 years

Interventional

45 min and 70% of VO2max ergometer cycling

Muscle biopsy, subcutaneous adipose tissue

Acute exercise had no effect on muscle FNDC5 mRNA

−

Hofmann et al. [32]

Anorexic patients with moderate and high activity (BMI 14.2 ± 0.4 versus 15.0 ± 0.4 kg/m2 )

n = 39

Female

28.6 ± 3.1 year (purging type AN) 27.8 ± 2.4 years (restricting type AN) 25.4 ± 1.7 years (atypical AN)

Cross-sectional

According to physical activity

Plasma

No correlation between irisin and activity parameters

−

The studies are displayed in the order of publication. +: stimulation, −: no effect. Abbreviations: BMI, body mass index; AN, anorexia nervosa.

T. Hofmann et al. / Peptides 54 (2014) 89–100

Reference

T. Hofmann et al. / Peptides 54 (2014) 89–100

elevation of FNDC5 mRNA expression levels was observed [72]. Similarly, a German study did not detect any changes of serum irisin concentrations after 6 months of aerobic or strength exercise trainings in a randomized controlled trial (Table 1) [30]. In line with the other reports describing negative data, a study investigating young healthy males did not find any changes in circulating irisin following an 8-week exercise program (Table 1) [35]. However, an increase was observed in a subpopulation of untrained subjects after short-term exercise (sprints) [35] suggesting a differential regulation dependent on acute or chronic forms of exercise with a possible counter-regulation/adaptation over time. This hypothesis is in line with results from recent studies that observed elevated circulating irisin levels following short-term exercise. Kraemer et al. detected transiently increased irisin levels during a 90-min treadmill aerobic exercise [38] and Norheim et al. described an elevation immediately following 45 min of ergometer cycling [51]. This was followed by decline to baseline levels at the end of the exercise (90 min) [38]. Since PGC-1␣-levels are reported to increase in later stages (>2 h) of exercise [51,67,76], one could speculate that the observed early elevation of irisin might be independent of PGC1␣ [38] and possibly also independent of FNDC5 since irisin but not FNDC5 was reported to be elevated in early stages of cycling [51]. This regulation may be different under conditions of chronic exercise, a hypothesis that warrants further investigation. However, comparing four different conditions with acute lowintensity aerobic exercise, heavy-intensity resistance exercise, and long-term endurance exercise without and combined with a resistance exercise training over a period of 21 weeks in untrained men of different age groups, no differences were observed for irisin serum levels and in skeletal muscle FNDC5 or PGC-1␣ mRNA expression with large inter- and intra-individual variations (Fig. 1 and Table 1) [52]. Yet, a 4-fold increase of PGC-1␣ in young and a 2-fold increase in older men was observed following single resistance exercise [52] adding more complexity to the regulatory mechanisms investigated. Moreover, differences in PGC-1␣ mRNA expression or irisin serum levels were not associated with alterations in FNDC5 mRNA leading to the suggestion that other factors apart from PGC-1␣ – e.g. some of the various other exerciseregulated myokines – might contribute to changes in FNDC5 and irisin [52]. The lacking association of FNDC5 expression and serum irisin in this study may indicate additional regulatory mechanisms for the circulating levels of irisin [52]. As also pointed out by the authors, small differences in the exercise protocols of the various studies performed so far could contribute to the in part conflicting results (Table 1). Lastly, also our group was unable to detect differences in circulating irisin in anorexic patients with high physical activity compared to a group with moderate physical activity [32]. Whether this is a special characteristic of this patient population or points toward a dissociation of exercise and irisin levels warrants to be further investigated. Although 100% identity between human and rodent irisin was detected, the controversial findings may also be attributed to different start codons as recently described [58] leading to a differential induction of this peptide by exercise. However, negative data have been recently obtained in animals as well. In pigs, no increase of FNDC5 gene or protein expression was observed after 16–20 weeks of aerobic exercise training, while an increase of circulating irisin was seen in familial hypercholesterolemic but not in healthy pigs [20]. The authors speculate that only low and hardly detectable amounts of FNDC5 could account for the observed elevation of irisin or that exercise might primarily affect the cleavage of FNDC5 to irisin and that genetic dyslipidemia could influence irisin levels [20]. In summary, these findings indicate that the proposed beneficial role of irisin may be relevant only in a highly selective population of subjects. Whether species differences, the time point of assessment

