Irisin: Still chasing shadows

Abstract

Objective: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin.

Methods: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin.

Results: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement.

Conclusion: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.

Keywords: Adipose tissue; FNDC5; Mass spectrometry; Plasma; Skeletal muscle; Transcript.

Figure 1

Annotated and expressed FNDC5 transcripts in human skeletal muscle. T1-T3: Transcripts annotated in GenBank. T1: NM_001171941.2. T2: NM_153756.2. T3: NM_001171940.1. T4-T6: Additional transcripts expressed in human skeletal muscle in this study. Exon numbering (circles) refers to data published by Albrecht et al. [6]. The GenBank-derived exons correspond to the following exons in Ensembl Release 98 (09/19): 1a: ENSE00001811973. 1b: ENSE00001862258. 2: ENSE00001751279. 3: ENSE00003591295. 4: ENSE00003704806. Annotation of exons 5a-6b differs between GenBank and Ensembl.

Figure 2

Detection of FNDC5 in different species with antibody E. A: FNDC5 in human and mouse muscle and adipose tissue. B: The antibody was pre-incubated with a blocking peptide in a parallel blot. C: FNDC5 in bovine muscle and adipose tissues. D: FNDC5 in four human and one mouse muscle sample before and after deglycosylation. M: molecular weight marker. SAT: subcutaneous adipose tissue. AT: adipose tissue. Regions of interest are marked by boxes or a red arrow.

Figure 3

Sensitivity and specificity of irisin antibodies. A-C: Detection of different amounts of recombinant irisin with antibodies A, B, and C. A: Irisin amounts per lanes 1–8: 20, 4, 2, 1, 0.5, 0.25, 0.125, and 0.062 ng. The first lane, containing 20 ng recombinant irisin, was excluded to calculate the linearity (when included: R² = 0.9781). B: Irisin amounts per lane 1–7: 4, 2, 1, 0.5, 0.25, 0.125, and 0.062 ng. C: Irisin amounts per lane 1–8: 4, 2, 1, 0.5, 0.25, 0.125, 0.062, and 0.031 ng. Linearity of detection is shown for each antibody in the lower panel (details in Supplementary Figs. S2A–C). D: Detection of non-glycosylated and glycosylated recombinant irisin, and FNDC5/irisin in human*, mouse and bovine serum/plasma, and muscle with antibody C (upper panel). The antibody was pre-incubated with recombinant irisin in a parallel blot (lower panel). ^∗Human serum sample: HS 1 (acute exercise; details in Supplementary Table S1). M: molecular weight marker. Regions of interest are marked by boxes.

Figure 4

Detection of irisin before and after deglycosylation. A: Deglycosylation of recombinant glycosylated irisin and human serum samples* with PNGase F or protein deglycosylation Mix II (NEB) and detection with antibody C. *Human samples: HS 2a, HS 3 (acute exercise; details in Supplementary Table S1). B: Detection of different amounts of recombinant glycosylated irisin (2, 1, 0.5, 0.25, 0.125, and 0.062 ng) with antibody B before (lanes 1–6, left panel) and after deglycosylation (lanes 1–6, right panel). Glycosylated recombinant irisin was added to albumin-depleted bovine plasma. Linearity of detection is shown in the right panel. Glycosylated irisin (left panel) could not be quantified (details in Supplementary Figure S2D). M: molecular weight marker. Regions of interest are marked by boxes.

Figure 5

Detection of recombinant and native irisin in different species with antibody B. A: Recombinant irisin (lanes 1–8: 4, 2, 1, 0.5, 0.25, 0.125, 0.062, and 0.031 ng) and native irisin in sera of human*, rat, and horse detected with antibody C. All sera were albumin- and IgG-depleted, deglycosylated and 80 μg protein were resolved per lane. *Human samples: HS 4b and HS 5b (12 w training; details in Supplementary Table S2). B: Recombinant irisin (lane 1); recombinant glycosylated irisin before (lane 2) and after (lane 3) deglycosylation. Bovine plasma was spiked with 50, 100, 200, and 400 ng/ml irisin, albumin- and IgG-depleted, deglycosylated and 120 μg protein were resolved (lanes 4–7) and detected with antibody B. C: Human*, rat, mouse (mou.), baboon (bab.), and bovine (bov.) plasma/serum samples were albumin- and IgG-depleted and deglycosylated and 120 μg total protein were resolved per lane. Native baboon plasma (2 μL with ∼100 μg protein) was applied to lane 9. Lane 1: 20 ng deglycosylated recombinant irisin. Detection with antibody B. *Human samples: HS 6a and HS 7a (12 w training + acute exercise; details in Supplementary Table S1). M: molecular weight marker. Regions of interest are marked by boxes.

Figure 6

Detection of irisin in rat and human serum by mass spectrometry (PRM mode). A: Coomassie-stained gels with boxed regions of interest removed for targeted mass spectrometry in the PRM mode; 300 μg total protein per sample was subjected to electrophoresis (5 × 60 μg). Only ∼270 μg total protein was available for sample HS 7b. B: PRM elution profile for the heavy AQUA irisin peptide (FIQEDVTTTR) in the rat (upper panel) and human (lower panel) sera. C: Corresponding profiles of the tryptic irisin peptide (FIQEDVTTTR). *Human samples: HS 6b (12 w training) and HS 7b (12 w training + acute exercise; details in Supplementary Table S1).