Irisin: fat or artefact

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1 Clinical Endocrinology (2015) 82, doi: /cen REVIEW ARTICLE Irisin: fat or artefact A.B. Crujeiras*,,1, M. Pardo*,,1 and F.F. Casanueva*, *Laboratory of Molecular and Cellular Endocrinology, Health Research Institute of Santiago (IDIS), University Hospital of Santiago (XXIS/SERGAS), Santiago de Compostela University (USC), and CIBER Fisiopatologıa de la Obesidad y la Nutricion (CIBERobn), Santiago de Compostela, Spain Summary Soon after the discovery of the muscle-derived factor irisin, a great controversy arose in the literature regarding certain inconsistencies in the regulation of the fibronectin type III domain containing 5 protein (FNDC5/irisin) after exercise, as well as the unpredicted association of circulating irisin levels with parameters of adiposity in humans. Due to these questionable findings, doubts as to the identity of the soluble portion of FNDC5 as well as the real role of irisin and its possible therapeutic applications in the treatment of obesity and diabetes have proliferated. We recently postulated that FNDC5/irisin is an adipokine expressed and secreted by white adipose tissue in rats and humans. Its circulating concentration correlates with adiposity in humans among independent cohorts of patients. Further analysis, focused on obesity-related metabolic disorders, has shown that irisin could play a role in promoting insulin resistance or act as an adaptive response to counteract disturbances in glucose and lipid homoeostasis in obesity. Overall, this leads us to raise the question whether the new factor, increased in circulation of obese patients, is really irisin-reflecting fat mass or it is an artefact. Therefore, the current review is focused on the potential participation of adipose tissue in irisin circulating levels, and the role of irisin in metabolic pathologies associated with obesity in an attempt to clarify the controversy generated by these recently published reports. (Received 7 July 2014; returned for revision 28 July 2014; finally revised 29 September 2014; accepted 1 October 2014) Introduction In pursuing the molecular events that are responsible for the metabolic benefits of exercise, the peptide irisin was recently Correspondence: Felipe F. Casanueva, Molecular and Cellular Endocrinology Area (Lab. 2). Instituto de Investigacion Sanitaria, Complejo Hospitalario Universitario de Santiago (CHUS), C/Choupana, s/n Santiago de Compostela, Spain. Tel.: ; Fax: ; endocrine@usc.es 1 Both authors equally contributed to this work. identified as a muscle-derived factor that is presumably secreted after cleavage of the extracellular portion of the type I membrane protein fibronectin type III domain containing 5 (Fndc5). 1 The relevance of this discovery is due to the beneficial effects attributed to this myokine. Thus, it was described that on exercise stimulation, and through PGC1a, the expression of FNDC5 in muscle is augmented and irisin is secreted, inducing the stimulation of thermogenesis genes in certain adipocytes. 2 As a result, irisin may act as a muscle-derived energy-expenditure signal that directly communicates with adipose tissue, inducing browning. This effect improves the white adipose tissue (WAT) metabolic profile and enhances whole-body energy expenditure, making irisin a potential new target for the treatment of metabolic diseases. In this regard, different evidence suggests an antidiabetic role of irisin, improving glucose homoeostasis, 3 5 an inverse association of irisin with liver fat content; 5 and a positive correlation with follistatin, a peptide that regulates muscle growth. 4 Controversy has arisen because many investigations have questioned the primary beneficial role of irisin. On the one hand, there are inconsistencies regarding the regulation of FNDC5/irisin by exercise 6,7 and, unexpectedly, different reports have indicated that circulating irisin levels in humans are positively correlated with parameters of adiposity, such as BMI, which is highest in obese individuals. 6,8 11 Further analysis, focused on obesity-related metabolic disorders, showed an association of irisin with markers of glucose and lipid homoeostasis disturbance in obesity On this matter, differences between healthy and diseased situations may explain these discrepancies. However, the theoretical 12 kda form of irisin has not yet been identified in the circulation in humans; and the ability of available antibodies to detect FNDC5/irisin has also been put into doubt. Taking into account the huge controversy in the literature regarding the association between irisin and metabolic disease, and considering the lack of consensus concerning the identity of the soluble portion of the FNDC5, the mechanism of its secretion, and the presence of diverse target epitopes among manufacturers, we assess whether the new factor that is increased in obese patients really is irisin, reflecting fat mass, or whether it is an artefact. The current review is focused on the potential participation of adipose tissue on circulating irisin levels, and its role in metabolic pathologies associated with obesity according to current 467