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or age-related alterations of regulatory processes account for the observed differences and controversial findings has to be further investigated. 2. Alteration of FNDC5/irisin in different diseases and possible implications 2.1. Alteration under conditions of long-term changes in body weight Several studies investigated the relationship between body weight and body mass index (BMI) on one hand and irisin on the other. Early on, a trend toward a positive correlation was observed in a study with a BMI range of 20–48 kg/m2 [35]. These results were extended by our group investigating subjects with anorexia nervosa, normal weight and obesity, thereby representing a very broad BMI range of 9–85 kg/m2 which indicated a significant positive correlation of irisin and BMI (Table 2) [69]. In addition, a positive correlation of irisin and fat-free mass was observed [69]. In line with these results, several other studies subsequently confirmed the positive association of circulating irisin with BMI (Table 2) [12,16,31,45]. However, one study in normal weight and overweight men did not detect any association [52] and another study investigating 29 morbidly obese subjects reported a negative correlation of irisin with BMI [48], which may be due to other – yet to be uncovered – factors influencing the expression of the rather pleiotropic myokine, irisin. Overall, most studies support the notion that irisin levels are associated with increased body weight. Whether this elevation is primarily due to an increase in muscle or fat mass will have to be further established as both tissues were shown to express FNDC5 mRNA. 2.2. Alteration under conditions of impaired glucose homeostasis Since a beneficial effect of irisin was proposed for obese patients, the regulation of this hormone has been studied with respect to obesity-associated diseases, predominantly type 2 diabetes mellitus (Table 2). A Korean study conducted in subjects with new-onset type 2 diabetes mellitus reported decreased serum irisin levels compared to matched controls with normal glucose tolerance [12]. In addition, 2-h plasma glucose was independently inversely correlated with circulating irisin levels after adjustment for age, sex and BMI [12]. Moreover, increased irisin levels were associated with reduced odds (OR 0.64) for a prevalent newly diagnosed type 2 diabetes mellitus [12]. Investigating healthy and normal weight Saudi Arabian children, irisin levels were also negatively associated with fasting blood glucose and with the HOMA-IR Index in girls but not in boys [1]. These results are in line with two other studies that also reported plasma irisin to be significantly lower in individuals diagnosed with type 2 diabetes mellitus versus non-diabetic controls (Table 2) [45,48]. Aydin et al. detected irisin also in breast milk in higher concentrations compared to plasma [3]. These levels along with circulating irisin concentrations were reported to be lower in lactating women with gestational diabetes mellitus than in healthy lactating women [3]. The authors speculated that the mammary gland could be a direct source of irisin besides skeletal muscle and adipose tissue [3], a hypothesis to be confirmed by expression and immunohistochemical studies. Taken together, these observational findings point toward a downregulation of irisin under conditions of impaired glucose control or diabetes mellitus. However, it has to be noted that the causality of this association has to be further investigated in exploratory studies. In contrast to the findings described above, two studies did not detect any correlation of irisin with glucose homeostasis

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Table 2 Studies in humans investigating other parameters influencing FNDC5/irisin. Population

Group size

Sex

Age

Study design

Intervention/grouping

Analyzed material

Key findings

Timmons et al. [72]; Gallagher et al. [24]

Obese subjects, with T2D, IGT, controls with NGT

n = 118

∼65% male, 35% female

∼55 years

Cross-sectional

Comparison over a BMI spectrum

Muscle biopsy

FNDC5 mRNA expression in muscle positively correlated with BMI, not correlated with glucose homeostasis

Huh et al. [35]

healthy subjects with different BMI

n = 18

No information provided

No information provided

Cross-sectional

Comparison over a BMI spectrum

Muscle biopsy

FNDC5 mRNA expression in muscle tended to positively correlate with BMI (no significance)

Huh et al. [35]

Obese subjects undergoing bariatric surgery (BMI 50.2 ± 10.6 kg/m2 )

n = 14

Male (n = 8) and female (n = 6)

