Irisin in humans: recent advances and questions for future research
2013; Elsevier BV; Volume: 63; Issue: 2 Linguagem: Inglês
10.1016/j.metabol.2013.11.009
ISSN1532-8600
AutoresPontus Boström, José Manuel Fernández‐Real, Christos S. Mantzoros,
Tópico(s)Exercise and Physiological Responses
ResumoIrisin is a muscle-derived factor, secreted from muscle after shedding of the extracellular portion of the type I membrane protein Fndc5. After its release, Irisin signals to the adipose tissue for the promotion of brown-like adipocytes ("beige cells"). Irisin was characterized two years ago and since its discovery a large number of studies have reported on the biology of Irisin, with the majority being studies of plasma Irisin in human cohorts. Importantly, the key function of Irisin, beige cell formation, has been observed in several studies [1Wu J. Bostrom P. Sparks L.M. et al.Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.Cell. 2012; 150: 366-376Abstract Full Text Full Text PDF PubMed Scopus (2260) Google Scholar, 2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 3Shan T. Liang X. Bi P. et al.Myostatin knockout drives browning of white adipose tissue through activating the AMPK-PGC1alpha-Fndc5 pathway in muscle.FASEB J. 2013; 27: 1981-1989Crossref PubMed Scopus (229) Google Scholar, 4Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar]. Moreover, the release of Irisin from Fndc5 to the extracellular space has also been reproduced in a number of studies [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 5Wrann C.D. White J.P. Salogiannnis J. et al.Exercise induces hippocampal bdnf through a PGC-1alpha/Fndc5 pathway.Cell Metab. 2013; 18 (http://dx.doi.org/10.1016/j.cmet.2013.09.008. [Epub 2013 Oct 10].): 649-659Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 6Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar, 7Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar]. The regulation of Irisin by exercise, however, has been reproduced only in some cohorts [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 5Wrann C.D. White J.P. Salogiannnis J. et al.Exercise induces hippocampal bdnf through a PGC-1alpha/Fndc5 pathway.Cell Metab. 2013; 18 (http://dx.doi.org/10.1016/j.cmet.2013.09.008. [Epub 2013 Oct 10].): 649-659Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 8Aydin S. Aydin S. Kuloglu T. 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 (http://dx.doi.org/10.1016/j.peptides.2013.09.011. [Epub 2013 Oct 1].): 13-18Crossref PubMed Scopus (81) Google Scholar, 9Kraemer R.R. Shockett P. Webb N.D. et al.A transient elevated irisin blood concentration in response to prolonged, moderate aerobic exercise in young men and women.Horm Metab Res. 2013; ([Epub ahead of print])PubMed Google Scholar, 10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 11Lecker S.H. Zavin A. Cao P. et al.Expression of the irisin precursor Fndc5 in skeletal muscle correlates with aerobic exercise performance in patients with heart failure.Circ Heart Fail. 2012; 5: 812-818Crossref PubMed Scopus (160) Google Scholar], whereas a lack of regulation was seen in others [12Pekkala S. Wiklund P. Hulmi J.J. et al.Are skeletal muscle Fndc5 gene expression and irisin release regulated by exercise and related to health?.J Physiol. 2013; 591 (http://dx.doi.org/10.1113/jphysiol.2013.263707. [Epub 2013 Sep 2].): 5393-5400Crossref PubMed Scopus (207) Google Scholar, 13Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar, 14Moraes C. Leal V.O. Marinho S.M. et al.Resistance exercise training does not affect plasma irisin levels of hemodialysis patients.Horm Metab Res. 2013; 45 (http://dx.doi.org/10.1055/s-0033-1354402. [Epub 2013 Sep 6].): 900-904Crossref PubMed Scopus (55) Google Scholar] done under different physiological and/or experimental conditions. Another aspect that remains to be clarified is the timing of Irisin increase after exercise; since the above studies have tested Irisin in different time points before and after exercise the possibility exists that Irisin increases for a finite period of time after exercise but its levels do not stay elevated for a prolonged period of time. An important advance in the understanding of Irisin biology was recently made when Schumacher et al. recently published the structure of Irisin demonstrating dimerization and the authors suggested a model for receptor activation [[15]Schumacher M.A. Chinnam N. Ohashi T. et al.Structure of irisin reveals a novel intersubunit beta-sheet fibronectin (FNIII) dimer; implications for receptor activation.J Biol Chem. 2013; 288 (http://dx.doi.org/10.1074/jbc.M113.516641. [Epub 2013 Oct 10].): 33738-33744Crossref PubMed Scopus (144) Google Scholar].Two recent studies, Polyzos et al. and Park et al., published in this issue of Metabolism, have addressed the relation between Irisin and two different aspects in the realm of metabolic dysfunction in humans [[16]Polyzos S.A. Kountouras J. Anastasilakis A.D. et al.Irisin in patients with nonalcoholic fatty liver disease.Metabolism. 