2 468 A. B. Crujeiras et al. published reports in an attempt to explain the controversy (Table 1). Conceptual aspects of FNDC5/irisin and the controversy surrounding its secretion and circulating levels It is noteworthy that since the discovery of FNDC5/irisin by Bostr om and colleagues in January 2012, the potential roles attributed to this myokine have still not been consolidated, despite the numerous reports that have been published. 38,39 The reason for this may lie in certain aspects of the irisin protein that have not been fully clarified, either in the original manuscript or since. 1,40,41 In the initial report, FNDC5 was considered to be synthesized as a Type I membrane protein that is cleaved proteolytically, releasing the 12 kda amino terminal portion into the extracellular space. However, to demonstrate secretion of this myokine, an Abcam antibody directed against the endogenous portion of the protein was used (Abcam Cterminal, Abcam, Cambridge, UK) (Fig. 1a). Using this antibody (which is no longer available from Abcam), substantial amounts of FNDC5 at a molecular weight of at least 32 kda were detected in the cell culture media of HEK 293 cells transfected with the C-terminal flag-tagged FNDC5. Utilizing the same antibody, however, a 22-kDa band was also detected in the plasma of mice that were forced to express FNDC5 in the liver through the adenoviral intravenous delivery of a full-length FNDC5 vector. Although the divergence between the theoretical and practical molecular weight could be explained by glycosylation, we must consider that full-length FNDC5/irisin may be secreted, as recently annotated in the Uniprot protein Knowledgebase (Uni- ProtKB Ref. Q8NAU1 Fibronectin Type III domain-containing Protein 5). Reinforcing this idea, and in accordance with Bostrom s results, we described a predominant band of approximately 25 kda in a rat model using the aforementioned Abcam antibody against the endogenous form of the protein (anti- FNDC5 Abcam C-terminal), and an additional antibody directed against the theoretically soluble secreted form of 12 kda (anti-irisin Phoenix Pharmaceuticals amino acids , Phoenix Pharmaceuticals, CA, USA) (Fig. 1c). At this point, there is no doubt regarding the need to determine which fraction of FNDC5, if any, is cleaved to produce the soluble portion of irisin and to discover its receptor. These data are crucial because all the published results regarding irisin, particularly regarding its circulating levels, have fallen under a cloud of suspicion. 41,42 The difficulty of identifying which part of FNDC5 is recognized by the commercially available detection kits, which is not indicated in the literature, only exacerbates this situation. Indeed, great uncertainty exists about the accuracy of commercial antibodies in detecting true irisin; huge inconsistencies in irisin levels are exposed in the bibliography. 41,42 In this regard, our experience has shown that despite the variations in detected irisin concentrations among different ELISA kit lots, the significant differences between the analysed groups of subjects are consistent across independent studies with different cohorts of patients. 8,11,13,14 On the other hand, studies that predict irisin secretion levels by analysing FNDC5 mrna expression should be interpreted very cautiously because expression does not always parallel secretion, particularly when it is regulated proteolytically. In this regard, it is still questionable whether a portion or the whole FNDC5 protein is secreted 9 (Fig. 1b), and whether the extracellular portion of irisin is secreted as a dimer 17 (Fig. 1). Finally, it has to be taken into account that despite the great homology among species, indicated by sequence analysis, a recent study has suggested that the human gene for FNDC5 carries a mutation in the start codon that results in lower translational efficiency and expression of smaller truncated forms 16 (Fig. 1c). Table 1. Concluding facts Fact Positive evidence Negative evidence