53.1 ± 8.9 years

Interventional

Bariatric surgery

Serum

Decrease of circulating irisin levels following bariatric surgery

Huh et al. [35]

Healthy volunteers

n = 117

Female

49.3 ± 8.6 years

Cross-sectional

Comparison over a BMI spectrum with different biceps circumference and fat-free mass

Plasma

Correlation of plasma irisin with biceps circumference, fat-free mass and BMI

Stengel et al. [69]

Patients with anorexia nervosa (n = 8); normal weight (n = 8); obese patients (n = 8); morbidly obese patients (n = 8); super obese patients (n = 8)

n = 40

Female (n = 24) and male (n = 16)

45–48 years (normal weight and obese); 26 years (anorexic)

Cross-sectional

Comparison over a very broad BMI spectrum

Plasma

Correlation of plasma irisin with BMI, fat mass, fat free mass and insulin

Choi et al. [12]

Subjects with NGT (n = 104) and subjects with new-onset T2D (n = 104)

n = 208

38/66 (NGT), 35/69 (T2D)

65.1 ± 9.6 (NGT) and 64.3 ± 4.0 (T2D) years

Cross-sectional

According to glucose tolerance

Serum

Decrease of serum irisin in subjects with T2D compared to controls with NGT

Moreno-Navarrete et al. [48]

Subjects with wide range of BMI (20–58 kg/m2 )

n = 125

Male (n = 28) and female (n = 97)

43.9 ± 10.4 to 55.5 ± 13.2 years

Cross-sectional

Comparison over a BMI spectrum

Adipose tissue, subgroup: muscle tissue

FNDC5 mRNA in adipose tissue negatively correlated with BMI; FNDC5 mRNA expression in muscle was decreased in association with T2D in obese subjects

Moreno-Navarrete et al. [48]

Overweight subjects with T2D and NGT

n = 76

Male

50.9 ± 10.9 (control); 53.6 ± 10.3 (T2D) years

Cross-sectional

Comparison over a BMI spectrum

Plasma

Plasma irisin negatively correlated with BMI; plasma irisin decreased in subjects with T2D

Swick et al. [70]

Post-menopausal women (BMI 24–45 kg/m2 )

n = 17

Female

50–70 years

Cross-sectional

24-h whole room indirect calorimetry

Plasma

Plasma irisin is correlated with energy expenditure only in a subgroup with high energy expenditure

Liu et al. [45]

Subjects with T2D (n = 96); controls with NGT (n = 60)

n = 156

NGT: female (n = 35) and male (n = 25); T2D: female (n = 38) and male (n = 38)

40.6 ± 12.7 (NGT) 58.7 ± 9.3 (T2D)

Cross-sectional

According to glucose tolerance

Plasma

Decrease of plasma irisin in subjects with T2D compared to non-diabetic controls; plasma irisin associated with BMI in non-diabetic subjects

Staiger et al. [68]

37 young subjects with increased risk of T2D and 14 elderly subjects (including 6 subjects with T2D)

n = 51

Female (n = 18) and male (n = 33)

28 ± 7 and 62 ± 4 years

Cross-sectional

Correlation with fasting insulin concentrations

Muscle biopsies

Positive association between FNDC5 mRNA and fasting insulin concentration

T. Hofmann et al. / Peptides 54 (2014) 89–100

Reference

Obese subjects

n = 296

Female (n = 199) and male (n = 97)

53.1 ± 7.3–55.0 ± 6.6 years

Cross-sectional

Four quartiles according to intrahepatic triglyceride content

Plasma

Decreased plasma irisin in non-alcoholic fatty liver disease

Wen et al. [77]

38 patients with stage 5 chronic kidney disease and 19 age- and sex-matched controls

n = 57

Female (n = 20) and male (n = 18)

57.4 ± 2.5 years

Cross-sectional

According to renal function

Plasma

Decreased plasma irisin in chronic kidney disease

Aydin et al. [3]

15 healthy lactating women; 15 lactating women with GDM; 14 age matched female controls

n = 44

Female

28.1 ± 1.9 (healthy lactating); 29.4 ± 2.3 (lactating with GDM); 27.1 ± 2.4 (healthy non-lactating)

