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Forum
Circulating irisin detection: Does it really work? Fabian Sanchis-Gomar1, Rafael Alis2, and Giuseppe Lippi3 1
Research Institute of Hospital 12 de Octubre (‘i+12’), Avda. de Co´rdoba s/n, 28041 Madrid, Spain Research Institute ‘‘Dr. Vin˜a Giner’’, Molecular and Mitochondrial Medicine, School of Medicine, Catholic University of Valencia San Vicente Ma´rtir, Valencia, Spain 3 Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy 2
The recent discovery of irisin has generated considerable interest in the scientific community. However, many studies on the biochemistry and biology of this intriguing hormone yielded controversial results in humans, which were mostly attributable to a number of drawbacks in the methods used for its detection and measurement.
A myokine named irisin Innovative biomarkers with clinically meaningful applicability generate a rush to develop new methods for their measurement. Nevertheless, these first assays are often unreliable and not ready for prime time [1]. In 2012, Bostro¨m and collaborators discovered a new myokine they called irisin [2], with putative efficacy for the treatment of obesity or type 2 diabetes [3,4]. Soon after, inconsistencies between results in circulating irisin values were obtained in several follow-up studies [5]. Moreover, Eckel and colleagues also suggested that the positive effects of irisin on glucose homeostasis and obesity that were observed in mice, were less predictable or even unlikely in humans [6]. Some concerns thus emerged as to whether: (i) the antibodies used in the enzyme-linked immunosorbent assays (ELISAs) for measuring circulating irisin levels exhibited acceptable analytical performance [6]; (ii) irisin was present or not in humans [7]; (iii) irisin plays any significant physiological role in humans [8]. Initially, Bo¨strom et al. described that FNDC5 (fibronectin domain-containing protein 5), a member of fibronectin type III repeat containing gene family that is prevalently synthesized in muscles after exercise, apparently promotes healthy metabolism in mice [2]. They also showed that irisin, a specific FNDC5 fragment generated by proteolytic processing, was secreted into the bloodstream and was effective to prime energy-storing white fat cells to act as energy-burning brown fat cells [2]. However, both biochemistry and biology of irisin remain largely debated. According to the study performed by Lee et al. [9], the protein could be detected in human serum, but cleavage site(s) and exact size both remain a matter of debate in the scientific community.
Corresponding author: Sanchis-Gomar, F. ([email protected]). Keywords: irisin; FNDC5; exercise; ELISA kit; antibodies; mass spectrometry. 1043-2760/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2015.05.004
Technical drawbacks in irisin detection methods To clarify these issues, Albrecht and co-workers analyzed the specificity and sensitivity of currently available antibodies for detecting irisin [10]. They reported a significant cross-reactivity with other proteins in both serum and plasma of different species, and ultimately failed to identify a protein of the same size as irisin (i.e., from 13 to 32 kDa, depending on glycosylation and means of detection) in blood samples [10]. Even the antibody that was originally used for the identification of irisin by Bostro¨m and collaborators was found to recognize the C-terminal sequence of the FNDC5 protein, which is not part of the irisin cleaved peptide [10]. Furthermore, Albrecht et al. immunoprecipitated the samples with the Adipogen polyclonal antibody raised against recombinant irisin, and the bands detected at 25 and 16 kDa could not be identified as irisin by mass spectrometry (MS) analysis, and they were predominantly represented by Apoliprotein A1 (ApoA1) [10]. It is noteworthy that the concentration of ApoA1 in serum is several orders of magnitude higher than the measurable concentration of irisin with ELISA (i.e., mg/ ml versus mg/ml). It is hence predictable that irisin ELISA kits may be saturated or exhibit a substantial deviation from linearity if ApoA1 or other abundant proteins are nonspecifically recognized by the antibodies. Therefore, although it might be concluded that the study by Albrecth et al. highlighted significant drawbacks in detection of irisin by the antibodies included in available the commercial ELISA kits, some technical issues can be highlighted in this investigation: (i) An epitope of a protein can be masked in serum or plasma if present in complex with other proteins (prostate specific antigen is a paradigmatic example) [11]. The ELISA kits produced by different manufacturers usually contain different antibodies, so that the relative detection of the free and complexed forms may vary widely among the different assays, and this may impact the final amount of the protein being measured. The use of different calibrators (i.e., recombinant or purified protein, free or complexed form, degree of glycosylation) may also profoundly impair detectability. (ii) Albrecht et al. used Western Blot (WB) in their analysis, which entails protein denaturation, and this may lead to detection of a discrete number of nonspecific bands attributable to abundant denatured peptides. Additionally, whether FNDC5/Irisin is cleaved at different sequences and migrates at 13, 20, 25, and/or 32 kDa is still unclear, and this adds uncertainty to irisin detection by WB. Trends in Endocrinology and Metabolism xx (2015) 1–2
