the benefits of exercise have been widely recognized , indeed the physical activity is reported as the better nonpharmacological treatment for cardiovascular , metabolic , and bone diseases [ 1 , 2 ] . however , for long time , the molecular mechanisms by which exercise exerts its healthful effects remained mostly unknown . a successful deal for
researchers and clinicians should be to reveal these mechanisms , encouraging practicing physical activity and promoting the development of exercise - mimetic drugs .
recently , several lines of evidence are suggesting that skeletal muscle is crucial in the regulation of energy homeostasis . therefore , the skeletal muscle is now considered an endocrine organ that secretes a number of myokines including the newly identified irisin . in this work ,
bostrm and colleagues have reported that physical exercise activity induces an increase of the transcriptional regulator peroxisome proliferator - activated receptor- coactivator 1 ( pgc-1 ) in the skeletal muscle , which in turn drives the production of the membrane protein fibronectin type iii domain - containing protein 5 ( fndc5 ) .
this is subsequently cleaved as the myokine irisin , which acts on white adipose tissue ( wat ) , stimulating uncoupling protein 1 ( ucp1 ) expression , one of the master genes of brown adipose tissue ( bat ) , and activating the browning response .
the authors showed that , after 3 weeks of free wheel running , plasma irisin levels in mice were increased by 65% and , in healthy humans , plasma irisin levels were found to double after 10 weeks of endurance exercise .
these results opened new frontiers for searching the involvement of irisin in a broader network , suggesting the intriguing feasibility that this myokine might represent an endocrine molecule that could target other organs besides the adipose tissue .
indeed , the hypothesis that muscle supports bone mass is confirmed by studies of microgravity and bed rest , as well as results showing the associated development of sarcopenia and osteoporosis at the same time .
for instance , the exercise strengthens bones , given the evidence that tennis players develop high bone mass in the playing arm compared to the nonplaying arm .
on the other hand , the absence of physical exercise or , even worse , the complete disuse of muscles , ensuing by severe pathological conditions , leads to a likewise severe bone loss .
for instance , child growing with congenital neuromuscular diseases develop fragile long bones with reduced periosteal circumference .
furthermore , long bones in a paralyzed limb do not achieve their normal ossification and show delay of mineralization in newly laid - down bone matrix .
consequently , this paralytic phenotype , associated with decreased bone mass , is prone to severe fractures as frequently occurs in progressive disease such as spina bifida .
moreover , the propensity of sarcopenic patients to falls can often degenerate into an osteoporotic hip fracture .
this event may reduce the life expectancy of up to two years and this corresponds to increased mortality of 2025% in the first year after the fracture .
for this reason , the onset of simultaneous sarcopenia and osteoporosis , also described as  the hazardous duet ,  has been defined as one of the most devastating threats during old age . although this tight relationship between skeletal muscle and bone is well recognized , it has hitherto been mainly explained concerning mechanical loading , as overall effect .
therefore , the description of bone response to mechanical load is currently defined as the ability of bone cells to perceive paracrine signals produced by mechanical stimulus .
hence , it could be extremely useful to deepen the understanding of the molecular mechanism involved , revealing the identity of all these signalling - paracrine molecules able to affect bone metabolism .
for this reason , we propose a model where exercise induces muscle to release myokines , which regulate mechanotransduction in bone , basing on the physical proximity of these two tissues .
noteworthy , we postulated that the protective effect of muscles on bone could be dependent on the paracrine action of the myokine irisin . to validate the potential role of irisin on bone metabolism
, we investigated whether irisin targets bone cells directly , demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts .
the relevance of these findings opens new frontiers in searching the irisin mechanism of action on bone metabolism .
the in vivo data , confidently obtained in future , could further correlate the well - known beneficial effects of physical exercise with bone recovery and improvements .
antibody anti - fndc5 ( irisin cleaved form ) was from abcam ; antibody anti - collagen i and -actin were from santa cruz , antibody anti-  -tubulin was from origene technologies .
ascorbic acid , b - glicerophosphate and alkaline phosphatase ( alp ) staining kit were from sigma aldrich . primers for qpcr are alp / s - aaacccagacacaagcattcc ; alp / as - tccaccagcaagaagaagcc ; coll i / s - ggctcctgctcctcttag ; coll i / as - acagtccagttcttcattgc .