2013; 63: 178-180Google Scholar] and Park et al. analyzed Irisin levels in subjects with different dietary preferences. For this purpose, the Alternate Healthy Eating Index (AHEI) and Alternate Mediterranean Diet Score (aMED) were used. In a total of 151 subjects, Irisin did not demonstrate any relation to either AHEI or aMED scores suggesting that healthier dietary patterns do not influence circulating Irisin levels. Similarly to previous studies [10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 17Park K.H. Zaichenko L. Brinkoetter M. et al.Circulating irisin in relation to insulin resistance and the metabolic syndrome.J Clin Endocrinol Metab. 2013; ([Epub ahead of print])Crossref Scopus (355) Google Scholar], however, Irisin displayed a positive relation to BMI and fat mass. These results thus suggest that dietary factors do not impact regulation of Irisin or vice versa. Interestingly though, the authors found that plasma CRP levels negatively correlated with AHEI and aMED.Polyzos and colleagues focused instead on Irisin levels in subjects with fatty liver disease. 16 subjects with nonalcoholic simple steatosis (NAFLD) and steatohepatitis (NASH) were compared to 24 lean and 28 obese controls [[16]Polyzos S.A. Kountouras J. Anastasilakis A.D. et al.Irisin in patients with nonalcoholic fatty liver disease.Metabolism. 2013; 63: 178-180Google Scholar]. Irisin levels were significantly lower in subjects with NAFL and NASH compared to both the lean controls, but obese controls displayed similar Irisin levels as the diseased cohort. Also, Irisin levels were higher in subjects with portal inflammation. This study partially confirms the previous findings from Zhang et al. [[18]Zhang H.J. Zhang X.F. Ma Z.M. et al.Irisin is inversely associated with intrahepatic triglyceride contents in obese adults.J Hepatol. 2013; 59 (http://dx.doi.org/10.1016/j.jhep.2013.04.030. [Epub 2013 May 9].): 557-562Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar] where an inverse relation was observed between Irisin and hepatic triglyceride levels. The relation between Irisin and serum AST/ALT levels, however, were different between the two studies. In the discussion of the present paper, Polysos et al. address the fact that Irisin has been negatively associated with BMI in some studies, and positively in others. The authors conclude that consistently Irisin was positively correlated with BMI in healthy subjects [10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 19Stengel A. Hofmann T. Goebel-Stengel M. et al.Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity—correlation with body mass index.Peptides. 2013; 39: 125-130Crossref PubMed Scopus (325) Google Scholar, 20Liu J.J. Wong M.D. Toy W.C. et al.Lower circulating irisin is associated with type 2 diabetes mellitus.J Diabetes Complications. 2013; 27: 365-369Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar] and negatively in subjects with metabolic disease [7Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar, 21Choi Y.K. Kim M.K. Bae K.H. et al.Serum irisin levels in new-onset type 2 diabetes.Diabetes Res Clin Pract. 2013; 100: 96-101Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar]. Thus, they propose that differences between the healthy and diseased state might thus explain these previous discrepancies, an important notion that needs to be confirmed or refuted by further studies.Park et al. and Polyzos et al. both analyzed human Irisin levels using a validated ELISA. The measurements of Irisin in humans were recently challenged however [13Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar, 22Erickson H.P. Irisin and Fndc5 in retrospect: an exercise hormone or a transmembrane receptor?.Adipocyte. 