Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼3.6 ng/ml in sedentary individuals; this level is increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise.

Human FNDC5 has an atypical translation start codon, ATA, in place of the more typical ATG. While it is now known that many eukaryotic mRNAs begin translation with non-ATG start codons (), two recent papers have claimed that this ATA codon in human FNDC5 represents a null mutation and therefore human irisin would not be produced (). These authors argue that if FNDC5 exists in humans, it is translated from a downstream ATG, and hence the irisin polypeptide is a “myth” and does not exist. In addition, these authors claim that the many papers measuring human irisin are all artifacts of poor antibody specificity (); this is despite the fact that Lee et al. had previously detected an irisin peptide in human plasma with mass spectrometry (). In this paper we have investigated the presence of human irisin in blood using quantitative mass spectrometry. As internal standards, we synthesized irisin peptides and included a valine enriched in stable isotopes (sixC atoms). The peptides were used to develop a quantitative platform for the measurement of human irisin; these data should facilitate future studies of this molecule in both mice and humans.

The health benefits of physical activity and exercise are well recognized (). Exercise is the first line of therapy for various metabolic diseases like diabetes and obesity, but exercise also improves outcomes in diseases involving other tissues, such as the heart and brain. We recently described a novel polypeptide that is secreted from skeletal muscle and is increased with exercise. Irisin is the shed extracellular domain of a transmembrane protein called FNDC5. FNDC5, when expressed from adenoviral vectors in mice, causes an elevation of irisin in the blood and improved metabolic health in recipient animals (). It also stimulates the expression of a potential neuroprotective gene program in the brain, particularly in the hippocampus (). Several papers have studied the effects of exercise on circulating irisin in humans; positive associations between irisin plasma level and exercise have been observed in some but not all cohorts and modes of exercise (). Data suggest that early sampling after exercise and high-intensity training protocols are particularly effective at raising circulating irisin levels. Most of these studies have relied on commercial antibodies and ELISA assays.

The effects of acute and chronic exercise on PGC-1α, irisin and browning of subcutaneous adipose tissue in humans.

Next, for the quantification of irisin in human plasma by mass spectrometry, albumin- and immunoglobulin-depleted plasma from four sedentary and six aerobically interval-trained subjects was deglycosylated and resolved by SDS-PAGE prior to in-gel trypsin digestion. After this, 12.5 femtomoles of each heavy peptide were spiked into the sample prior to absolute quantification (AQUA) of irisin ( Figure 2 A) (). Of note, often with enzymatic deglycosylation of proteins there is a propensity for deamidation occurring on asparagine residues, increasing the mass of the residue by 0.984 Da and slightly delaying the reverse phase retention (). Therefore, successful identification of human irisin peptides (as for other N-glycosylated plasma proteins) must take into account this mass shift. Deamidation modifications for both endogenous plasma irisin peptides are observed without dramatically changing the MSspectra ( Figure S1 A) nor altering the PRM rank order elution profile ( Figure 2 B). Fragment ions for both peptides were quantified using Skyline version 3.1 (), and comparable levels of quantification for both peptides, downstream of the ATA start codon and the later ATG, suggest irisin is mainly translated from its non-canonical start codon ( Table 1 Figures S1 B and S2 ). We found that irisin levels are present at ∼3.6 ng/ml in sedentary individuals and are significantly increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training ( Figure 2 C, Table 1 ).

Skyline software was used to quantify absolute amounts of irisin peptides from the plasma of sedentary and aerobically trained subjects. The 25 kDa glycosylated bioactive form of irisin was used to calculate its ng/ml concentrations in plasma.

(D) Depicted are several plasma proteins and their circulating concentrations ranging from the μg/ml (red), ng/ml (yellow), and pg/ml (blue) levels. We quantify circulating plasma irisin at a 3–5 ng/ml. See also Figure S2

(C) Irisin levels in plasma from sedentary subjects (Sedentary) or subjects undergoing aerobic interval training (Aerobic). Values are shown as mean ± SEM; n = 4 (Sedentary) and n = 6 (Aerobic). ∗ p = 0.0411 compared to sedentary subject group as determined by unpaired t test, two-tailed.

(B) PRM elution profile for internal tryptic irisin peptide (FIQEVNTTR) using Skyline software found in sedentary subject 1. Top panel is the deamidated asparagine form of the peptide found in the plasma, middle panel is the unmodified peptide found in the plasma, and the bottom panel is 12.5 femtomoles of heavy internal standard (IS) AQUA peptide.

(A) SDS-PAGE separation of 50 μg of plasma from each subject and visualized by Coomassie staining. Molecular mass regions corresponding to completely deglycosylated irisin (10–15 kDa) were excised from six separate gels (300 μg from the original 100 μl plasma) for each subject and digested in-gel in the presence of 12.5 femtomoles of each internal standard AQUA peptide.

Two peptides were chosen as standards for this mass spectrometric analysis. These were both chosen because they are unique to the irisin sequence (FNDC5 ectodomain) and not encoded in any other proteins in the annotated human genome. As shown in Figure 1 A, one peptide represents the most extreme N-terminal 12 amino acids (DSPSAPVNVT) of the processed irisin molecule, coming immediately after the signal peptide ( Figure 1 A). Importantly, this peptide is downstream of the non-canonical ATA codon but upstream of the first ATG codon in the FNDC5 mRNA. Therefore detection of this peptide would demonstrate use of the non-canonical start codon. A second tryptic peptide (FIQENTTTR) was chosen from the central portion of irisin, three amino acids downstream of the ATG. Plasma samples from human volunteers who had undergone aerobic interval training (see Experimental Procedures ) were used to develop this assay. These plasma samples were first treated with a commercial affinity resin to remove the very abundant albumin and immunoglobulins, so that these proteins would not hinder analysis of less abundant proteins (see Experimental Procedures ). Samples were then deglycosylated with the Protein Deglycosylation Mix from New England Biolabs (NEB), which contains PNGase F, O-glycosidase, neuraminidase, β1-4-galactosidase, and β-N-acetylglucosaminidase and results in complete deglycosylation. After electrophoresis, the anti-irisin antibody detected a band running at ∼12 kDa, the predicted size of the irisin polypeptide ( Figure 1 B). To characterize the synthetic heavy irisin, peptides were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis in both data-dependent and parallel reaction monitoring (PRM) acquisition modes. As shown in Figure 1 C, the intensity of the y ions series from the MSspectra for both peptides corresponds to the rank order elution profile in the PRM acquisition mode ( Figure 1 D), validating that these ions can be used for identification and quantification of irisin.

(D) PRM elution profile for the y-ions for the AQUA peptides using Skyline software. Retention times for each peptide are labeled on the x axis, and y axis represents the relative intensity for each y-ion peak. See also Figure S1

(C) MS 2 spectra acquired using a Q Exactive mass spectrometer for the two synthetic AQUA peptides and their b-, y-ion series m/z values. Mass accuracy values are given in PPMs and “#” denotes the heavy valine residue.

(B) Immunoblotting of irisin plasma samples from three subjects undergoing aerobic interval training with or without deglycosylation enzyme (Protein Deglycosylation Mix [NEB]) and deglycosylated recombinant irisin.

(A) Schematic representation of the FNDC5 protein structure (top) and irisin (bottom). SP, signal peptide; H, hydrophobic domain; C, C-terminal domain. Below is shown the human FNDC5 sequence with corresponding domains colored. Human irisin sequence is underlined as are synthetic AQUA peptides used in this study (red).

Discussion

Bostrom et al., 2012 Bostrom P.

Wu J.

Jedrychowski M.P.

Korde A.

Ye L.

Lo J.C.

Rasbach K.A.

Bostrom E.A.

Choi J.H.

Long J.Z.

et al. A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Albrecht et al., 2015 Albrecht E.

Norheim F.

Thiede B.

Holen T.

Ohashi T.

Schering L.

Lee S.

Brenmoehl J.

Thomas S.

Drevon C.A.

et al. Irisin - a myth rather than an exercise-inducible myokine. Lee et al., 2014 Lee P.