Cross-sectional

According to lactation and GDM

Breast milk, plasma

Higher irisin concentration in colostrum than in transitional and mature milk; lower irisin concentration in breast milk in women with GDM compared to healthy lactating women; lower plasma irisin in women with GDM than non-lactating or healthy lactating women

Hee Park et al. [31]

Caucasian and African American subjects

n = 151

Female (n = 80) and male (n = 71)

46.0 (range 43.0–47.0) years

Cross-sectional

According to metabolic syndrome

Plasma

Increased irisin plasma levels in subjects with metabolic syndrome and insulin resistance, positive association with cardiovascular disease

Aydin et al. [2]

Obese (n = 7) and normal weight (n = 7) subjects

n = 14

Male

Obese (40.5 ± 6.5) and normal weight (44.1 ± 6.8) years

Interventional

Turkish bath

Serum and saliva

Elevation of irisin levels in serum and saliva

Ebert et al. [18]

Healthy pregnant women before and after cesarean section (BMI 22.9 (5.1))a

n = 40

Female

31 (6)a

Longitudinal

Postpartum to prepartal

Serum

Positive association of insulin and irisin during pregnancy

Piya et al. [56]

Pregnant women (37.7 ± 7.6 kg/m2 )

n = 91

Female

32 ± 8.3 years

Cross-sectional

According to diabetes mellitus

Serum and cerebrospinal fluid

Lower levels of serum irisin in pregnant women with gestational diabetes mellitus; correlation of serum irisin with levels in cerebrospinal fluid

Crujeiras et al. [16]

Obese subjects (BMI 35.6 ± 4.5 kg/m2 ); normal weight subjects (BMI 22.9 ± 2.2 kg/m2 )

n = 94 (obese); n = 48 (normal weight)

Obese: female (n = 44) and male (n = 50); normal weight (n = 32) and male (n = 16)

Obese: 49.4 ± 9.4 years; normal weight: 35.7 ± 8.8 years

Interventional

Weight loss program following an 8-week hypocaloric diet

Plasma

Higher irisin levels in obese subjects compared to normal weight; higher in men than women; decrease following weight loss and restoration after weight regain

Ebert et al. [18]

Pregnant women with (BMI 24.5 (6.6) before pregnancy) or without gestational diabetes mellitus (BMI 22.4 (6.7) before pregnancy)a

n = 148 (n = 74 each group)

Female

31.0 (7.5) (GDM) 28.9 (4.5) (controls)a

Cross-sectional

According to diabetes mellitus

Serum

Higher irisin levels in women with gestational diabetes mellitus compared to controls after delivery

Liu et al. [44]

Subjects with diabetes mellitus and different stages of kidney dysfunction

n = 365

∼61% male; ∼39% female

∼60 ± 10 years

Cross-sectional

According to renal function

Plasma

Decrease of circulating irisin with renal insufficiency

T. Hofmann et al. / Peptides 54 (2014) 89–100

Zhang et al. [83]

The studies are displayed in the order of publication. Abbreviations: BMI, body mass index; FNDC5, fibronectin type III domain containing 5; GDM, gestational diabetes mellitus; T2D, type 2 diabetes mellitus; NGT, normal glucose tolerance, IGT, impaired glucose tolerance. a Values for median (interquartile range). 97

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[52,72]. Similarly, in rats, insulin sensitivity achieved by caloric restriction was independent of alterations of circulating irisin levels [65]. Adding more complexity to the regulation of irisin, a recent study reported irisin levels to be positively associated with insulin resistance per se in obese subjects [31]. The authors speculated that obesity and the metabolic syndrome might cause irisin resistance similar to those known for insulin and leptin [31], a hypothesis that questions the expected therapeutic use of this peptide for the treatment of obesity. Similarly, also pregnant women with gestational diabetes mellitus displayed higher circulating levels of irisin compared to non-diabetic pregnant controls [18,56]. The divergent results observed in the studies described above may be due to the degree of glucose homeostasis impairment (insulin resistance versus fully developed type 2 diabetes mellitus). Moreover, one cannot exclude that different ethnic backgrounds (Caucasian versus African American [31]), sex (higher circulating irisin levels in females versus males [1,35]) and also different antibodies and assay kits (Abcam versus Aviscera versus Adipogen versus Acris versus USCN Life Sciences versus Phoenix Pharmaceuticals [19]) influence these results.