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TEM-1036; No. of Pages 2
Forum (iii) Due to the complex biochemical structure of irisin, the comparison of data obtained by immunoprecipitation (IP) with those generated using irisin ELISA kits requires similar sample treatment to limit the likelihood of protein modification. Indeed, the IP performed by Albrecht et al. entailed serum denaturation at 958C, whereas this step is unnecessary with conventional ELISAs [10]. In addition, anti-irisin antibodies are chemically cross-linked to magnetic beads, and this binding might affect the avidity of the antibodies. Ideally, an unrelated antibody should also be used as a negative control in IP studies, but this step has been overlooked [10].
Considerations moving forward Overall, the biochemical and biological properties of irisin remain largely debated, and additional studies would be needed to clarify the nature, metabolism and biological activity of this intriguing hormone. Indeed, development and characterization of highly specific (monoclonal) antibodies against recombinant (both glycosylated and nonglycosylated) human irisin is a mainstay. Identification of a candidate reference material and a reference method is also necessary for the development of commercial immunoassays, as well as for improving consistency in findings across different methods and enabling comparisons among different experimental studies. Finally, defining and standardizing preanalytical requirements (i.e., sample matrix, sample stability, optimal storage conditions) will be crucial for preventing protein degradation or modification, which may ultimately impair irisin detection. Specific and
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Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
reliable assays are needed to validate the recent evidence in mice suggesting that irisin is secreted by the muscle and acts on fatty tissue, thus exerting autocrine, paracrine, and endocrine activity [2,12]. Until then, the biology and function of irisin in human physiology will remain unknown. Disclaimer statement The authors declare no competing interests.
References 1 Servick, K. (2015) Biomedicine. Woes for ‘exercise hormone’. Science (New York, N.Y.) 347, 1299 2 Bostrom, P. et al. (2012) A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463–468 3 Sanchis-Gomar, F. et al. (2012) Irisin: a new potential hormonal target for the treatment of obesity and type 2 diabetes. J. Diabetes 4, 196 4 Sanchis-Gomar, F. and Perez-Quilis, C. (2013) Irisinemia: A novel concept to coin in clinical medicine? Ann. Nutr. Metab. 63, 60–61 5 Sanchis-Gomar, F. et al. (2014) Inconsistency in circulating irisin levels: what is really happening? Horm. Metab. Res. 46, 591–596 6 Elsen, M. et al. (2014) Browning of white fat: does irisin play a role in humans? J. Endocrinol. 222, R25–R38 7 Erickson, H.P. (2013) Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor? Adipocyte 2, 289–293 8 Timmons, J.A. et al. (2012) Is irisin a human exercise gene? Nature 488, E9–10 9 Lee, P. et al. (2014) Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Cell Metab. 19, 302–309 10 Albrecht, E. et al. (2015) Irisin - a myth rather than an exerciseinducible myokine. Sci. Rep. 5, 8889 11 Lippi, G. et al. (2009) Prostate-specific antigen-based screening for prostate cancer in the third millennium: useful or hype? Ann. Med. 41, 480–489 12 Irving, B.A. et al. (2014) Does IRISIN Have a BRITE future as a therapeutic agent in humans? Curr. Obes. Rep. 3, 235–241
Forum
Circulating irisin detection: Does it really work? Fabian Sanchis-Gomar1, Rafael Alis2, and Giuseppe Lippi3 1
Research Institute of Hospital 12 de Octubre (‘i+12’), Avda. de Co´rdoba s/n, 28041 Madrid, Spain Research Institute ‘‘Dr. Vin˜a Giner’’, Molecular and Mitochondrial Medicine, School of Medicine, Catholic University of Valencia San Vicente Ma´rtir, Valencia, Spain 3 Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy 2
The recent discovery of irisin has generated considerable interest in the scientific community. However, many studies on the biochemistry and biology of this intriguing hormone yielded controversial results in humans, which were mostly attributable to a number of drawbacks in the methods used for its detection and measurement.
