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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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Identification of proteins by mass spectrometry.(a) Western blot of human and caprine serum before (lanes 1, 4) and after (lanes 2, 3) immuno-precipitation with pAb-C. Bands of interest are within white boxes. (b) Coomassie-stained gel with the same samples. After immuno-precipitation, target bands (black boxes) were cut out of the gel and analyzed. (c) Coomassie-stained gel with 500 ng rNG-irisin in PBS (lane 1), 500 ng and 100 ng rNG-irisin added to human serum (lanes 2 and 3) and human serum without addition (lane 4). Serum samples were albumin-depleted prior to electrophoresis. Gel pieces within black boxes were cut out and analyzed. (d) Addition of rG-irisin instead of rNG-irisin. Samples and procedure are as in (c). M: molecular weight marker.
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f5: Identification of proteins by mass spectrometry.(a) Western blot of human and caprine serum before (lanes 1, 4) and after (lanes 2, 3) immuno-precipitation with pAb-C. Bands of interest are within white boxes. (b) Coomassie-stained gel with the same samples. After immuno-precipitation, target bands (black boxes) were cut out of the gel and analyzed. (c) Coomassie-stained gel with 500 ng rNG-irisin in PBS (lane 1), 500 ng and 100 ng rNG-irisin added to human serum (lanes 2 and 3) and human serum without addition (lane 4). Serum samples were albumin-depleted prior to electrophoresis. Gel pieces within black boxes were cut out and analyzed. (d) Addition of rG-irisin instead of rNG-irisin. Samples and procedure are as in (c). M: molecular weight marker.

Mentions: The immuno-reactive bands at ~25 kDa stained by pAb-C in human (Supplementary Fig. 1b, Fig. 3b) and goat serum (Fig. 3b) were purified by immuno-precipitation with pAb-C cross-linked to magnetic beads (Fig. 5a,b). An additional band at ~16 kDa observed earlier in human serum (Fig. 3b) was also included in subsequent mass spectrometric analysis. No peptide corresponding to FNDC5 or its irisin-part was identified in any of the precipitated samples in 2 repeats of the experiment. Instead, apolipoprotein A1 was identified as a predominant protein in both human and goat bands. The human band of lower molecular weight included mainly Ig kappa chains and hemoglobin subunits.


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)

Identification of proteins by mass spectrometry.(a) Western blot of human and caprine serum before (lanes 1, 4) and after (lanes 2, 3) immuno-precipitation with pAb-C. Bands of interest are within white boxes. (b) Coomassie-stained gel with the same samples. After immuno-precipitation, target bands (black boxes) were cut out of the gel and analyzed. (c) Coomassie-stained gel with 500 ng rNG-irisin in PBS (lane 1), 500 ng and 100 ng rNG-irisin added to human serum (lanes 2 and 3) and human serum without addition (lane 4). Serum samples were albumin-depleted prior to electrophoresis. Gel pieces within black boxes were cut out and analyzed. (d) Addition of rG-irisin instead of rNG-irisin. Samples and procedure are as in (c). M: molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352853&req=5

f5: Identification of proteins by mass spectrometry.(a) Western blot of human and caprine serum before (lanes 1, 4) and after (lanes 2, 3) immuno-precipitation with pAb-C. Bands of interest are within white boxes. (b) Coomassie-stained gel with the same samples. After immuno-precipitation, target bands (black boxes) were cut out of the gel and analyzed. (c) Coomassie-stained gel with 500 ng rNG-irisin in PBS (lane 1), 500 ng and 100 ng rNG-irisin added to human serum (lanes 2 and 3) and human serum without addition (lane 4). Serum samples were albumin-depleted prior to electrophoresis. Gel pieces within black boxes were cut out and analyzed. (d) Addition of rG-irisin instead of rNG-irisin. Samples and procedure are as in (c). M: molecular weight marker.
Mentions: The immuno-reactive bands at ~25 kDa stained by pAb-C in human (Supplementary Fig. 1b, Fig. 3b) and goat serum (Fig. 3b) were purified by immuno-precipitation with pAb-C cross-linked to magnetic beads (Fig. 5a,b). An additional band at ~16 kDa observed earlier in human serum (Fig. 3b) was also included in subsequent mass spectrometric analysis. No peptide corresponding to FNDC5 or its irisin-part was identified in any of the precipitated samples in 2 repeats of the experiment. Instead, apolipoprotein A1 was identified as a predominant protein in both human and goat bands. The human band of lower molecular weight included mainly Ig kappa chains and hemoglobin subunits.

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