Irisin is elevated in skeletal muscle and serum of mice immediately after acute exercise

Abstract

Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.

Keywords: Fndc5; Ppargc1a.; endurance exercise; irisin; myokine.

Figure 1

Detection of irisin in mouse serum and muscle. (A) Western Blot of each 2 mouse serum (lane 2 and 3) and muscle samples (lane 4 and 5) and recombinant irisin (lane 6) incubated with either (left) antibody directed against irisin (aa 32-143) or (right) antibody against irisin previously blocked with the recombinant protein. A specific band was detected at approximately ~12 kDa in serum and muscle tissue as indicated by the white frame. Images were identically enhanced in contrast. (B) Irisin band of mouse serum (left) without or (right) with PNGase treatment indicating that circulating irisin was partly glycosylated. Images were identically enhanced in contrast and cropped to the specific ~12 kDa band.

Figure 2

Protein abundance of irisin in (A) serum and (B) muscle tissue of mice after 3 weeks of voluntary exercise in a running wheel (serum and FM: n = 12, CM: n = 8) or one bout of treadmill exercise (serum, FM, and CM: n = 12) relative to a sedentary control (serum and FM: n = 11, CM: n = 8). Representative parts of the respective western blots for determination of irisin protein expression are shown above the graphs. Different letters indicate significant differences between groups (P < 0.05). CO - control group, RW - running wheel group, TM - treadmill group.

Figure 3

Abundance of Fndc5 mRNA and protein in femoral (FM) and crus muscle (CM) tissue of mice after 3 weeks of voluntary exercise in a running wheel (FM: n = 12, CM: n = 8) or one bout of treadmill exercise (FM and CM: n = 12) relative to a sedentary control (FM: n = 11, CM: n = 8). (A) mRNA abundance normalized to B2m and Hprt1. Bars represent means of fold changes compared to control group with 95 % confidence intervals marked as vertical lines. (B) Western blot of each 2 mouse serum (lane 2 and 3) and muscle samples (lane 4 and 5) incubated with either (left) antibody directed against Fndc5 (aa 149-178) or (right) the antibody against Fndc5 previously blocked with the corresponding blocking peptide. The marker lane is laid on the image to determine the protein size. A specific band was detected at ~25 kDa exclusively in muscle tissue as indicated by the white frame. Images were identically enhanced in contrast. (C) Protein abundance of Fndc5 determined by quantitative western blot analysis. Representative parts of respective western blots are shown above the graph. CO - control group, RW - running wheel group, TM - treadmill group.

Figure 4

Cellular localization of (A) Fndc5 and (B) irisin in muscle cross sections of rectus femoris of a treadmill exercised mouse. Cryo-sections were immunostained with anti-Fndc5 and irisin primary antibodies and a MFP488 labeled goat anti-rabbit IgG secondary antibody. Fndc5 immunoreactivity was detected at the muscle fiber membrane (A, arrowheads) and punctuate in the cytoplasm, as well as in additional cells in the connective tissue (A, arrows). Irisin was mainly located in the intercellular space (B, arrows).

Figure 5

Relative mRNA expression of Ppargc1a transcripts 1, 3, and 4 in femoral (A) and crus (B) muscle tissue of mice after 3 weeks of voluntary exercise in a running wheel (n = 8) or one bout of treadmill exercise (n = 12) compared to a sedentary control (n = 8). The values were normalized to B2m and Hprt1. Bars represent means of fold changes compared to control group with 95 % confidence intervals marked as vertical lines. Asterisks indicate significant differences to control (*P < 0.05, **P < 0.01). (C) Relationship between Ppargc1a4 mRNA abundance, normalized to Hprt1 (2^-ΔCp), in femoral and crus muscles of mice and running distance during one bout of treadmill exercise (r = 0.63 and 0.69, respectively, P < 0.05).

Figure 6

Protein abundance of Ppargc1a4 in femoral (FM) and crus muscle (CM) tissue of mice after 3 weeks of voluntary exercise in a running wheel (FM: n = 12, CM: n = 8) or one bout of treadmill exercise (FM and CM: n = 12) relative to a sedentary control (FM: n = 11, CM: n = 8). Representative parts of respective western blots are shown above the graph. CO - control group, RW - running wheel group, TM - treadmill group.