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Irisin - a myth rather than an exercise-inducible myokine.

Albrecht E, Norheim F, Thiede B, Holen T, Ohashi T, Schering L, Lee S, Brenmoehl J, Thomas S, Drevon CA, Erickson HP, Maak S - Sci Rep (2015)

Bottom Line: The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism.Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples.A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested.

View Article: PubMed Central - PubMed

Affiliation: Institute for Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology, D-18196 Dummerstorf, Germany.

ABSTRACT
The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

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FNDC5 transcripts and deduced peptides.(a) Transcript structure of human FNDC5 (T1: NM_001171941.2, T2: NM_153756.2, T3: NM_001171940.1) and deduced peptide structure (P1–P3). Numbers refer to nucleotides (T1-3) or amino acids (P1–3). Black bars represent exons. Exon numbers are given in circles. Start and stop codons are indicated above the bars. The irisin peptide is marked by a bold box. The open box marks the truncated irisin peptide in P1 theoretically resulting from transcript T1. The size of irisin and FNDC5 protein variants is given. The peptide signature identified by mass spectrometry is marked in red. SP: signal peptide. (b) Example for detection of FNDC5 transcripts by RNA-sequencing of skeletal muscle biopsies. Exon 1a is specific for transcript T1 whereas exon 1b is part of transcripts T2 and T3. The panels represent results for one individual before (A1–A3) and after 12 weeks of training intervention (B1–B3). A1 and B1 were measured before, A2 and B2 immediately after, and A3 and B3 2 hours after acute exercise13.
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f6: FNDC5 transcripts and deduced peptides.(a) Transcript structure of human FNDC5 (T1: NM_001171941.2, T2: NM_153756.2, T3: NM_001171940.1) and deduced peptide structure (P1–P3). Numbers refer to nucleotides (T1-3) or amino acids (P1–3). Black bars represent exons. Exon numbers are given in circles. Start and stop codons are indicated above the bars. The irisin peptide is marked by a bold box. The open box marks the truncated irisin peptide in P1 theoretically resulting from transcript T1. The size of irisin and FNDC5 protein variants is given. The peptide signature identified by mass spectrometry is marked in red. SP: signal peptide. (b) Example for detection of FNDC5 transcripts by RNA-sequencing of skeletal muscle biopsies. Exon 1a is specific for transcript T1 whereas exon 1b is part of transcripts T2 and T3. The panels represent results for one individual before (A1–A3) and after 12 weeks of training intervention (B1–B3). A1 and B1 were measured before, A2 and B2 immediately after, and A3 and B3 2 hours after acute exercise13.

Mentions: We then spiked rNG- and rG-irisin into human sera and analyzed bands matched to the size of the recombinant control peptides by HPLC/mass spectrometry (Fig. 5c,d). Samples of human serum without addition of rNG- or rG-irisin were applied to the same gels. Gel pieces at the positions corresponding to the size of the controls were cut out and analyzed by HPLC/mass spectrometry even though no staining with Coomassie Blue was observed. Unique peptide signatures for FNDC5 or irisin (MLRFIQEVNTTTR or FIQEVNTTTR, Fig. 6a) were detected in samples with added 100 ng or 500 ng rNG-irisin in bands at ~13 kDa. The doublet band visible in human serum without addition (lane 4 in Fig. 5c) contained no FNDC5 or irisin. Specific signatures for FNDC5 or irisin were found at ~20 kDa in human serum with 100 ng or 500 ng rG-irisin added. The respective gel piece from a serum sample without added rG-irisin (lane 4 in Fig. 5d) revealed no visible band but contained a single peptide signature unique for FNDC5 or irisin (FIQEVNTTTR) among 13 other proteins. Besides this, the results from mass spectrometry supported the assumption of massive non-specific reaction of proteins with irisin antibody pAb-C.


Irisin - a myth rather than an exercise-inducible myokine.

Albrecht E, Norheim F, Thiede B, Holen T, Ohashi T, Schering L, Lee S, Brenmoehl J, Thomas S, Drevon CA, Erickson HP, Maak S - Sci Rep (2015)

FNDC5 transcripts and deduced peptides.(a) Transcript structure of human FNDC5 (T1: NM_001171941.2, T2: NM_153756.2, T3: NM_001171940.1) and deduced peptide structure (P1–P3). Numbers refer to nucleotides (T1-3) or amino acids (P1–3). Black bars represent exons. Exon numbers are given in circles. Start and stop codons are indicated above the bars. The irisin peptide is marked by a bold box. The open box marks the truncated irisin peptide in P1 theoretically resulting from transcript T1. The size of irisin and FNDC5 protein variants is given. The peptide signature identified by mass spectrometry is marked in red. SP: signal peptide. (b) Example for detection of FNDC5 transcripts by RNA-sequencing of skeletal muscle biopsies. Exon 1a is specific for transcript T1 whereas exon 1b is part of transcripts T2 and T3. The panels represent results for one individual before (A1–A3) and after 12 weeks of training intervention (B1–B3). A1 and B1 were measured before, A2 and B2 immediately after, and A3 and B3 2 hours after acute exercise13.
© Copyright Policy - open-access
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f6: FNDC5 transcripts and deduced peptides.(a) Transcript structure of human FNDC5 (T1: NM_001171941.2, T2: NM_153756.2, T3: NM_001171940.1) and deduced peptide structure (P1–P3). Numbers refer to nucleotides (T1-3) or amino acids (P1–3). Black bars represent exons. Exon numbers are given in circles. Start and stop codons are indicated above the bars. The irisin peptide is marked by a bold box. The open box marks the truncated irisin peptide in P1 theoretically resulting from transcript T1. The size of irisin and FNDC5 protein variants is given. The peptide signature identified by mass spectrometry is marked in red. SP: signal peptide. (b) Example for detection of FNDC5 transcripts by RNA-sequencing of skeletal muscle biopsies. Exon 1a is specific for transcript T1 whereas exon 1b is part of transcripts T2 and T3. The panels represent results for one individual before (A1–A3) and after 12 weeks of training intervention (B1–B3). A1 and B1 were measured before, A2 and B2 immediately after, and A3 and B3 2 hours after acute exercise13.
Mentions: We then spiked rNG- and rG-irisin into human sera and analyzed bands matched to the size of the recombinant control peptides by HPLC/mass spectrometry (Fig. 5c,d). Samples of human serum without addition of rNG- or rG-irisin were applied to the same gels. Gel pieces at the positions corresponding to the size of the controls were cut out and analyzed by HPLC/mass spectrometry even though no staining with Coomassie Blue was observed. Unique peptide signatures for FNDC5 or irisin (MLRFIQEVNTTTR or FIQEVNTTTR, Fig. 6a) were detected in samples with added 100 ng or 500 ng rNG-irisin in bands at ~13 kDa. The doublet band visible in human serum without addition (lane 4 in Fig. 5c) contained no FNDC5 or irisin. Specific signatures for FNDC5 or irisin were found at ~20 kDa in human serum with 100 ng or 500 ng rG-irisin added. The respective gel piece from a serum sample without added rG-irisin (lane 4 in Fig. 5d) revealed no visible band but contained a single peptide signature unique for FNDC5 or irisin (FIQEVNTTTR) among 13 other proteins. Besides this, the results from mass spectrometry supported the assumption of massive non-specific reaction of proteins with irisin antibody pAb-C.

Bottom Line: The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism.Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples.A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested.

View Article: PubMed Central - PubMed

Affiliation: Institute for Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology, D-18196 Dummerstorf, Germany.

ABSTRACT
The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Show MeSH