Not defined FNDC5 is cleaved to liberate a smaller 12-kDA peptide named Irisin In mice 1,15 In humans 16 In rats 9 In vitro 17 FNDC5/Irisin is augmented with activity or exercise training Acute exercise: In mice 15,18 Long-term training: In mice 19 In rats 6,9 In humans 7,11,16,18,20 23 In vitro 24 In pigs 25 Irisin circulating levels are correlated with muscle mass In humans 6 In humans 22 FNDC5/Irisin is also an adipokine secreted by the adipose tissue In humans 8,11,12,29 In rats 9 Irisin has a positive effect on glucose homeostasis In humans 8,14,29 31 In humans 7,12,32 34 Irisin circulating levels are correlated with BMI In humans 6 8,11 In humans 35 Irisin circulating levels are elevated in obesity In humans 6,8 11 In humans 29 In rats 9,36 Irisin correlates with fat mass In humans 6,8,11,29 In rats 9 Irisin correlates with metabolic syndrome In humans 14,32 In humans 37 In humans 26 28

3 Irisin: Fat or artefact 469 (a) (b) Fig. 1 FNDC5 and irisin structure and secretion. Schematic representation of the FNDC5 protein showing the location of its amino acids, including those epitopes recognized by different commercial antibodies (a); schematic representation of the FNDC5 protein located in the cell membrane, encompassing the secretion of irisin portion and two of the truncated isoforms predicted to be expressed in humans by Raschke and collaborators due to a mutation on the start codon of the protein (b); representative immunodetection by Western blot of FNDC5/irisin in the muscle (soleus and gastrocnemius) and adipose tissue (subcutaneous and visceral) secretomes, using the same antibody used in the original paper by Bostrom and collaborators (Abcam) against the intracellular portion of the protein (upper panel) and another antibody against the extracellular portion (Phoenix pharmaceuticals) to recognize the soluble 12-kDa part of the protein (c). SP, signalling peptide; N, n-terminal end; C, c-terminal end; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; 1W/3W, one or 3 weeks of voluntary exercise. (c) FNDC5/irisin and metabolic pathology: an adaptive response mediated by adipose tissue? Notwithstanding the fact that the nature and concentration of circulating FNDC5 remains unclear, 40 irisin has attracted much attention since its discovery due to its proposed therapeutic potential for the treatment of obesity and diabetes; indeed, it has even been suggested to improve life expectancy. 3,43,44 However, contrary to expectations, obese individuals have been found to have higher circulating levels of irisin than normal weight or anorexic patients (Fig. 2a). 10,11 Significantly, higher irisin levels were also described in subjects with metabolic syndrome (MetS) compared to subjects without MetS. 32 Moreover, a positive correlation between FNDC5 muscle gene expression and BMI in humans was found, 7 and higher FNDC5 gene expression in the triceps muscles of obese/diabetic-prone rats vs their lean/healthy counterparts was observed, even though no statistically significant differences were found in the circulating levels between the groups. 36 Reinforcing this finding, weight loss, either by bariatric surgery or energy restriction, has been shown to decrease circulating levels of irisin. 6,8 Interestingly, this reduction of irisin after weight loss was reversed in those patients who regained their original weight (Fig. 3). 8 However, other studies reported no correlation or lower irisin concentrations in obese compared to lean individuals as well as a negative correlation with BMI. 29,35 Intriguingly, the levels of irisin in these studies diverged enormously (from 24 ng/l to 2 mg/l), creating inconsistency in the results. 42 This discrepancy could be based on any of the following reasons: methodological differences in the assay because, as mentioned above, there is no consensus concerning the identity of the soluble portion of the FNDC5 membrane protein; differing mechanisms of secretion; and the varying presence of target epitopes among manufacturers (Fig. 1a). In spite of the existing controversy, most of the published reports have indicated correlations between irisin and different markers of adiposity, suggesting that plasma irisin levels reflect net body adiposity. Because irisin was initially discovered to be secreted by muscle after aerobic exercise-training, 1 it could be possible that the increased processing and release of muscle irisin is induced by