2013; 2: 289-293Crossref PubMed Google Scholar]. Moreover, the expression of Irisin in humans was questioned by Raschke et al. [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar] as they highlighted that Fndc5 in humans is utilizing an alternative start codon. Several lines of evidence, however, strongly support the notion that Irisin is expressed, secreted and metabolically regulated in humans: I. Although not mentioned clearly by Raschke et al., the alternative start codon for Fndc5 was already described previously, where an unbiased search found Fndc5 amongst many other expressed non-pseudogenes [[23]Ivanov I.P. Firth A.E. Michel A.M. et al.Identification of evolutionarily conserved non-aug-initiated n-terminal extensions in human coding sequences.Nucleic Acids Res. 2011; 39: 4220-4234Crossref PubMed Scopus (147) Google Scholar]. II. Studies have compared Fndc5 mRNA levels and plasma Irisin levels and found clear covariation [[7]Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar] or concordant regulation after intervention [[10]Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar]. Such co-variation would not be expected for a pseudogene, and/or if the ELISA was unspecific. III. We and others have clearly demonstrated the validity of the Aviscera Irisin ELISA kit (now sold by other vendors), which correctly detects spiked Irisin in physiological concentrations [[10]Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar]. Western blot analysis using antibody from this ELISA, also detects a 25 kDa band in plasma, which is the same size as where fully glycosylated recombinant Irisin migrates [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar]. Importantly, this antibody detects native, glycosylated Irisin when spiked to human plasma (PB, data not shown) at physiological concentrations. Thus, this antibody (which is the most predominantly used in the field currently) detects endogenous and exogenous Irisin in human plasma at physiological concentrations.Erickson and Raschke point out that the early studies of Irisin were performed using an older antibody sold by Abcam [[2]Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar]. The annotation of this antibody suggested that it would be directed primarily against the c-terminus of Fndc5. We and others, however, have observed immunoreactivity using this antibody against recombinant N-terminal Fndc5 (Irisin) and questioned the original annotation. The antibody is now discontinued (possibly for this reason) making it impossible to pinpoint the epitope. Zhang et al. have recently [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar] utilized a different irisin antibody provided by Sigma-Aldrich that identified recombinant irisin separated by SDS-PAGE at 25, 22, and 15 kDa. They further confirmed that all bands represent Irisin with different glycosylation. Thus, it is clear that Irisin is present in human plasma and that its concentrations are altered in the metabolically diseased state [6Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar, 17Park K.H. Zaichenko L. Brinkoetter M. et al.Circulating irisin in relation to insulin resistance and the metabolic syndrome.J Clin Endocrinol Metab. 2013; ([Epub ahead of print])Crossref Scopus (355) Google Scholar, 19Stengel A. Hofmann T. Goebel-Stengel M. et al.Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity—correlation with body mass index.Peptides. 2013; 39: 125-130Crossref PubMed Scopus (325) Google Scholar, 21Choi Y.K. Kim M.K. Bae K.H. et al.Serum irisin levels in new-onset type 2 diabetes.Diabetes Res Clin Pract. 2013; 100: 96-101Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 24Zhang H.J. Zhang X.F. Ma Z.M. et al.Irisin is inversely associated with intrahepatic triglyceride contents in obese adults.J Hepatol. 2013; 59: 557-562Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 25Wen M.S. Wang C.Y. Lin S.L. et al.Decrease in irisin in patients with chronic kidney disease.PLoS One. 2013; 8 (http://dx.doi.org/10.1371/journal.pone.0064025. [Print 2013].): e64025Crossref PubMed Scopus (136) Google Scholar, 26Vamvini M.T. Aronis K.N. Panagiotou G. et al.Irisin mrna and circulating levels in relation to other myokines in healthy and morbidly obese humans.Eur J Endocrinol. 2013; 169: 829-834Crossref PubMed Scopus (54) Google Scholar]. Not all ELISAs and antibodies, however, have been validated. Studies of different commercially available assays give very different baseline values [[12]Pekkala S. Wiklund P. Hulmi J.J. et al.Are skeletal muscle Fndc5 gene expression and irisin release regulated by exercise and related to health?.J Physiol. 