2-month - old c57bl/6 male mice were subjected to 3 weeks of rest activity or free wheel running activity , as described previously .
the rest activity was performed isolating each mouse in one cage , in order to avoid their tendency to fight with cage mates , preventing any exercise - mimetic activity .
the wheel mice were individually housed , in order to avoid that the dominant mouse in the cage inhibited other mice in the free use of wheel .
primary myoblasts were obtained from digestion of vastus lateralis specimens with a solution of trypsin , collagenase , and cacl2 .
the isolated cells were preplated on an uncoated petri dish for 1 hour to remove fibroblasts and then transferred on tissue culture plate and cultured with -mem/10% fcs .
cells were then cultured for 14 days until multinucleated , spontaneously contracting myotubes were formed .
then , cm were purified by centrifugation at 13  k  rmp to eliminate debris . bone marrow stromal cells , obtained by flushing bone marrow of 2-month - old c57bl/6 mice , were cultured to induce osteoblast differentiation with -mem/5% fcs in the presence of 50  g / ml ascorbic acid and 10  m -glycerophosphate or with 1/2  cm from primary myoblast + 1/2-mem/10% fcs in the presence of 50  g / ml ascorbic acid and 10  m -glycerophosphate .
qpcr was carried out after rna extraction using spin columns ( rnasy , qiagen ) according to the manufacturer 's instruction . by using superscript first - strand synthesis system kit ( invitrogen ) , the resulting cdna ( 20  ng ) was subjected to quantitative pcr and , thereafter , to itaq sybr green supermix with rox kit ( bio - rad ) on an icycler iq5 cromo4 ( biorad ) .
each transcript was assayed 3 times , and cdna was normalized to murine gapdh , 18s or -actin and quantitative measures were obtained using the ct method .
analyses were performed using unpaired student 's t - tests ( excel ) for significant differences at p < 0.05 .
protein amounts from all samples were assessed using the bca - kit ( biorad ) followed by protein concentration normalization before all western blot experiments .
the blots were probed using primary antibodies , described in materials section , and irdye - labeled secondary antibodies ( 680/800  cw ) ( li - cor biosciences ) . for immunodetection ,
therefore , we confirmed the effects of exercise on fndc5 expression in our exercise regimen , based on 3 weeks of voluntary free wheel running . by qpcr analysis , we detected a 2-fold increase in fndc5 mrna of wheel myoblasts ( figure 1(a ) ) .
this result was confirmed by the analysis of fndc5/irisin protein expression ( figure 1(b ) ) .
indeed , we were able to detect a stronger band , corresponding to fndc5/irisin , in cell lysates of myoblasts from wheel mice compared with those from rest mice ( figure 1(b ) ) .
these data are , according to previous observations , showing an increase of about 65% in muscle of mice subjected to three weeks of voluntary exercise .
it should be further noted that fndc5/irisin is slightly detectable also in rest myoblasts , suggesting a constitutive expression of this myokine even in nonexercised state that might be related to a basal metabolism . in the last years , accumulating evidences have shown that skeletal muscle release hormone - like substances .
. our model of primary murine skeletal muscle cells allowed us to recapitulate in vitro what occurs in vivo when muscle is subjected to exercise and releases these circulating myokines . the conditioned medium ( cm ) collected from these myoblasts , which likely contained several released myokines , was used to evaluate its ability in regulating maturation of undifferentiated bone marrow stromal cells toward osteoblast differentiation .
our result shows that the cm obtained from wheel myoblasts increased by 2.5-fold the number of alkaline phosphatase ( alp ) positive colonies compared to control medium ( figure 2 ) .
this assay , based on a histochemical staining for alp , is the first evidence of osteoblast differentiation , since the alp enzyme is established as the osteoblastogenesis relevant marker .
the result suggests , for the first time , that muscles could exert a direct anabolic effect on osteoblasts through the paracrine action of released myokines , rather than the solely mechanotransduction action on osteocytes , the mechanosensor cells of bone .
