Irisin enhances osteoblast differentiation in vitro

Abstract

It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM) collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

Figure 1

qPCR analysis of FNDC5 in mRNA extracts (a) and western blot analysis of Irisin/FNDC5 in total cell lysates (b) from primary culture of myoblasts obtained from mice subjected to 3 weeks of rest activity or free wheel running activity. N = 8 for each group, repeated in 3 separate experiments. Data is presented as mean ± SEM.  **P < 0.001 and ^∧ P < 0.05 compared to rest group. Student's t-test was used for single comparison.

Figure 2

Histochemical staining for ALP in osteoblasts primary culture obtained from mouse bone marrow stromal cells treated with α-MEM/5% FCS in the presence of 50 μg/mL ascorbic acid and 10⁻² M β-glycerophosphate (CTR) or with 1/2 CM from primary myoblast (rest or wheel) +1/2α-MEM/10% FCS in the presence of 50 μg/mL ascorbic acid and 10⁻² M β-glycerophosphate. The graph shows quantification of ALP positive colonies as percentage (*P < 0.01) compared to control and is representative for 3 independent experiments. Data is presented as mean ± SEM. Student's t-test was used for single comparisons.

Figure 3

qPCRanalysis of ALP and Collagen I in mRNA extracts from osteoblasts treated with conditioned medium (CM) of myoblasts from rest and wheel mice. N = 8 for each group, repeated in 3 separate experiments. Data is presented as mean ± SEM. *P < 0.01 compared to rest CM. Student's t-test was used for single comparisons.

Figure 4

Western blot analysis of Collagen I in total cells lysates (a) and qPCR analysis of ALP in mRNA extracts (b) from osteoblasts treated with conditioned medium (CM) of myoblasts from rest and wheel mice ± Irisin/FNDC5 neutralizing antibody. The graph (a) shows quantification of OD ratio Collagen I/β -Tubulin as percentage (*P < 0,01) compared to rest CM and is representative for 3 independent experiments. Data is presented as mean ± SEM. Student's t-test was used for single comparisons.

Figure 5

The muscle-fat-bone axis. It is been ascertained that a local network exists between the adipose and the bone tissue, which creates what has been defined as the fat-bone axis. In this paracrine circuit, fat influences bone both positively and negatively by secreting Leptin [11] and Adiponectin [12], respectively. Recently, it has been emphasized the function of brown adipocytes which also affect bone tissue byproducing factors that may be secreted to circulation or act directly in the bone marrow environment to induce osteoblast differentiation and osteocyte support for bone formation and bone turnover. Two of these factors, insulin-like growth factor binding protein 2 (IGFBP2) and wingless related MMTV integration site 10b (WNT10b), gather considerable interest because they regulate both bone remodelling and energy metabolism [13]. Moreover, beside its classical functions, bone acts in turn as endocrine organ secreting Osteocalcin, a hormone active on glucose and fat metabolism, stimulating insulin secretion and β-cell proliferation [14]. Of further significance, the discovering of Irisin, which is released from muscle, acts as endocrine molecule targeting adipose tissue by increasing energy expenditure [3] and bone by enhancing osteoblast differentiation. As shown in this work, Irisin is a new protagonist of the axis, which now could be considered as the muscle-fat-bone axis.