2013; 591 (http://dx.doi.org/10.1113/jphysiol.2013.263707. [Epub 2013 Sep 2].): 5393-5400Crossref PubMed Scopus (207) Google Scholar]. IV. Lastly, direct measures of Irisin levels in cell culture media from human cells also have resulted in clear signal [[6]Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar]. In fact, even Raschke et al. found detectable Irisin in the media of human primary myocytes [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar]. The comparison to a murine cell line kept under completely other conditions, however, did not allow for any conclusions as to differences between the species [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar]. In summary, these converging pieces of evidence, when put together, strongly suggest that Irisin is synthesized from Fndc5 in humans and released to plasma where they are measurable by western or appropriate ELISA methods.In this context, the two clinical papers published in this issue of Metabolism advance significantly our clinical understanding of Irisin in humans. One major challenge for future advances in the understanding of Irisin biology is identification of the cellular receptor. The existence of a specific, high-affinity receptor is supported by the low-nanomolar activity of the protein [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 4Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar], and knowledge of its identity would likely unravel new possible target cells/tissues for Irisin actions. Moreover, the notion of a potential "irisin-resistance" in various disease states, as suggested by various authors, could be directly tested if the receptor system is known. Loss-of-function models will also be important to understand Irisin biology. Genetic mouse models with conditional ablation of the Fndc5 could unravel new, key biological functions for Irisin. Such mice will also reveal potential redundancies in Irisin signaling possibly mediated by the paralog Fndc4. Zhang et al. recently showed that recombinant yeast-produced Irisin could induce UCP-1 25-fold when injected to mice [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar], which led to reduced body weight and improved glucose control. These findings are exciting as they suggest that Irisin could be tested therapeutically and raise the hope that Irisin might come of use in obese and diabetic patients in the future.Conflict of interestThe authors do not have any conflict of interest related to this manuscript. Irisin is a muscle-derived factor, secreted from muscle after shedding of the extracellular portion of the type I membrane protein Fndc5. After its release, Irisin signals to the adipose tissue for the promotion of brown-like adipocytes ("beige cells"). Irisin was characterized two years ago and since its discovery a large number of studies have reported on the biology of Irisin, with the majority being studies of plasma Irisin in human cohorts. Importantly, the key function of Irisin, beige cell formation, has been observed in several studies [1Wu J. Bostrom P. Sparks L.M. et al.Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.Cell. 2012; 150: 366-376Abstract Full Text Full Text PDF PubMed Scopus (2260) Google Scholar, 2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 3Shan T. Liang X. Bi P. et al.Myostatin knockout drives browning of white adipose tissue through activating the AMPK-PGC1alpha-Fndc5 pathway in muscle.FASEB J. 2013; 27: 1981-1989Crossref PubMed Scopus (229) Google Scholar, 4Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar]. Moreover, the release of Irisin from Fndc5 to the extracellular space has also been reproduced in a number of studies [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 5Wrann C.D. White J.P. Salogiannnis J. et al.Exercise induces hippocampal bdnf through a PGC-1alpha/Fndc5 pathway.Cell Metab. 2013; 18 (http://dx.doi.org/10.1016/j.cmet.2013.09.008. [Epub 2013 Oct 10].): 649-659Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 6Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar, 7Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar]. The regulation of Irisin by exercise, however, has been reproduced only in some cohorts [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 5Wrann C.D. White J.P. Salogiannnis J. et al.Exercise induces hippocampal bdnf through a PGC-1alpha/Fndc5 pathway.Cell Metab. 2013; 18 (http://dx.doi.org/10.1016/j.cmet.2013.09.008. [Epub 2013 Oct 10].): 649-659Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 8Aydin S. Aydin S. Kuloglu T. 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 (http://dx.doi.org/10.1016/j.peptides.2013.09.011. [Epub 2013 Oct 1].): 13-18Crossref PubMed Scopus (81) Google Scholar, 9Kraemer R.R. Shockett P. Webb N.D. et al.A transient elevated irisin blood concentration in response to prolonged, moderate aerobic exercise in young men and women.Horm Metab Res. 2013; ([Epub ahead of print])PubMed Google Scholar, 10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 11Lecker S.H. Zavin A. Cao P. et al.Expression of the irisin precursor Fndc5 in skeletal muscle correlates with aerobic exercise performance in patients with heart failure.Circ Heart Fail. 2012; 5: 812-818Crossref PubMed Scopus (160) Google Scholar], whereas a lack of regulation was seen in others [12Pekkala S. Wiklund P. Hulmi J.J. et al.Are skeletal muscle Fndc5 gene expression and irisin release regulated by exercise and related to health?.J Physiol. 