A myokine named irisin Innovative biomarkers with clinically meaningful applicability generate a rush to develop new methods for their measurement. Nevertheless, these first assays are often unreliable and not ready for prime time [1]. In 2012, Bostro¨m and collaborators discovered a new myokine they called irisin [2], with putative efficacy for the treatment of obesity or type 2 diabetes [3,4]. Soon after, inconsistencies between results in circulating irisin values were obtained in several follow-up studies [5]. Moreover, Eckel and colleagues also suggested that the positive effects of irisin on glucose homeostasis and obesity that were observed in mice, were less predictable or even unlikely in humans [6]. Some concerns thus emerged as to whether: (i) the antibodies used in the enzyme-linked immunosorbent assays (ELISAs) for measuring circulating irisin levels exhibited acceptable analytical performance [6]; (ii) irisin was present or not in humans [7]; (iii) irisin plays any significant physiological role in humans [8]. Initially, Bo¨strom et al. described that FNDC5 (fibronectin domain-containing protein 5), a member of fibronectin type III repeat containing gene family that is prevalently synthesized in muscles after exercise, apparently promotes healthy metabolism in mice [2]. They also showed that irisin, a specific FNDC5 fragment generated by proteolytic processing, was secreted into the bloodstream and was effective to prime energy-storing white fat cells to act as energy-burning brown fat cells [2]. However, both biochemistry and biology of irisin remain largely debated. According to the study performed by Lee et al. [9], the protein could be detected in human serum, but cleavage site(s) and exact size both remain a matter of debate in the scientific community.
Corresponding author: Sanchis-Gomar, F. ([email protected]). Keywords: irisin; FNDC5; exercise; ELISA kit; antibodies; mass spectrometry. 1043-2760/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2015.05.004
Technical drawbacks in irisin detection methods To clarify these issues, Albrecht and co-workers analyzed the specificity and sensitivity of currently available antibodies for detecting irisin [10]. They reported a significant cross-reactivity with other proteins in both serum and plasma of different species, and ultimately failed to identify a protein of the same size as irisin (i.e., from 13 to 32 kDa, depending on glycosylation and means of detection) in blood samples [10]. Even the antibody that was originally used for the identification of irisin by Bostro¨m and collaborators was found to recognize the C-terminal sequence of the FNDC5 protein, which is not part of the irisin cleaved peptide [10]. Furthermore, Albrecht et al. immunoprecipitated the samples with the Adipogen polyclonal antibody raised against recombinant irisin, and the bands detected at 25 and 16 kDa could not be identified as irisin by mass spectrometry (MS) analysis, and they were predominantly represented by Apoliprotein A1 (ApoA1) [10]. It is noteworthy that the concentration of ApoA1 in serum is several orders of magnitude higher than the measurable concentration of irisin with ELISA (i.e., mg/ ml versus mg/ml). It is hence predictable that irisin ELISA kits may be saturated or exhibit a substantial deviation from linearity if ApoA1 or other abundant proteins are nonspecifically recognized by the antibodies. Therefore, although it might be concluded that the study by Albrecth et al. highlighted significant drawbacks in detection of irisin by the antibodies included in available the commercial ELISA kits, some technical issues can be highlighted in this investigation: (i) An epitope of a protein can be masked in serum or plasma if present in complex with other proteins (prostate specific antigen is a paradigmatic example) [11]. The ELISA kits produced by different manufacturers usually contain different antibodies, so that the relative detection of the free and complexed forms may vary widely among the different assays, and this may impact the final amount of