given the ability of cm from wheel myoblasts to enhance osteoblast differentiation , we investigated which bone proteins were upregulated .
by qpcr analysis , we demonstrated that osteoblasts treated for 3 days with cm from wheel myoblasts have an increased expression of alp and collagen i mrna ( figure 3 ) .
these data , further confirming the previously shown increased number of alp positive colonies ( figure 2 ) , is supported by an enhanced expression of alp mrna , the marker gene of osteoblasts
. moreover , the upregulation of collagen i , the most abundant bone protein , greatly corroborates the beneficial effect on osteoblasts exerted by molecules released from the exercised muscle .
subsequently , our effort has been to obtain evidence about the involved myokine , present in cm , responsible for such a great effect on osteoblast differentiation .
given the increased expression of fndc5/irisin seen in myoblasts from wheel mice ( figures 1(a ) and 1(b ) ) , we choose irisin as candidate myokine .
for this challenge , we cultured osteoblasts with cm from myoblasts in presence of a neutralizing antibody direct against irisin .
we showed that the increase in collagen i and alp was completely reversed by neutralizing irisin in wheel cm used to treat osteoblasts ( figures 4(a ) and 4(b ) ) .
this finding demonstrated that the enhanced osteoblastogenesis , induced by exercised muscle , is irisin - dependent .
it should be noted that the molecular weight of the secreted form of fndc5/irisin has remained for long time controversial .
now it is well ascertained that the sequence of mouse fndc5 is cleaved from aa 29 to aa 151 to give its released form , as irisin .
being aware of this , we used an anti - fndc5/irisin antibody ( amino acids 50150 from abcam ) directed against the predicted irisin cleaved form .
bostrm and colleagues have recently reported that physical exercise activity induces the release , from skeletal muscle to bloodstream , of the myokine irisin which was so called ( from iris , the messenger goddess ) to highlight its role as positive messenger which targets an endocrine signal from skeletal muscle to adipose tissue ( wat ) .
irisin induces browning of wat ( i.e. , conversion from wat to bat ) , that is a well - known new avenue for its great therapeutic potential in diabetes and obesity . in the present study
, we show that mature exercised primary myoblasts and myotubes secrete factor(s ) , which increase osteoblast differentiation in vitro .
we also establish that this enhanced osteoblastogenesis , induced by exercised muscle , is mediated through irisin .
these results presented here add new insights in the complex relationship between muscle and bone tissue , indeed the mechanical influence of skeletal muscle on bone has long been documented but the molecular mechanisms involved remain still poorly understood . skeletal muscle and bone
are tightly connected : they have a common origin , share the same integrated system that provides shape and physical function , and display significant changes across the lifespan . in elderly ,
the severe decline of skeletal muscle function , known as sarcopenia , is associated with impaired function of bone ( osteopenia ) .
these two simultaneous losses of function lead to increased risk of falls and bone fractures .
therefore , developing a better understanding of the complex relationship between these important components of the musculoskeletal system may reveal new strategies for early identification , prevention , and treatment of sarcopenia and osteopenia , as well as their consequences [ 2 , 8 , 17 , 18 ] .
currently , the most effective measure to counteract both diseases is exercise , but not all patients can perform a physical exercise program ; thus , our evidence that exercise - induced irisin could account for this effect greatly improves the chances of achieving this goal .
we show here that irisin directly targets osteoblasts , enhancing their differentiation in vitro , proving that myokines , produced by exercised muscle , might be among the molecules regulating mechanotransduction in bone . in our system
, mice subjected to three weeks of voluntary exercise showed an increased expression of irisin / fndc5 in skeletal muscles .
this result confirms previously published data demonstrating that endurance exercise training for 10 weeks increased plasma irisin levels in healthy adult .
conversely , timmons et al . were not able to confirm fndc5 gene activation by aerobic exercise in younger subjects .
these discrepancies have been explained by another study , which demonstrated that irisin levels increase only when more energy is needed , such as in circumstances where atp concentration in muscle is strongly decreased .
moreover , we achieved evidence that conditioned medium from primary culture of myoblasts and myotubes , obtained from exercised muscles , were able to enhance the number of alkaline phosphatase ( alp ) positive colonies in culture of undifferentiated bone marrow stromal cells .