Linderman J.D.

Smith S.

Brychta R.J.

Wang J.

Idelson C.

Perron R.M.

Werner C.D.

Phan G.Q.

Kammula U.S.

et al. Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Bostrom et al., 2012 Bostrom P.

Wu J.

Jedrychowski M.P.

Korde A.

Ye L.

Lo J.C.

Rasbach K.A.

Bostrom E.A.

Choi J.H.

Long J.Z.

et al. A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. We have developed here a quantitative, precise, and unbiased assay for the detection of human irisin in plasma. This assay definitively shows that human irisin circulates and has a very similar or identical architecture to the mouse protein (). Human irisin circulates at a level at or above the levels observed for many other important biological hormones, as shown in Figure 2 D. It confirms earlier reports that have identified a unique peptide in human plasma by untargeted mass spectrometry (), but provides quantitation of the circulating levels of human irisin in an unbiased and antibody-independent manner. Irisin concentrations are present at ∼3.6 ng/ml in sedentary individuals and are significantly increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. We therefore also confirm our earlier report of irisin being regulated by endurance exercise in humans ().

Albrecht et al., 2015 Albrecht E.

Norheim F.

Thiede B.

Holen T.

Ohashi T.

Schering L.

Lee S.

Brenmoehl J.

Thomas S.

Drevon C.A.

et al. Irisin - a myth rather than an exercise-inducible myokine. Raschke et al., 2013 Raschke S.

Elsen M.

Gassenhuber H.

Sommerfeld M.

Schwahn U.

Brockmann B.

Jung R.

Wisløff U.

Tjønna A.E.

Raastad T.

et al. Evidence against a beneficial effect of irisin in humans. Vanin, 1985 Vanin E.F. Processed pseudogenes: characteristics and evolution. Chang and Wang, 2004 Chang K.J.

Wang C.C. Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. Starck et al., 2012 Starck S.R.

Jiang V.

Pavon-Eternod M.

Prasad S.

McCarthy B.

Pan T.

Shastri N. Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Several papers have called the start codon of the human FNDC5 gene, which is an ATA, rather than the more common ATG, a mutation. Indeed, these authors concluded that human FNDC5 is a non-coding “pseudogene” or that “the human species has an effective gene knockout of FNDC5” (). This claim was based on a transfection assay expressing human FNDC5 from a CMV-promoter-driven plasmid, which yielded protein levels lower than human FNDC5 expressed with an ATG instead of an ATA from the same plasmid. However, several lines of reasoning stand against that claim. First, the high degree of conservation of the irisin amino acid sequence across most mammalian species (including humans) strongly argues against FNDC5 in humans being a pseudogene. Second, the simple fact that Raschke et al. detect human FNDC5 protein made from the ATA-FNDC5 sequence proves that human FNDC5 is not a pseudogene; these are generally defined as genes that have lost their protein-coding ability (). Third, their conclusion that low protein production from CMV-promoter-driven plasmid expressed in HEK293 cells translates to inefficient FNDC5 translation in vivo is completely speculative, since this experiment did not consider endogenous regulation of human FNDC5 in its native state. Indeed, non-canonical starts of translation are often indicative of complex regulation of translation (). Fourth, as mentioned above, our detection here of equal amounts of peptide 1 and 2 in human plasma demonstrates that human irisin is, in fact, mainly translated from its non-canonical start codon and not the further downstream ATG.

Albrecht et al., 2015 Albrecht E.

Norheim F.

Thiede B.

Holen T.

Ohashi T.

Schering L.

Lee S.

Brenmoehl J.

Thomas S.

Drevon C.A.

et al. Irisin - a myth rather than an exercise-inducible myokine. Wrann et al., 2013 Wrann C.D.

White J.P.

Salogiannnis J.

Laznik-Bogoslavski D.

Wu J.

Ma D.

Lin J.D.

Greenberg M.E.

