# Molecular Basis of Irisin Regulating the Effects of Exercise on Insulin Resistance
## 2 Biology and Physiology of FNDC5/Irisin
### Consequence, Structure, and Detection of FNDC5/Irisin  
The FNDC5 locus contains six exons and encodes a 209-aa protein, including a 28-aa signal peptide, a 93-aa fibronectin type III (FNIII) domain, a 30-aa linker, a 19-aa transmembrane region, and a 39-aa intracellular domain [17]. This transmembrane protein's FNIII domain plus the linker has been called irisin and is thought to be cleaved (Figure 1). The molecular weight (MW) of the FNDC5 protein is predicted to be about 20,300, and without glycosylation, irisin (112 aa peptide) has a MW of 12,600.  
Figure 1: Structure of irisin. The FNDC5 locus has six exons and encodes a 209-aa protein. The FNIII domain plus the linker, containing 112-aa, is thought to be split off this transmembrane protein and given the name irisin. Irisin can alleviate IR by promoting tissues or organs including skeletal muscle, adipose, or liver more sensitive or responsive to insulin, such as increasing glucose uptake, glycogenesis, and energy expenditure, and decreasing gluconeogenesis. Abbreviations: aa, amino acid.  
Irisin is highly conserved, with 100 percent identity in its amino acid sequence across humans, mouse, rat, and bovines [18]. However, the start codons of humans are different from those of animals, with the human FNDC5 gene having a mutated start codon ATA rather than the standard ATG frequently seen in rodents. To test if the ATA codon has an effect on FNDC5 expression, [PERSON] and colleagues [18] used HEK293 cells to transfect FNDC5 with either the conventional or noncanonical codon and found that the protein level of FNDC5 in constructs with the ATA codon is only 1% that of constructs with the ATG codon. As a result, they proposed that the human FNDC5 gene would lose its ability to translate to a full-length FNDC5 protein. Under the circumstances, PCR and other more specific and sensitive procedures are more effective compared to enzyme-linked immunosorbent assays (ELISA) kits or Western blot analyses [19].  
FNDC5 is proposed to be cut at the N-terminal site, and released as irisin into circulation targeting various tissues. Irisin has the typical FNIII domain structure, which is a beta-sandwich with three beta-strands on one side and four on the other [17]. Irisin can form a tight dimer through the 2 C\({}^{\prime}\) strands correlating antiparallel to form a beta zipper, giving an extended eight-strand beta construct. [PERSON] et al. [19] determined non-glycosylated irisin with Western blots running as a band at an MW of 13,000, approaching 12,600 for this 112-aa peptide. Though the details and the mechanism of the cleavage of FNDC5 are still unclear, there has been clear evidence to demonstrate that Irisin is found in human and mouse body fluids, and the effects of r (recombinant)-irisin treatment in cells and animal models have been extensively reported.  
As an irisin receptor, \(\alpha\)V\(\beta\)5 was first reported by [PERSON] [20], which involved the action of irisin on both osteocyte survival and sclerostin production to regulate bone remodeling. Since then, \(\alpha\)V\(\beta\)5 was detected in other tissues, such as the brain and gut [21; 22]. However, as pointed out by [PERSON] [20], \(\alpha\)V\(\beta\)5 may not be the only irisin receptor, so further studies on irisin receptors are still needed to address the concern.  
There are mainly three methods, i.e., Western blot, ELISA, and qualitative and quantitative mass spectrometry (MS) used for the assessment of circulating irisin. Among those methods, MS with quantification peptides is recognized as the \"holy grail\" [23]. So far, the currently available reference values for circulating irisin in humans [24] and mice [20] were 3-4 ng/mL and 0.3 ng/mL, respectively.