2.3. Alteration under other conditions Irisin has also been studied in various other conditions. Due to irisin’s proposed ability to dissipate energy via the browning of white adipose tissue it is likely to assume that alterations in energy expenditure correlate with FNDC5/irisin expression. So far, there is one study investigating the relationship of irisin with resting energy expenditure in 17 postmenopausal women using whole room indirect calorimetry. No correlation was detected in the whole sample (although the authors described a correlation in a very special subgroup whose energy expenditure was greater than predicted by an equation based on the amount of fat free mass, Table 2) [70]. Interestingly Aydin et al. reported an increase of serum and saliva irisin levels following a Turkish bath which may point toward an induction of irisin by higher temperature and a possible implication in thermoregulation [2]. This finding will have to be confirmed in future studies and the underlying pathways investigated. Since browning of white adipose tissue is also known to be induced by cold exposure [15,43] it will be interesting to investigate whether a decrease in ambient temperature affects irisin levels. A Taiwanese study investigated irisin plasma levels in patients with stage 5 chronic kidney disease. Irisin levels were significantly decreased and inversely correlated with urea nitrogen and creatinine compared to healthy, age- and sex-matched subjects [77], a finding that was recently confirmed [18,44]. Whether these alterations mainly reflect diminished muscle mass in these patients remains to be investigated. Furthermore, in this study, circulating irisin was positively associated with high-density lipoprotein [77]. Lastly, lower blood triglycerides and transaminases were associated with higher irisin levels and independent of lower intrahepatic triglyceride content in Chinese obese subjects with non-alcoholic fatty liver disease [83] suggesting a beneficial effect of irisin on lipogenesis (Table 2). As an additional function besides energy expenditure, several studies reported the involvement of FNDC5/irisin in the development of the nervous system [28,35,47], suggesting the implication of FNDC5/irisin in the regulation of cognitive functions possibly via expression of brain-derived neurotrophic factor (BDNF) in the brain [79]. These results might support the assumption of a beneficial or protective effect of exercise for brain functions in neuropsychiatric conditions such as depression [14,66] or Alzheimer’s disease [11,21].

3. Gaps to fill Although our knowledge on the regulation and effects of irisin greatly expanded during the past two years, several questions remain to be answered. The major gaps of knowledge to be filled in the future are discussed in the following paragraphs. 3.1. Establishment and validation of antibodies Different commercially available antibodies or ELISA kits have been used in the various studies to describe and quantify circulating irisin levels [31,35,60]. As extensively discussed by Erickson [19], several antibodies used have not been well validated. Huh et al. used an antibody sold by Aviscera (Santa Clara, CA, USA) they tested before against other antibodies that were not named in the paper and reported it to be the most reliable one [35]. Hee Park et al. [31] compared the mentioned kit by Aviscera with those from Phoenix Pharmaceuticals (Burlingame, CA, USA) and Adipogen (San Diego, CA, USA). Single values and the range measured with the Adipogen kit were higher than those measured with Phoenix or Aviscera kits. Roca-Rivada et al. [60] used the antibody by Abcam (Cambridge, UK) and the Phoenix antibody that are directed against different sequences of the FNDC5/irisin peptide. Since the different antibodies used could likely contribute to the lacking consistency of the results (at least regarding circulating irisin levels), the thorough validation of antibodies by Western blot analysis to confirm the correct length of the peptide and mass spectrometry to investigate whether the correct structure of irisin is contained in the band of interest should precede the extensive use of the commercially available antibodies and assay kits. 3.2. Evaluation of the translation of human versus mouse FNDC5/irisin As mentioned before in this paper, there is 100% identity between human and murine irisin amino acid sequences. However, Raschke et al. [58] reported a different start codon section in the human irisin sequence, questioning effective translation and therefore transferability of data from mouse to human. This could be an explanation for the largely consistent data in rodents, whereas in humans conflicting results have been reported. Thus, future studies should investigate the translation of human versus murine FNDC5/irisin in vitro under different stimulated conditions to further clarify this potentially relevant difference. 3.3. Identification and regulation of the irisin receptor Despite the increasing knowledge on effects of circulating irisin, the receptor(s) mediating these effects remain to be discovered. In their initial study, Boström and colleagues suggested the existence of a cell surface receptor [10]. Interestingly, irisin itself was recently shown to form dimers [62], which may be important for the ligand–receptor interaction. The identification of the irisin receptor along with the subsequent description of the expression sites and regulation will be a big leap forward in the understanding of irisin’s physiology. 3.4. Identification of the molecular pathways regulating the processing of FNDC5 to irisin The original study of Boström et al. introduced a pathway of exercise-induced stimulation of FNDC5 expression followed by the cleavage and release of irisin [10]. However, this concept was recently challenged by the observation of an increase of circulating irisin levels following exercise while FNDC5 mRNA or protein expression was not altered in pigs [20]. This could point toward a