4 470 A. B. Crujeiras et al. (a) (b) (c) Fig. 2 Circulating irisin levels in situations of altered BMI and its correlation with fat mass. The circulating levels of Irisin were assayed with Phoenix Pharmaceuticals ELISA-52 (old Aviscera) against aminoacids in different cohorts of patients at extreme BMI conditions 11 (a). Correlation of circulating irisin with fat mass (kg) and fat mass (%) in the same patients (b,c); figure modified from Pardo et al. 11 (***) denotes statistically significant differences versos normal weight and anorexia nervosa. Anorexia Nervosa (9), Normal weight (white dot), Obesity (black dot). signals originating in adipose tissue, contributing to the increased irisin levels in circulation observed in obesity. Moreover, the irisin plasma levels observed in obese patients could be explained by the high physical cost associated with elevated body weight. Still, varying data have suggested that despite the positive association of irisin and BMI, which is at least in part explained by muscle mass in humans, other tissues, including adipose tissue, may be involved in this matter. 6,29 Indeed, in a cohort ranging from anorexia to morbid obesity, fat mass was revealed to be the main factor explaining approximately 30% of the variability in plasma irisin levels, independent of age, fat-free mass, daily physical activity, resting energy expenditure (REE) and BMI (Fig. 2b,c). 11 Accordingly, irisin levels correlated with body weight and fat mass reduction after an intervention of 8 weeks on a hypocaloric diet in obese patients. 8 Later, during this study and after a follow-up period, circulating levels of irisin returned to baseline only in obese patients who regained the lost weight (Fig. 3b). Taking into account that no evident changes in fat-free mass were observed in these patients, fat mass can be considered the main factor explaining irisin s circulating levels. 8 Importantly, the contribution of adipose tissue to circulating levels of irisin was confirmed by a molecular animal-based study demonstrating that adipose tissue secretion could contribute to circulating FNDC5/irisin. 9 This work identifies for the first time the expression and secretion of FNDC5/irisin by the visceral adipose tissue and, to a greater extent, by the subcutaneous adipose tissue in ad libitum rats. Moreover, we investigated how shortterm endurance exercise increased FNDC5/irisin secretion by adipose tissue, even as food restriction decreased its secretion in animal models. Consequently, fat from activity-based anorectic animals showed sparse secretion of FNDC5/irisin although, surprisingly, adipose tissue from obese individuals over-secreted this protein. 9 Further, adipose tissue of human origin has been demonstrated to be able to express and secrete FNDC5/irisin. This initial report led us to hypothesize that adipose tissue may play a role in determining circulating FNDC5/irisin levels in co-operation with muscle and that this contribution may vary depending on the physiological or pathological situation. Hence, the participation of adipose tissue in determining irisin levels may be at its highest in situations of increased adiposity, such as obesity. In the light of these findings, it could be postulated that the muscle/adipose secretion ratio is affected by the pathophysiological situation. Therefore, along with muscle, irisin is also generated by adipose tissue, responding to direct alterations in adipose tissue mass. Under this scenario, whether the increased circulating levels of irisin observed in obesity represent an adaptive response to counteract the metabolic disturbances associated with obesity or play a role in promoting these metabolic disturbances remains an open question, because no functional data on irisin have yet been confirmed unequivocally. However, a direct association was observed between irisin and insulin, alongside an inverse correlation between irisin and ghrelin, a circulating orexigenic hormone with an opposite action to insulin or leptin. 8 Interestingly, the potential effect of irisin on glucose homoeostasis-related parameters was revealed in a study of obese subjects with features of the metabolic syndrome. 14 Thus, after treatment designed to lose weight, it was found that higher irisin concentrations at the beginning of the intervention were associated with greater reductions in glucose