Spiegelman B.M. Exercise induces hippocampal BDNF through a PGC-1α/FNDC5 pathway. The earlier report () had several serious methodological deficiencies. First, their failure to detect irisin in human serum at 12 kDa by western blotting relied on deglycosylation by only one enzyme, namely PNGase F; however, this leads to only incomplete deglycosylation. PNGase F is an effective enzymatic method for removing almost all N-linked oligosaccharides, but not other oligosaccharides. Hence, with PNGase F, no visible band will appear at 12 kDa and the irisin signal will be diluted across the lane, leading to apparent lower levels. In our previously published method (), we used the Protein Deglycosylation Mix from NEB, which contains, in addition to PNGase F, O-glycosidase, neuraminidase, β1-4-galactosidase, and β-N-acetylglucosaminidase; this leads to complete deglycosylation and the appearance of 12 kDa bands in recombinant mammalian irisin and human plasma by immunoblot ( Figure 1 ).

Albrecht et al., 2015 Albrecht E.

Norheim F.

Thiede B.

Holen T.

Ohashi T.

Schering L.

Lee S.

Brenmoehl J.

Thomas S.

Drevon C.A.

et al. Irisin - a myth rather than an exercise-inducible myokine. Second, these authors () used a method of protein mass spectrometry called “shotgun proteomics,” which randomly samples peptides for detection from all the peptides contained in the sample. While the method has the potential to detect irisin, it would be suboptimal for detection because the peptides of interest can be missed in complex samples due to their low abundance. In these cases targeted proteomics is required. This allows the mass spectrometer to focus on the targeted peptides and ignore signal from co-eluting peptides. AQUA-based quantification concomitantly with PRM produces spectra that are highly specific because all potential product ions of a peptide and elution profile confirm the identity of the peptide.

Kraemer et al., 2014 Kraemer R.R.

Shockett P.

Webb N.D.

Shah U.

Castracane V.D. A transient elevated irisin blood concentration in response to prolonged, moderate aerobic exercise in young men and women. Kurdiova et al., 2014 Kurdiova T.

Balaz M.

Vician M.

Maderova D.

Vlcek M.

Valkovic L.

Srbecky M.

Imrich R.

Kyselovicova O.

Belan V.

et al. Effects of obesity, diabetes and exercise on Fndc5 gene expression and irisin release in human skeletal muscle and adipose tissue: in vivo and in vitro studies. Moraes et al., 2013 Moraes C.

Leal V.O.

Marinho S.M.

Barroso S.G.

Rocha G.S.

Boaventura G.T.

Mafra D. Resistance exercise training does not affect plasma irisin levels of hemodialysis patients. Wang et al., 2015 Wang H.H.

Zhang X.W.

Chen W.K.

Huang Q.X.

Chen Q.Q. Relationship between serum irisin levels and urinary albumin excretion in patients with type 2 diabetes. Zhang et al., 2014 Zhang M.

Chen P.

Chen S.

Sun Q.

Zeng Q.C.

Chen J.Y.

Liu Y.X.

Cao X.H.

Ren M.

Wang J.K. The association of new inflammatory markers with type 2 diabetes mellitus and macrovascular complications: a preliminary study. Third, and perhaps most importantly, the authors report their own detection limits for irisin at about 100 ng/ml. However, many reports of human irisin fall below this level (). Hence it is rather surprising that these authors concluded that human irisin did not exist or was a “myth.”

It is worth noting that limitations of own study include that the AQUA heavy peptides were added to the irisin preparations after the extraction of the proteins from the SDS-PAGE gel; we therefore cannot account for how much irisin protein was lost during the sample preparation (albumin/IgG removal, deglycosylation, and retrieval from the gel band, etc.); the numbers reported here must therefore be considered a slight underestimation of the irisin levels. In our experience, typical losses during sample preparation range between 10% and 30%. In addition, this assay is relatively costly and relies on available mass spectrometry instrumentation and capabilities. However, while this assay is relatively low throughput, it should prove useful for benchmarking more high-throughput assays as they are developed. Taken together, targeted mass spectrometry with the use of heavy irisin AQUA peptides settles the existence, the overall architecture of human irisin in the plasma, and its regulation by exercise.