T. Hofmann et al. / Peptides 54 (2014) 89–100

selective induction of processing following exercise, a hypothesis to be further corroborated. Moreover, a recent review also challenged the function of irisin as a circulating hormone and proposed the action as a membrane-bound peptide [19]. Studies using mass spectrometry and studying the membrane-bound and soluble fractions in detail should be performed to address this point. 3.5. Identification of other functions of irisin Additional confounding factors could be another explanation for the controversial findings on the exercise-dependent production of irisin in skeletal muscle and its secretion into the circulation. Several peptides before were introduced with a specific function and later appeared to be additionally – or even predominantly – involved in other physiological processes. For example, ghrelin, initially described to be a hunger-inducing hormone [80] was subsequently shown to be also implicated in several gastrointestinal functions [49], the regulation of mood [46] and stress [39] as well as sleep [25], and immunomodulation [5]. Likewise, nesfatin-1 was described first as a regulator of food intake [61] but seems to be also implicated in mood disorders [8], stress [26] and anxiety [33] as well as the regulation of sleep [75]. Likewise, as reported above, also irisin – besides the possible implication in energy expenditure – has already been shown to be involved in neuronal processes [17,28,47] and proposed to mediate beneficial exercise-induced effects on cognitive functions [79]. Due to the recent amount of negative data and the need for highly selective patient populations in order to observe irisin’s proposed main effect on energy expenditure, it may be speculated that the major function of irisin is yet to be uncovered. This should be investigated in future studies. 4. Summary and conclusion Irisin’s discovery two years ago and its proposed mediation of the browning of white adipose tissue and therefore increasing energy expenditure by enhanced thermogenesis has attracted a lot of attention regarding its potential use as a new treatment option in obesity and diabetes mellitus. However, subsequent studies in humans have shown inconsistent results primarily related to the proposed induction of FNDC5 and irisin by exercise. Besides methodological inconsistencies, results might be influenced by yet to be better described additional parameters regulating irisin. Lastly, one has to consider that the effect on energy expenditure may not be the main/only function of irisin as described before for other peptide hormones. Conflicts of interest The authors have nothing to disclose. No conflicts of interest exist. Acknowledgements This work was supported by German Research Foundation STE 1765/3-1 (A.S.) and Charité University Funding UFF 89-441-176 (A.S. and T.H.). References [1] Al-Daghri NM, Alkharfy KM, Rahman S, Amer OE, Vinodson B, Sabico S, et al. Irisin as a predictor of glucose metabolism in children: sexually dimorphic effects. Eur J Clin Invest 2013. [2] Aydin S, Aydin S, Kuloglu T, Yilmaz M, Kalayci M, Sahin I, et al. Alterations of irisin concentrations in saliva and serum of obese and normal-weight subjects, before and after 45 min of a Turkish bath or running. Peptides 2013;50:13–8. [3] Aydin S, Kuloglu T, Aydin S. Copeptin, adropin and irisin concentrations in breast milk and plasma of healthy women and those with gestational diabetes mellitus. Peptides 2013;47:66–70.

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