2013; 591 (http://dx.doi.org/10.1113/jphysiol.2013.263707. [Epub 2013 Sep 2].): 5393-5400Crossref PubMed Scopus (207) Google Scholar, 13Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar, 14Moraes C. Leal V.O. Marinho S.M. et al.Resistance exercise training does not affect plasma irisin levels of hemodialysis patients.Horm Metab Res. 2013; 45 (http://dx.doi.org/10.1055/s-0033-1354402. [Epub 2013 Sep 6].): 900-904Crossref PubMed Scopus (55) Google Scholar] done under different physiological and/or experimental conditions. Another aspect that remains to be clarified is the timing of Irisin increase after exercise; since the above studies have tested Irisin in different time points before and after exercise the possibility exists that Irisin increases for a finite period of time after exercise but its levels do not stay elevated for a prolonged period of time. An important advance in the understanding of Irisin biology was recently made when Schumacher et al. recently published the structure of Irisin demonstrating dimerization and the authors suggested a model for receptor activation [[15]Schumacher M.A. Chinnam N. Ohashi T. et al.Structure of irisin reveals a novel intersubunit beta-sheet fibronectin (FNIII) dimer; implications for receptor activation.J Biol Chem. 2013; 288 (http://dx.doi.org/10.1074/jbc.M113.516641. [Epub 2013 Oct 10].): 33738-33744Crossref PubMed Scopus (144) Google Scholar]. Two recent studies, Polyzos et al. and Park et al., published in this issue of Metabolism, have addressed the relation between Irisin and two different aspects in the realm of metabolic dysfunction in humans [[16]Polyzos S.A. Kountouras J. Anastasilakis A.D. et al.Irisin in patients with nonalcoholic fatty liver disease.Metabolism. 2013; 63: 178-180Google Scholar] and Park et al. analyzed Irisin levels in subjects with different dietary preferences. For this purpose, the Alternate Healthy Eating Index (AHEI) and Alternate Mediterranean Diet Score (aMED) were used. In a total of 151 subjects, Irisin did not demonstrate any relation to either AHEI or aMED scores suggesting that healthier dietary patterns do not influence circulating Irisin levels. Similarly to previous studies [10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 17Park K.H. Zaichenko L. Brinkoetter M. et al.Circulating irisin in relation to insulin resistance and the metabolic syndrome.J Clin Endocrinol Metab. 2013; ([Epub ahead of print])Crossref Scopus (355) Google Scholar], however, Irisin displayed a positive relation to BMI and fat mass. These results thus suggest that dietary factors do not impact regulation of Irisin or vice versa. Interestingly though, the authors found that plasma CRP levels negatively correlated with AHEI and aMED. Polyzos and colleagues focused instead on Irisin levels in subjects with fatty liver disease. 16 subjects with nonalcoholic simple steatosis (NAFLD) and steatohepatitis (NASH) were compared to 24 lean and 28 obese controls [[16]Polyzos S.A. Kountouras J. Anastasilakis A.D. et al.Irisin in patients with nonalcoholic fatty liver disease.Metabolism. 2013; 63: 178-180Google Scholar]. Irisin levels were significantly lower in subjects with NAFL and NASH compared to both the lean controls, but obese controls displayed similar Irisin levels as the diseased cohort. Also, Irisin levels were higher in subjects with portal inflammation. This study partially confirms the previous findings from Zhang et al. [[18]Zhang H.J. Zhang X.F. Ma Z.M. et al.Irisin is inversely associated with intrahepatic triglyceride contents in obese adults.J Hepatol. 2013; 59 (http://dx.doi.org/10.1016/j.jhep.2013.04.030. [Epub 2013 May 9].): 557-562Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar] where an inverse relation was observed between Irisin and hepatic triglyceride levels. The relation between Irisin and serum AST/ALT levels, however, were different between the two studies. In the discussion of the present paper, Polysos et al. address the fact that Irisin has been negatively associated with BMI in some studies, and positively in others. The authors conclude that consistently Irisin was positively correlated with BMI in healthy subjects [10Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 19Stengel A. Hofmann T. Goebel-Stengel M. et al.Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity—correlation with body mass index.Peptides. 2013; 39: 125-130Crossref PubMed Scopus (325) Google Scholar, 20Liu J.J. Wong M.D. Toy W.C. et al.Lower circulating irisin is associated with type 2 diabetes mellitus.J Diabetes Complications. 2013; 27: 365-369Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar] and negatively in subjects with metabolic disease [7Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar, 21Choi Y.K. Kim M.K. Bae K.H. et al.Serum irisin levels in new-onset type 2 diabetes.Diabetes Res Clin Pract. 2013; 100: 96-101Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar]. Thus, they propose that differences between the healthy and diseased state might thus explain these previous discrepancies, an important notion that needs to be confirmed or refuted by further studies. Park et al. and Polyzos et al. both analyzed human Irisin levels using a validated ELISA. The measurements of Irisin in humans were recently challenged however [13Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar, 22Erickson H.P. Irisin and Fndc5 in retrospect: an exercise hormone or a transmembrane receptor?.Adipocyte. 2013; 2: 289-293Crossref PubMed Google Scholar]. Moreover, the expression of Irisin in humans was questioned by Raschke et al. [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar] as they highlighted that Fndc5 in humans is utilizing an alternative start codon. Several lines of evidence, however, strongly support the notion that Irisin is expressed, secreted and metabolically regulated in humans: I. Although not mentioned clearly by Raschke et al., the alternative start codon for Fndc5 was already described previously, where an unbiased search found Fndc5 amongst many other expressed non-pseudogenes [[23]Ivanov I.P. Firth A.E. Michel A.M. et al.Identification of evolutionarily conserved non-aug-initiated n-terminal extensions in human coding sequences.Nucleic Acids Res. 2011; 39: 4220-4234Crossref PubMed Scopus (147) Google Scholar]. II. Studies have compared Fndc5 mRNA levels and plasma Irisin levels and found clear covariation [[7]Moreno-Navarrete J.M. Ortega F. Serrano M. et al.Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.J Clin Endocrinol Metab. 2013; 98: E769-E778Crossref PubMed Scopus (567) Google Scholar] or concordant regulation after intervention [[10]Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar]. Such co-variation would not be expected for a pseudogene, and/or if the ELISA was unspecific. III. We and others have clearly demonstrated the validity of the Aviscera Irisin ELISA kit (now sold by other vendors), which correctly detects spiked Irisin in physiological concentrations [[10]Huh J.Y. Panagiotou G. Mougios V. et al.Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and II. mRNA expression and circulating concentrations in response to weight loss and exercise.Metabolism. 2012; 61: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar]. Western blot analysis using antibody from this ELISA, also detects a 25 kDa band in plasma, which is the same size as where fully glycosylated recombinant Irisin migrates [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar]. Importantly, this antibody detects native, glycosylated Irisin when spiked to human plasma (PB, data not shown) at physiological concentrations. Thus, this antibody (which is the most predominantly used in the field currently) detects endogenous and exogenous Irisin in human plasma at physiological concentrations. Erickson and Raschke point out that the early studies of Irisin were performed using an older antibody sold by Abcam [[2]Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar]. The annotation of this antibody suggested that it would be directed primarily against the c-terminus of Fndc5. We and others, however, have observed immunoreactivity using this antibody against recombinant N-terminal Fndc5 (Irisin) and questioned the original annotation. The antibody is now discontinued (possibly for this reason) making it impossible to pinpoint the epitope. Zhang et al. have recently [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar] utilized a different irisin antibody provided by Sigma-Aldrich that identified recombinant irisin separated by SDS-PAGE at 25, 22, and 15 kDa. They further confirmed that all bands represent Irisin with different glycosylation. Thus, it is clear that Irisin is present in human plasma and that its concentrations are altered in the metabolically diseased state [6Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar, 17Park K.H. Zaichenko L. Brinkoetter M. et al.Circulating irisin in relation to insulin resistance and the metabolic syndrome.J Clin Endocrinol Metab. 