noteworthy , this is the first study showing the osteogenic potential of irisin released from exercised skeletal muscle . from a physiological point of view , this result adds another explanation of the tightly connection between muscle and bone , which share a common fate even in positive scenarios , like in this concomitant gain of mass .
the effects of physical exercise are systemic and , obviously , can not be solely related to the energy expenditure in muscle .
reported a new mechanism that explains how the total body energy expenditure is increased by muscle activity , elucidating the molecular circuit triggered by exercised muscle .
analysis of subcutaneous fat tissue depots , in mice overexpressing the muscle - specific pgc-1 , revealed that white adipocytes displayed signatures of brown fat cells . delineating muscle genes activated by pgc-1
, authors identified the myokine irisin , able to drive the white - to - brown adipocyte transdifferentiation [ 3 , 22 ] . by considering the tight relationship between skeletal metabolism and energy homeostasis
, clinical and experimental results are giving great importance to the role of bat on bone metabolism .
due to the evidence that an inducible form of bat exists during adulthood and given the importance of its ability to dissipate the stored energy with thermogenesis [ 2326 ] , bat induction is becoming a significant promise for the treatment of obesity and metabolic syndrome .
a cross - sectional study , carried on 15 young women , has shown a positive correlation between the amount of bat and bone mineral density ( bmd ) .
data derived from experimental mice model showed that , foxc2(ad)(+/tg ) mice , overexpressing foxc2 as well - established model for induction of bat have high bone mass due to increased bone formation associated with high bone turnover . on the contrary
, mice named misty ( m / m ) , carrying a very low amount of bat , albeit partially functional , have accelerated age - related trabecular bone loss and impaired brown fat function , such as reduced temperature and lower expression of pgc-1 .
this growing body of evidences suggests a functional fat - bone axis , but the discovery of irisin , together with our new findings , add a new protagonist to this axis , allowing enlarging it as the muscle - fat - bone axis ( figure 5 ) .
based on the scenario described , in which both muscle and bat affect bone metabolism , it might be questioning whether the effect of physical activity on the skeleton could also be mediated by bat , which in turn has been affected by irisin .
this would imply double , direct and indirect , irisin action on bone in vivo . in our hands the irisin - dependent action on osteoblasts
is further supported by the increase of alp and collagen i expression , observed in osteoblasts cultured in the presence of cm from muscle cells of wheel mice .
the irisin involvement was proved by the fact that the cm - induced upregulation of alp and collagen i was abolished when cells were treated with cm containing anti - irisin antibody .
this suggests that irisin does not target only adipocytes but also other body compartments , according to recent data demonstrating that irisin could play a role in the central nervous system . in this context , recent studies revealed that cerebellar purkinje cells of rat and mice express irisin , which is also required for the proper neural differentiation of mouse embryonic stem cells .
hence , given that physical activity improves neurogenesis , by reducing risk of neurodegenerative diseases such as alzheimer and parkinson [ 33 , 34 ] , irisin could represent the molecular link between exercise and healthy brain .
in conclusion , we showed a novel role of irisin , adding new evidence to the complex muscle - fat - bone axis ( figure 5 ) .
this seems remarkably promising , considering the aforementioned tight relationships between skeletal muscle and bone .
our future efforts will focus on a deepen analysis of the molecular signalling triggered by its action and , in particular , on the overall effect of irisin in the bone context .
moreover , the characterization of its receptor will allow a more clear understanding of irisin - induced signalling . in this respect , we would also elucidate whether this myokine affects osteoclasts , the bone resorbing cells .
this might better explain the global regulation of skeletal homeostasis exerted by physical exercise or , conversely , by lack of mechanical loading .
future studies could reveal whether expectations on the potential role of irisin as pharmacological treatment will be confirmed .
hopefully , with regard to the skeleton , irisin could represent a new anabolic therapy to gain bone mass in osteopenia caused by muscle - disabling diseases , such as sarcopenia , tumor - associated cachexia , neuromuscular disease , or situations with forced lack of mechanical loading , such as absence of gravity which astronauts undergo .
 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions