classical understanding of skeleton is that bone functions as the structure scaffold in animals and contains three types of cells : osteoblast , osteocyte , and osteoclast .
osteoblasts come from mesenchymal stem cells and can differentiate to osteocytes , which stay in the bone matrix .
osteoblasts are responsible for bone formation , and its activity can be regulated by several pathways such as wnt signaling pathway .
osteoclasts are a type of bone cells , which resorb bone and derived from the fusion of mononuclear cells belonging to the monocyte / macrophage lineage .
osteocytes and osteoblasts can secrete some signaling factors , such as wnt , opg , and sost , in order to regulate the coupling function of osteoblast and osteoclast ; in this way bone obtains the ability to model and remodel itself .
recent reports indicate that sost , a wnt antagonist , downregulates the wnt signaling activity by crosstalk with parathyroid hormone ( pth ) and bone morphogenetic protein ( bmp ) in order to regulate bone cell differentiation , proliferation , bone formation , and bone resorption activities [ 14 ] . even though there are plenty of evidences indicating that bone can secrete protein factors in the circulation and regulate itself ,
all these secreted factors can not be called hormones . however , in the past decades with the development of technology we found that skeleton can also be an endocrine organ , which regulates not only itself but also other organs . the most well known and studied two hormones secreted by skeleton are ocn and fibroblast growth factor 23 ( fgf23 )   ( figure 1 ) . in this review
we would focus on ocn , especially on its function as a hormone in metabolism homeostasis .
ocn is specially synthesized and secreted by cells of osteoblast lineage , such as osteoblast and osteocyte [ 6 , 7 ] . in humans
ocn is also known as bone -carboxyglutamic acid - containing protein ( bgp ) and it is one of the richest noncollagenous proteins in bone matrix .
the most of ocn is found in bone matrix and only a small amount in blood , because it has strong affinity to bone matrix .
the ocn gene has 4 exons and 3 introns , and more than 70% of the exon sequences are conserved both in human and in mice .
the ocn protein contains 4650 amino acid residues ( varying from different species ) , the synthesis process of which is a little complicated ( figure 2 ) .
after the translation it is only preproosteocalcin , which contains 98 amino acid residues composed of three parts : a 23-residue signal protein , a 26-residue target propeptide , and a 49-residue mature protein .
proteolysis of the prepropeptide will form the mature ocn . at position 17 , 21 and 24 of the 49-residue peptide second carboxyl groups ( cooh )
are added to form -carboxyglutamyl residues , which are essential for its activity regulation . according to the carboxylation level
, ocn can be divided into two groups ; one is called undercarboxylated osteocalcin ( ucocn ) , and the other is carboxylated osteocalcin ( cocn ) .
the decarboxylated form ucocn is the active form of ocn as a hormone , which has little affinity to bone so that most of ucocn will circulate with the blood , while most of cocn will be stored in the bone matrix because of its strong affinity to bone matrix .
usually the concentration of ucocn is not controlled by the protein synthesis , but by the decarboxylation of ocn and its releasing from the bone matrix .
there are evidences indicating that in ocn knocked out mice the total amount of insulin in serum is downregulated , and the ocn knocked out mice have so - called impaired glucose - stimulated insulin secretion ( gsis ) and poor glucose tolerance phenotype . at the same time the serum adiponectin , a protein hormone that modulates several metabolic processes such as glucose regulation and fatty acid oxidation , was also reduced .
all these results suggest that ocn may target  cells and insulin targeting tissue such as muscle , liver , and adipocyte in order to regulate both insulin secretion and sensitivity [ 11 , 12 ] .
later studies show that the change of insulin sensitivity is probably mediated by adiponectin instead of direct interaction with ocn .
this result indicates that adipocytes are probably one target of ucocn , and ucocn functions as the activity form of ocn in circulation .
there are also many evidences indicating that ucocn can regulate  cells in pancreatic islets .
not only insulin 1 and insulin 2 can be upregulated by ucocn but also the proliferation of  cells .
further research indicates that cyclind1 , cyclind2 , and cdk4 in  cells can also be regulated by ucocn , and in this way it is not difficult to understand why ucocn can stimulate  cell proliferation [ 9 , 12 , 14 ] .
 cells proliferation and insulin secretion can be significantly affected at low concentrations of ucocn ( 660  pm ) .
the mice lacking osteoblasts have not only problems on bone density and strength but also impaired glucose metabolism , such as high blood glucose , low insulin secretion , and insulin resistance .
. however , administration of ocn can restore glucose and insulin level in circulation but only partially insulin sensitivity .
although more and more evidences show that ocn can regulate  cells and increase the insulin sensitivity of insulin targeting cells , the receptor of ocn or more correctly ucocn has not been identified yet .
what is more , we do not even know how ocn regulates the insulin sensitivity .
there are only evidences indicating adiponectin mediates the insulin sensitivity regulation of ocn . whether some other mechanisms involved in this progress is still not clear .
some in vitro studies indicated that cocn could regulate bone formation and resorption ; however , in the ocn knocked out mice no negative bone effects were observed [ 1719 ] .
these were observed from the experimental studies on esp and ocn knocked out mice . with or without ocn ,
mice with high ocn activity ( the esp knocked out mice ) had increased testicular volume and sperm count . on the contrary , the ocn absent mice ( the ocn knocked out mice ) showed shriveled testes , epididymis , and seminal vesicles . however , the female in both esp and ocn knocked out groups did not show any defects in reproduction .
this indicates that ocn secreted by skeleton regulates male reproduction activity as a hormone . and
it is a g protein - coupled receptor ( gpcr6a ) and localized on the surface of the leydig cell . this 7-transmembrane protein
was found because the gpcr6a knocked out mice copy the metabolic syndrome of the ocn knocked out mice [ 21 , 22 ] . in the past decades
it was demonstrated that gonadal hormone ( estrogen ) can regulate bone formation [ 23 , 24 ] , and now we know that bone can also regulate testicle and testosterone secretion .
this discovery suggested that there may be a novel mechanism regulating bone formation in some special stage , such as teenage time , in which rapid bone formation is associated with androgen secretion changing .
in general , ocn , particularly ucocn ( released during bone resorption ) targets directly  cells and stimulates insulin synthesis and secretion . at the same time it affects adipocytes and enhances adiponectin secretion .
interestingly , ocn also targets leydig cells and regulates testosterone production and male reproductive activity ( figure 3 ) .
vitamin d is a kind of fat - soluble secosteroids which plays an essential role in absorption of calcium , phosphorus , iron , zinc , and their metabolism . in humans and mice
recent studies showed that the ocn expression in osteoblasts can be partly stimulated by vitamin d3 .
more interestingly fibroblast growth factor 23 ( fgf23 ) , another bone secreted hormone , can affect kidney and inhibit vitamin d synthesis .
this loop suggested a feedback loop , in which bone can partly regulate its own hormone secretion .
studies showed that accumulation of osteocalcin in the ecm of human osteoblastic cultures stimulated by 1,25-dihydroxyvitamin d3 is inhibited by warfarin ( antagonist of vitamin k ) , while vitamin k2 enhanced the 1,25-dihydroxyvitamin d3 effect , and 1,25-dihydroxyvitamin d3 stimulated mineralization was significantly augmented by warfarin .
the biological active form of ocn is ucocn , while the cocn has a strong affinity to bone matrix and binds tightly to hydroxyapatite , as a result little cocn could be found in the serum .
vitamin k is a cofactor for the glutamate carboxylase , which is required for carboxylation . in the absence of vitamin
, high level of vitamin k can reduce ucocn level , which had been proved by vitamin k administration in daily diet . generally speaking , there are two ways controlling ocn signaling , one is on the transcription level and the other is on the posttranslational level .
the product of esp is an osteotesticular protein tyrosine phosphatase ( ost - ptp ) , which is found in embryonic cells , sertoli cells , and osteoblast .
karsenty et al . found esp knocked out mice have the opposite phenotype compared with the ocn knocked out group ( table 1 ) .
the phenotypes of the esp knocked out mice are as follows : hypoglycemia , high glucose - stimulated insulin secretion , glucose tolerance , pancreatic insulin content , and  cell proliferation .
esp deletion ( either totally knocked out or selectively knocked out in osteoblasts ) resulted in the exact opposite phenotype as found in the ocn knocked out mice . and
esp can rescue the phenotype of the high fat diet and gold thioglucose - induced obesity and diabetes , which mimics the phenotype of ucocn treated mice [ 9 , 12 ] .
indeed lacking one allele of ocn in the esp knocked out mice rescued the metabolic abnormality of the esp absent mice .
as the total ocn gene expression did not change in esp knocked out mice , ferron et al . suggested that ost - ptp was involved in the osteoclast - dependent ucocn formation and releasing .
in the recent years the molecular mechanism of esp regulation was revealed ( figure 4 ) .
two transcription factors , forkhead box protein o1 ( foxo1 ) and cyclic amp - dependent transcription factor ( atf-4 ) , are involved in the regulation of the total ocn .
foxo1 is a conservative modulator of glucose metabolism in many organs , as well as insulin synthesis in  cells , and atf4 regulates expression of many genes which mostly are important for adult bone formation and development [ 33 , 34 ] .
osteoblast specific foxo1 knocked out mice have low blood glucose level and high glucose tolerance , which is similar to the mice with high ucocn level .
molecular experimental studies showed that foxo1 could bind to a cognate foxo1 binding site in the esp promoter area and stimulate the expression of the downstream esp ; then ost - ptp would activate the carboxylation of ocn or ucocn .
