'Exercise Protein' Irisin Fades to Myth

March 16, 2015, MedPage Today

(view original)

Harold Erickson, PhD, let his curiosity get the best of him while preparing to teach a routine class, and the result has called into question the findings of more than 170 studies that held the promise of an obesity cure.

Erickson, a professor of cell biology and biochemistry at Duke University, was covering fat tissue for first-year medical students in 2012, was intrigued when a colleague showed him a paper relevant to the material. But when he read it, he noticed problems that, it now appears, were endemic to virtually the entire literature on the protein known as irisin.

Irisin had been billed as a substance that could turn white fat into brown fat -- potentially holding the key to weight loss and a myriad of other problems. Named after the Greek goddess and messenger Iris, it was supposed to relay directions from muscles to fat after exercise to burn, not store.

The study that caught Erickson's attention was published in Nature, and it wasn't even in his field. But Erickson said he remembers sitting in his office on a weekend, thinking about how it could be applicable to the work that he did with other proteins. But he saw that the study's methods were questionable, and began an investigation that has now culminated in a paper published, with colleagues from other laboratories, in Scientific Reports.

It indicates that the commercial kits used to measure irisin in the study and many others -- so-called ELISA assays that use antibodies used to measure the levels of irisin in the blood -- hadn't been rigorously tested and turned out to be nonspecific, reacting with other blood proteins in addition to irisin. Consequently, the assays were yielding irisin levels much higher than they actually were.

"The birth of the new, promising myokine irisin has been complicated from the beginning by multiple contradictions and obscurities," wrote Erickson and colleagues. "Hopefully, our findings will finally convince other researchers to stop chasing a myth."

The findings are potentially devastating to almost all of the previous work on irisin, and the researchers argue that a physiological role for the protein in humans is very unlikely.

The hoopla surrounding irisin began shortly after the publication of the 2012 Nature article. "A newly discovered hormone produced in response to exercise may be turning people's white fat brown, a groundbreaking new study suggests," led a story on The New York Times' website. For Bloomberg, it was a "Harvard study" that could hold the key to a new, effective obesity medication.

Other publications, unsurprisingly, were even less skeptical. "Miracle weight loss pill 'irisin' allows for easy workouts -- scientists claim they've created a pill that provides a workout without the sweat" was the headline of an article in Men's Fitness.

Researchers began to study the protein, though some were not convinced, even early on. Erickson told MedPage Today that he has been raising concerns ever since he realized something was wrong, and in 2013 he published a commentary in Adipocyte voicing those concerns. And the original study prompted controversy, as a 2012 letter to Nature shows. But dozens of studies went forward. Researchers looked at how the protein, whose parent gene is FNDC5, was affected by not just exercise, but by diabetes, obesity, and even cancer. And almost all of them took a fatal shortcut: they used commercial ELISA kits, which analyze proteins in a single dish.

Steffen Maak, PhD, at the Leibniz Institute for Farm Animal Biology in Germany, did a Western blot test for irisin, which was more accurate than the ELISA kit. His work with farm animals caught the attention of Erickson.

"I was excited," said Erickson. "I told him we can do an experiment that nobody's done before."

They started to collaborate, bringing in other labs and researchers. They ordered the antibodies used in the ELISA kits and analyzed them using a Western blot instead, and they noticed two things: no proteins were the size that irisin should have been, and the antibodies reacted with other blood proteins that were the wrong size for irisin. In other words, the commercial kits had been leading to false positive results.

Erickson's laboratory provided the necessary controls by synthesizing two different versions of irisin, one with sugar, and one without. They added the proteins to a sample and did a Western blot, finding that the antibodies detected both forms of irisin and that the irisin samples were the sizes they should have been.

"Our conclusions make sense, especially in light of the work of other researchers who have shown that the human version of the FNDC5 gene has a deleterious mutation at the beginning," Erickson said in a press release. "As a result, humans can produce less than 1% of the irisin present in other species. Humans are essentially a gene knock-out -- they can't produce FNDC5, and therefore they can't produce irisin."

Even recently, the results of the irisin studies were defended, Erickson told MPT. "But nobody has previously considered the fact that the proteins they're measuring may be nonspecific proteins," he said.

He also noted that most of the evidence that irisin acts after exercise has been from cell culture, and it has all been pretty indirect.

However, Bruce Spiegelman, PhD, the Harvard cancer biology professor who coined the term ‘irisin’ in the Nature paper, told MedPage Today that he disagreed with the Erickson group’s conclusions.

“The previous identification of human irisin in blood by mass spectrometry by Celi and colleagues has previously closed the question as to the existence of irisin [in humans],” he wrote in an email. “The lack of ability of the current investigators to find circulating irisin may reflect the sensitivity of the procedures.”

The authors of the Scientific Reports paper noted that previous researchers have asked the question: Is irisin a Greek goddess or a Greek myth? “Our results provide experimental evidence for irisin being a myth,” Erickson and colleagues concluded.