the protein being measured. The use of different calibrators (i.e., recombinant or purified protein, free or complexed form, degree of glycosylation) may also profoundly impair detectability. (ii) Albrecht et al. used Western Blot (WB) in their analysis, which entails protein denaturation, and this may lead to detection of a discrete number of nonspecific bands attributable to abundant denatured peptides. Additionally, whether FNDC5/Irisin is cleaved at different sequences and migrates at 13, 20, 25, and/or 32 kDa is still unclear, and this adds uncertainty to irisin detection by WB. Trends in Endocrinology and Metabolism xx (2015) 1–2
1
TEM-1036; No. of Pages 2
Forum (iii) Due to the complex biochemical structure of irisin, the comparison of data obtained by immunoprecipitation (IP) with those generated using irisin ELISA kits requires similar sample treatment to limit the likelihood of protein modification. Indeed, the IP performed by Albrecht et al. entailed serum denaturation at 958C, whereas this step is unnecessary with conventional ELISAs [10]. In addition, anti-irisin antibodies are chemically cross-linked to magnetic beads, and this binding might affect the avidity of the antibodies. Ideally, an unrelated antibody should also be used as a negative control in IP studies, but this step has been overlooked [10].
Considerations moving forward Overall, the biochemical and biological properties of irisin remain largely debated, and additional studies would be needed to clarify the nature, metabolism and biological activity of this intriguing hormone. Indeed, development and characterization of highly specific (monoclonal) antibodies against recombinant (both glycosylated and nonglycosylated) human irisin is a mainstay. Identification of a candidate reference material and a reference method is also necessary for the development of commercial immunoassays, as well as for improving consistency in findings across different methods and enabling comparisons among different experimental studies. Finally, defining and standardizing preanalytical requirements (i.e., sample matrix, sample stability, optimal storage conditions) will be crucial for preventing protein degradation or modification, which may ultimately impair irisin detection. Specific and
2
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
reliable assays are needed to validate the recent evidence in mice suggesting that irisin is secreted by the muscle and acts on fatty tissue, thus exerting autocrine, paracrine, and endocrine activity [2,12]. Until then, the biology and function of irisin in human physiology will remain unknown. Disclaimer statement The authors declare no competing interests.
References 1 Servick, K. (2015) Biomedicine. Woes for ‘exercise hormone’. Science (New York, N.Y.) 347, 1299 2 Bostrom, P. et al. (2012) A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463–468 3 Sanchis-Gomar, F. et al. (2012) Irisin: a new potential hormonal target for the treatment of obesity and type 2 diabetes. J. Diabetes 4, 196 4 Sanchis-Gomar, F. and Perez-Quilis, C. (2013) Irisinemia: A novel concept to coin in clinical medicine? Ann. Nutr. Metab. 63, 60–61 5 Sanchis-Gomar, F. et al. (2014) Inconsistency in circulating irisin levels: what is really happening? Horm. Metab. Res. 46, 591–596 6 Elsen, M. et al. (2014) Browning of white fat: does irisin play a role in humans? J. Endocrinol. 222, R25–R38 7 Erickson, H.P. (2013) Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor? Adipocyte 2, 289–293 8 Timmons, J.A. et al. (2012) Is irisin a human exercise gene? Nature 488, E9–10 9 Lee, P. et al. (2014) Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Cell Metab. 19, 302–309 10 Albrecht, E. et al. (2015) Irisin - a myth rather than an exerciseinducible myokine. Sci. Rep. 5, 8889 11 Lippi, G. et al. (2009) Prostate-specific antigen-based screening for prostate cancer in the third millennium: useful or hype? Ann. Med. 41, 480–489 12 Irving, B.A. et al. (2014) Does IRISIN Have a BRITE future as a therapeutic agent in humans? Curr. Obes. Rep. 3, 235–241