5 Irisin: Fat or artefact 471 (a) (b) Fig. 3 Circulating irisin levels in obese individuals after energy restriction-induced weight loss and following weight regain. Body weight (a) and plasma irisin levels (b) during 8 weeks of nutritional intervention and after a 4-month follow-up period from Crujeiras et al. 8 Patients were classified as nonregainers if they maintained weight loss after the dietary intervention and as regainers if they recovered at least 10% of the lost weight. Statistically significant differences from week 0 to week 8., statistically significant changes over the duration of the nutritional program (week 0, week 8, and week 24). Statistical significance for the time-weight maintenance group (regainers, nonregainers) interaction. * and #, Statically significant differences between regainers and non-regainers (P < 0.05 and P < 0.100, respectively). and insulin concentrations and in the homoeostasis model assessment of insulin resistance (HOMA-IR) index, independent of body weight loss. 14 Moreover, the improvement in the metabolic parameters induced by dieting was concomitant with a decrease in circulating levels of irisin. 14 Additionally, irisin was identified as a potential factor associated with progression of insulin resistance in patients who regain their diet-induced weight loss. 12 Hence, when the association between insulin sensitivity and irisin, among other hormones related to body weight homoeostasis, was explored under weight-regaining conditions, the findings identified irisin and leptin as relevant factors contributing to the onset of insulin resistance. The risk of insulin resistance during the weight maintenance period after a dietary intervention was higher among patients with high levels of irisin and leptin. 12 Similar results were found in previous reports showing a positive association between circulating irisin levels or FNDC5 expression in human myotubes and fasting insulin concentrations and HOMA- IR. 7,33 Additionally, a significant positive correlation has been reported between baseline circulating irisin levels and insulin resistance, as assessed by HOMA-IR. 32 Lipid metabolic disturbances in an excess body weight condition also appear to be regulated by irisin, as suggested by its association with triglycerides (TC) and the atherogenic index TC/HDL-c or Apo B levels. 13 Irisin levels were reduced in parallel with these atherogenic factors in metabolic syndrome patients following an energy-restricted program. 13 Together, this evidence supports the hypothesis that increased circulating irisin may be an adaptive response to compensate for decreased insulin sensitivity and other metabolic disturbances associated with obesity. 3,6 We suggest that irisin provides physiological feedback that is increased in unfavourable metabolic situations, triggering a compensatory mechanism that may recede once the altered metabolic state is restored after weight loss. 14 In contrast, it was first shown that forced expression of FNDC5 by adenoviral vectors in the livers of mice caused an increase in circulating levels of irisin and activated the browning program in specific depots of WAT. As a result, greater energy expenditure, improved glucose tolerance and significant weight loss were reported. 1 In agreement with this first study, decreased levels of irisin were associated with the risk of several diseases, such as chronic kidney disease, 45 heart failure, 46,47 preeclampsia and ageing-related processes, 46,48 compared with healthy subjects. These findings suggest that irisin may contribute to successful ageing and lifestyle improvements; however, this tautological explanation is not supported by the facts. In fact, both these are clinical situations that generate catabolism and a reduction of fat mass. Therefore, fat mass is among the simplest explanations. Regarding metabolic diseases, different reports have indicated lower irisin levels in patients with type 2 diabetes (T2D) which has been suggested to play a role in so-called exercise resistance. 3 Surprisingly, the negative relationship between irisin and T2D is not found in obese individuals, and it has been shown that irisin correlates with most signs of insulin resistance in nondiabetic populations. 12,31,32 Therefore, the adaptive response to compensate for the disturbed metabolic functions in obesity, as potentially mediated by irisin, could be triggered primarily by adipose tissue. Muscle tissue may strongly participate in the circulating levels of irisin after exercise training, although adipose tissue would be responsible for the described high irisin levels in atypical metabolic situations, such as obesity. This observed pattern of irisin secretion appears to resemble that of leptin in obese subjects. Like leptin, irisin has attracted considerable attention due to its proposed therapeutic potential for obesity and diabetes, even for improving life expectancy. However, and again like leptin, the circulating concentrations 6,8,10 and expression levels of irisin in muscle and adipose tissues 9,36 are increased in obesity, suggesting potential irisin resistance. 