Three years ago, Spiegelman’s team showed that the type I transmembrane protein FNDC5 is upregulated in muscle during exercise in both mice and humans. The researchers had also reported that, during physical activity, the protein’s extracellular portion—which they called irisin—is cleaved and released into the blood stream.

Spiegelman’s group first identified irisin as a hormone released from human muscle during physical activity in 2012. It has been a source of scientific contention ever since.

For their latest analysis, Spiegelman, Steven Gygi, and colleagues used tandem mass spectrometry—a method that does not rely on antibodies—to detect irisin.

“This study fairly clearly demonstrates the existence of irisin in circulation in humans and changes of its levels post-exercise,” Philipp Scherer, who studies adipogenesis at the University of Texas Southwestern Medical Center but was not involved in the work, told The Scientist in an email.

Sven Enerbäck, who studies adipose tissue at the Gothenburg University in Sweden and was also not involved in the work, agreed. “Using state-of-the-art mass spectrometry, the authors show irisin in circulation—the strongest evidence for irisin in humans,” he said.

“There is no next level of analysis,” said Spiegelman. “This is down to, literally, the atomic level.”

Following up on their 2012 work, for the present study, the team first removed the most abundant plasma proteins—albumin and immunoglobulin—from human plasma samples collected from sedentary volunteers as well as those who had aerobic interval training. The researchers then spiked the plasma with two irisin-specific peptides labeled with stable, heavy isotopes. These peptides, which do not map to any other proteins in the annotated human genome, allowed the researchers to identify the chemically identical—but physically different—native irisin. “[These heavy peptides] let us to follow exactly where irisin would go in the mass spec,” explained Spiegelman.

The team also used the two synthetic irisin peptides to investigate the irisin mRNA translation start site. One encoded part of the protein downstream from the ATA translation start site but upstream of the first ATG codon within the mRNA, while the other was downstream from both. Because both native peptides were present in circulation in similar quantities, the researchers confirmed the previously reported ATA start codon.

Circulating blood irisin levels were about 3.6 nanograms per milliliter (ng/mL) in sedentary individuals, and slightly higher—4.3 ng/mL—in those who did aerobic internal training. “This is the level at which many biologically active hormones circulate,” said Spiegelman.

“This quantitative mass spectrometry analysis is a major step forward to understand how this hormone affects human metabolism,” said Francesco Celi of the Virginia Commonwealth University Medical Center who also studies irisin but was not involved in the work.

But Harold Erickson, a cell biologist at Duke University in Durham, North Carolina, who was an author on a study disputing the existence of irisin, is still not entirely convinced. “The mass spectrometry seems to be done very correctly but I think it is still a possibility that the 3 ng/mL concentration [protein] is a contaminant of some sort,” he told The Scientist. Citing a 2013 paper that showed very low translation of the human FNDC5 gene from the ATA compared to the ATG codon, Erickson said he is skeptical that humans have retained functional FNDC5.

Spiegelman’s lab previously showed that irisin could induce the browning of white fat, turning fat from inert to metabolically active. Using knockout and transgenic mice, his team is now investigating the hormone’s mechanism of action and whether irisin has additional functions related to exercise. “How many other [exercise-related] things that can be embodied by irisin is what is exciting for me,” said Spiegelman.

With these latest results in hand, said Enerbäck, “the field can now turn to evaluating the effects of irisin on human physiology.”

M.P. Jedrychowski et al., “Detection and quantitation of circulating human irisin by tandem mass spectrometry,” Cell Metabolism, doi:10.1016/j.cmet.2015.08.001, 2015.