2013; ([Epub ahead of print])Crossref Scopus (355) Google Scholar, 19Stengel A. Hofmann T. Goebel-Stengel M. et al.Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity—correlation with body mass index.Peptides. 2013; 39: 125-130Crossref PubMed Scopus (325) Google Scholar, 21Choi Y.K. Kim M.K. Bae K.H. et al.Serum irisin levels in new-onset type 2 diabetes.Diabetes Res Clin Pract. 2013; 100: 96-101Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 24Zhang H.J. Zhang X.F. Ma Z.M. et al.Irisin is inversely associated with intrahepatic triglyceride contents in obese adults.J Hepatol. 2013; 59: 557-562Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 25Wen M.S. Wang C.Y. Lin S.L. et al.Decrease in irisin in patients with chronic kidney disease.PLoS One. 2013; 8 (http://dx.doi.org/10.1371/journal.pone.0064025. [Print 2013].): e64025Crossref PubMed Scopus (136) Google Scholar, 26Vamvini M.T. Aronis K.N. Panagiotou G. et al.Irisin mrna and circulating levels in relation to other myokines in healthy and morbidly obese humans.Eur J Endocrinol. 2013; 169: 829-834Crossref PubMed Scopus (54) Google Scholar]. Not all ELISAs and antibodies, however, have been validated. Studies of different commercially available assays give very different baseline values [[12]Pekkala S. Wiklund P. Hulmi J.J. et al.Are skeletal muscle Fndc5 gene expression and irisin release regulated by exercise and related to health?.J Physiol. 2013; 591 (http://dx.doi.org/10.1113/jphysiol.2013.263707. [Epub 2013 Sep 2].): 5393-5400Crossref PubMed Scopus (207) Google Scholar]. IV. Lastly, direct measures of Irisin levels in cell culture media from human cells also have resulted in clear signal [[6]Gouni-Berthold I. Berthold H.K. Huh J.Y. et al.Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.PLoS One. 2013; 8: e72858https://doi.org/10.1371/journal.pone.0072858Crossref PubMed Scopus (92) Google Scholar]. In fact, even Raschke et al. found detectable Irisin in the media of human primary myocytes [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar]. The comparison to a murine cell line kept under completely other conditions, however, did not allow for any conclusions as to differences between the species [[13]Raschke S. Elsen M. Gassenhuber H. et al.Evidence against a beneficial effect of irisin in humans.PLoS One. 2013; 8: e73680https://doi.org/10.1371/journal.pone.0073680Crossref PubMed Scopus (249) Google Scholar]. In summary, these converging pieces of evidence, when put together, strongly suggest that Irisin is synthesized from Fndc5 in humans and released to plasma where they are measurable by western or appropriate ELISA methods. In this context, the two clinical papers published in this issue of Metabolism advance significantly our clinical understanding of Irisin in humans. One major challenge for future advances in the understanding of Irisin biology is identification of the cellular receptor. The existence of a specific, high-affinity receptor is supported by the low-nanomolar activity of the protein [2Bostrom P. Wu J. Jedrychowski M.P. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (2956) Google Scholar, 4Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar], and knowledge of its identity would likely unravel new possible target cells/tissues for Irisin actions. Moreover, the notion of a potential "irisin-resistance" in various disease states, as suggested by various authors, could be directly tested if the receptor system is known. Loss-of-function models will also be important to understand Irisin biology. Genetic mouse models with conditional ablation of the Fndc5 could unravel new, key biological functions for Irisin. Such mice will also reveal potential redundancies in Irisin signaling possibly mediated by the paralog Fndc4. Zhang et al. recently showed that recombinant yeast-produced Irisin could induce UCP-1 25-fold when injected to mice [[4]Zhang Y. Li R. Meng Y. et al.Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.Diabetes. 2013; ([Epub ahead of print])Google Scholar], which led to reduced body weight and improved glucose control. These findings are exciting as they suggest that Irisin could be tested therapeutically and raise the hope that Irisin might come of use in obese and diabetic patients in the future. Conflict of interestThe authors do not have any conflict of interest related to this manuscript. The authors do not have any conflict of interest related to this manuscript.
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