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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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Related in: MedlinePlus

Western blot of serum or plasma samples of different species with irisin antibodies pAb-A (a) and pAb-C (b).Images were taken after 10 min (a) and 20 min (b) exposure and equally enhanced in contrast. Boxes indicate bands of rNG-irisin (rNGI), rG-irisin (rGI), and synthetic (SI) irisin. Samples in lanes 20–25 differ between (a) and (b).
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f3: Western blot of serum or plasma samples of different species with irisin antibodies pAb-A (a) and pAb-C (b).Images were taken after 10 min (a) and 20 min (b) exposure and equally enhanced in contrast. Boxes indicate bands of rNG-irisin (rNGI), rG-irisin (rGI), and synthetic (SI) irisin. Samples in lanes 20–25 differ between (a) and (b).

Mentions: Sera were analyzed from 3 horses undergoing extreme physical exercise (160 km endurance race) and from 1 horse with established metabolic syndrome. For comparison, samples from cattle, domestic pig, wild boar, donkey, goat, rabbit and mouse were included (Fig. 3). Human samples with irisin levels of 76 ng/mL and 864 ng/mL, previously measured with ELISA (pAb-A), were from 1 healthy and 1 pre-diabetic individual prior to or 2 hours after a single bout of acute exercise13. All samples were analyzed by western blot with antibodies pAb-A and pAb-C (Fig. 3a,b). Recombinant NG- and rG-irisin were used as positive controls. No antibody detected proteins at the size of the positive controls, indicating that neither non-glycosylated nor glycosylated irisin circulated in the serum/plasma of any species. However, immune-reactive bands were observed at ~16 kDa in a human sample and at ~25 kDa in both human and goat samples with pAb-C. These bands were consequently further analyzed by HPLC/mass spectrometry.


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)

Western blot of serum or plasma samples of different species with irisin antibodies pAb-A (a) and pAb-C (b).Images were taken after 10 min (a) and 20 min (b) exposure and equally enhanced in contrast. Boxes indicate bands of rNG-irisin (rNGI), rG-irisin (rGI), and synthetic (SI) irisin. Samples in lanes 20–25 differ between (a) and (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Western blot of serum or plasma samples of different species with irisin antibodies pAb-A (a) and pAb-C (b).Images were taken after 10 min (a) and 20 min (b) exposure and equally enhanced in contrast. Boxes indicate bands of rNG-irisin (rNGI), rG-irisin (rGI), and synthetic (SI) irisin. Samples in lanes 20–25 differ between (a) and (b).
Mentions: Sera were analyzed from 3 horses undergoing extreme physical exercise (160 km endurance race) and from 1 horse with established metabolic syndrome. For comparison, samples from cattle, domestic pig, wild boar, donkey, goat, rabbit and mouse were included (Fig. 3). Human samples with irisin levels of 76 ng/mL and 864 ng/mL, previously measured with ELISA (pAb-A), were from 1 healthy and 1 pre-diabetic individual prior to or 2 hours after a single bout of acute exercise13. All samples were analyzed by western blot with antibodies pAb-A and pAb-C (Fig. 3a,b). Recombinant NG- and rG-irisin were used as positive controls. No antibody detected proteins at the size of the positive controls, indicating that neither non-glycosylated nor glycosylated irisin circulated in the serum/plasma of any species. However, immune-reactive bands were observed at ~16 kDa in a human sample and at ~25 kDa in both human and goat samples with pAb-C. These bands were consequently further analyzed by HPLC/mass spectrometry.

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
Related in: MedlinePlus