49 Thus, as irisin is also secreted by adipose tissue, 9 reduced irisin levels indicate type 2 diabetes and may be explained by the decrease in body fat stores in cases of uncontrolled insulin-deficient diabetes, similar to leptin. 50 Alternatively, similar to leptin, 51 it could also be speculated that long-term exposure to elevated irisin levels in obesity leads to pathological conditions that promote insulin insensitivity and atherogenic processes. The potential resistance to irisin displayed in obesity could promote insulin secretion, resulting in hyperin-

6 472 A. B. Crujeiras et al. Fig. 4 Summary chart. Representation of the hypothesized functions of FNDC5/irisin in both healthy and obese subjects. FNDC5/irisin is expressed and secreted mostly by muscle tissues on stimulation with exercise through PGC1a. An increase in circulating FNDC5/irisin levels would induce the browning of certain adipose cells within white adipose tissue. Activation of the thermogenic genes in these cells would improve the metabolic profile of white adipose tissue (WAT), enhance whole-body energy expenditure, augment glucose tolerance and produce significant weight loss. On the other hand, we propose that, in situations of obesity, WAT is the major contributor to circulating FNDC5/irisin levels. Therefore, FNDC5/irisin levels are correlated with BMI and other adiposity parameters. In this pathological situation, weight loss and fat mass reduction would be responsible for the decreased FNDC5/irisin levels observed after this type of intervention. In this context, irisin could be part of an adaptive response to counteract the metabolic homoeostasis disturbances induced by obesity; resistance to irisin is thus suggested. sulinaemia and leading to insulin resistance and other obesityassociated disorders. This hypothesis could additionally be supported by the described association in human muscle 52 and liver 53 between increased irisin levels and oxidative stress and inflammation; processes that are involved in the onset of insulin resistance and cardiovascular disease 54 and represent a potential link between obesity and its comorbidities. 55,56 Concluding remarks The possible beneficial effect of irisin in the treatment of obesity and diabetes has been challenged by recent reports showing unexpectedly high levels of irisin in obese animals and humans. Although these contradictory findings could illustrate methodological features related to the undetermined nature and concentration of circulating FNDC5/irisin and to the difficulty of translating the effects observed in murine models to humans, several reports have demonstrated an association between irisin levels and adiposity markers. This suggests a potential contribution of adipose tissue to circulating levels of the protein (Fig. 4). In fact, after demonstrating that adipose tissue expresses and secretes irisin, the results of the ensuing literature, including several independent studies and our research, have led us to hypothesize that adipose tissue secretes irisin in obese subjects in an effort to compensate for the metabolic disturbances triggered by excess body weight (Fig. 4). Additionally, resistance to irisin action appears to occur in the same way as other known hormones, such as insulin and leptin (Fig. 4). Further mechanistic studies are needed to elucidate whether irisin is an adaptive response to counteract the disturbances in metabolic homoeostasis induced by obesity or whether irisin levels represent a promoter of such disturbances. In any case, the translational potential of recent reports is evident, and evaluation of circulating irisin values at baseline could predict future disturbances in metabolic homoeostasis among obese patients. In other words, because irisin appears to be increased in metabolically altered situations related to obesity, circulating levels of irisin could be used as a biomarker of poor metabolic function in obese patients. Acknowledgements The authors laboratory work is supported by CIBERobn (CB06/ 03), the INTRASALUD proyect (PI10/02464) and the PI13/01915, Instituto de Salud Carlos III (ISCIII) initiatives as well as the Xunta de Galicia and Fundacion Lilly. AB. Crujeiras. is funded by the ISCIII through a research contract Sara Borrell (C09/00365). M. Pardo is a Miguel Servet Felow (ISCIII/SERGAS). Conflict of interest The authors declare no conflict of interests.

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