The myokine irisin increases cortical bone mass

Abstract

It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity.

Keywords: mechanical loading; osteoporosis; sarcopenia.

Fig. 1.

Anabolic action of low-dose recombinant irisin (r-irisin) on cortical bone. (A) Contact radiographs of selected long bones from mice treated with vehicle or low-dose r-irisin (100 µg kg⁻¹ per week for 28 d, mice killed 24 h after last dose). Arrows indicate areas of increased radiodensity. Representative micro-CT–generated section images of cortical (midshaft, B) and trabecular (metaphyseal, C) bone in tibia harvested from vehicle- or irisin-injected mice. (D) Calculated cortical and trabecular parameters at the tibial midshaft and metaphysis of vehicle- or r-irisin–injected mice. Cortical bone parameters included tissue mineral density (TMD-Cortical), polar moment of inertia (pMOI), cortical bone surface (Ct.BS), cortical bone perimeter (Ct.Pm), total cross-sectional area (Tt.Area), marrow cross-sectional area (Marrow Area), and cortical thickness (Ct.Th). Trabecular bone parameters for tibial epiphyses included bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb Sp). (E) von Kossa-stained vertebral sections from vehicle- or irisin-injected mice, together with trabecular bone parameters as in D. Data are presented as mean ± SEM. Statistics: Unpaired Student’s t test, n = 5–7 mice per group. *P ≤ 0.05, or shown in D.

Fig. 2.

Low-dose r-irisin increases bone strength by stimulating bone formation. (A) Three-point bending test on tibia from mice treated with vehicle or low-dose r-irisin (100 µg kg⁻¹ per week for 28 d, mice killed 24 h after last dose). Bending strength and force-at-break are noted. (B) Dynamic histomorphometry on tibial sections following timed injections (2 and 7 d before sacrifice) of xylelol orange and calcein, respectively. Representative images are shown, together with calculated indices of bone formation, including mineralized surface/total bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR). Also shown are representative images of toluidine blue-stained osteoblasts (C) and tartrate-resistant acid phosphatase-stained osteoclasts (D) in tibial diaphyseal sections, together with cell counts (Ob and Oc, respectively) per bone perimeter (BPm). Data are presented as mean ± SEM. Statistics: Unpaired Student’s t test, n = 4–5 mice per group. P values as shown.

Fig. 3.

Low-dose r-irisin modulates osteoblast gene expression in vivo without causing a browning response. (A) Mice injected with low-dose r-irisin (100 µg kg⁻¹ per week for 28 d, mice killed 24 h after last dose) do not display a difference in tissue weight/body weight of interscapular brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT). (B) r-irisin injection also does not affect uncoupling protein-1 (Ucp1) mRNA expression in BAT or iWAT. (C) Photomicrographs of hematoxylin and eosin stained sections of iWAT from vehicle- and r-irisin–injected mice (magnification: 40×), showing no difference. (D) Pparγ and Atf4 mRNA expression (qPCR) in whole bone marrow isolated from tibiae of vehicle- or r-irisin–injected mice. (E) Spp1 and Sost mRNA expression (qPCR) in whole tibia (depleted of bone marrow) harvested from vehicle- or r-irisin–injected mice. (F) Fluorescent micrographs of metatarsal sections of vehicle- or r-irisin–injected mice immunolabeled for Spp1 (green; nuclei labeled blue; magnification: 20×). Spp1 positivity was measured as percentage of green fluorescence area/total bone area (Spp1/BS). Data are presented as mean ± SEM. Statistics: Unpaired Student’s t test, n = 4–5 mice per group. *P ≤ 0.05.

Fig. 4.

Recombinant irisin enhances osteoblast differentiation. Bone marrow stromal cells were grown in osteoblast differentiation medium in the presence of r-irisin (100 ng/mL). The percentage of alkaline phosphatase-positive colonies (10 d) (A) and von Kossa-positive mineralized colonies (21 d) (B) were calculated versus untreated cultures. Representative wells are shown. (C) mRNA expression levels of osteoblast marker genes (Bmp2, Spp1, Bglap, Runx2, Alp, Opg, Bmp4, Ibsp, Sp7, and Atf4) were assayed (qPCR) after 10-d culture with r-irisin. Osteoblast transcription regulators (Atf4, Runx2, and Sp7) and osteogenesis genes (Alp, Col1a1, Bglap, Ibsp, Spp1, Lrp5, Wnt7b, and β-catenin) were evaluated (qPCR) following culture with r-irisin for 3, 8 (D), or 48 h (E). (F) Western immunoblotting showing Erk phosphorylation (p-Erk) triggered by r-irisin (100 ng/mL) in osteoblast cultures. Data are presented as mean ± SEM. Statistics: Unpaired Student’s t test. *P ≤ 0.05; **P ≤ 0.01.

Fig. 5.

Irisin expression beyond muscle. (A) Fibronectin type III domain-containing protein-5 (Fndc5) mRNA levels in different tissues, namely muscle, iWAT (inguinal white adipose tissue), eWAT (epididymal white adipose tissue), BAT (brown adipose tissue), bone, brain, kidney, and liver (qPCR). (B) Immunofluorescence micrograph of muscle fibers from mice injected with vehicle or r-irisin (100 µg kg⁻¹ per week for 28 d) costained for Fndc5 (green) and dystrophin (red) (Magnification: 20×). Fndc5-positive fibers were quantitated as percentage of green fluorescent fibers/total fibers. (C) Effect of r-irisin on Fndc5 mRNA in C2C12 myoblasts (qPCR). Data are presented as mean ± SEM. Statistics: Unpaired Student’s t test, n = 4–5 mice per group. *P ≤ 0.05.

Fig. S1.

Specificity of the anti-irisin antibody (Abcam). Western immunoblot showing that the antibody not only recognized recombinant irisin (ng), but also, in C2C12 myoblast supernatants, its high-molecular-weight glycosylated form.