the new formed cocn would bind to hydroxyapatite and stay in bone matrix , and as a result the ucocn would be downregulated .
now it is not surprising that atf4 can suppress obesity in mice and involve in the glucose homeostasis in human [ 3638 ] .
in fact even though atf4 is mainly expressed in osteoblast , it functions quite similar to foxo1 . via stimulating ost - ptp transcription atf4 suppress the activity of ocn , in this way suppressing insulin secretion and insulin sensitivity [ 39 , 40 ] .
more details of ocn regulation are still unknown , and the endocrine regulation of ocn will be discussed in the later section of this review .
as mentioned in the sections above , in the ocn knocked out mice the blood glucose concentration was higher than that of the wild type .
serum insulin in the knocked out mice would be downregulated and the mice tended to be obesity . all these indicate that ocn may be essential in energy metabolism , especially in glucose metabolism .
with administration of ocn or deletion of esp ( ost - ptp increases ucocn , the active form of ocn ) , the mice achieved higher activity of insulin , lower blood glucose , higher glucose tolerance , and higher insulin sensitivity with normal glucagon levels .
this abnormality is mainly caused by the increased number or mass of pancreatic islets and  cells .
this means that ocn can increase insulin secretion and insulin sensitivity in some tissues such as muscles and adipose tissue .
the increasing sensitivity of insulin is probably mediated by adiponectin , which can be upregulated by ucocn [ 9 , 12 , 41 ] .
cocn would be stored in the bone matrix and released during bone resorption [ 10 , 42 ] .
osteoclasts would secret protons and decrease the ph value during resorption ; this would facilitate the decarboxylation of cocn and by the way the active form ucocn would enter the circulation to stimulate  cells proliferation and insulin secretion [ 31 , 43 ] . however , more details of the mechanism need to be revealed .
insulin , synthesized and modified in  cells in pancreas , is an essential protein hormone in almost all vertebrates . through clinical observation
insulin receptor ( insr ) is required for osteoblast survival , proliferation , and differentiation [ 44 , 45 ] .
studies showed that the expression of runx2 was decreased in osteoblasts lacking insr [ 45 , 46 ] .
instead , insulin downregulates twist2 , an inhibitor of runx2 activity , to suppress the activity of runx2 .
insulin signaling can decrease osteoprotegerin ( opg ) expression in osteoblasts via binding to insr .
opg , a key regulator of osteoclasts , together with receptor activator of nuclear factor- ligand ( rankl ) via receptor activator of nuclear factor- ( rnak ) regulates the differentiation and activity of osteoclasts [ 2 , 47 , 48 ] . during bone resorption
in turn ucocn increases  cells proliferation , insulin secretion , and insulin sensitivity [ 16 , 49 ] .
thus , insulin signaling and ocn form a feed - forward loop , in which insulin affects osteoblasts and in turn increases its own secretion and sensitivity via ucocn .
insr is downstream molecule of ost - ptp , which inhibits phosphorylation of insr and stops insulin signaling in osteoblasts [ 50 , 51 ] .
this feed - forward model explains why the osteoblastic insr knocked out mice have low serum insulin level and high insulin resistance . as mentioned above
, the relationship between ocn and insulin is not a feedback relationship , so there must be some other signaling pathways to regulate the ost - ptp activity . if not , the serum insulin concentration can not stay in a certain level with only the feed - forward loop between bone and pancreatic islets .
some studies suggested that sympathetic nervous system ( sns ) may control the expression of esp via leptin , a hormone secreted by adipocytes .
the main function of leptin is regulating the amount of fat stored in the body .
it inactivates the 5-hydroxylase 2 ( tph2 ) , which is known as the rate - limiting enzyme of serotonin synthesis .
tph2 knocked out mice have osteoporotic and anorectic phenotype , indicating this source of serotonin can regulate osteoblasts .
have proved that leptin - dependent regulation of ocn carboxylation appeared to be through the hypothalamus [ 53 , 54 ] .
the sns acts through 2-adrenergic receptors ( adrb2 ) in osteoblasts to upregulate the esp expression , and in so doing the activity of ucocn would be suppressed .
the esp gene in human is a so - called pseudogene which does not have a functional product .
years ago a study showed that in human dephosphorylation of insr can be achieved by protein - tyrosine phosphatase 1b ( ptp1b ) instead of ost - ptp .
so it seems that the mechanism of ocn regulation in human is different from that in mice . in human the concentration of ocn
will arrive at its peak in the early morning and its lowest point in the afternoon , while the mice will have a high level of ocn in the day and the level of ocn reaches its lowest point at night during sleep .
in the older studies the researchers could only test the concentration of total ocn , now there are several ways to test the level of ucocn , such as ucocn specific elisa and hydroxyapatite ( hap ) binding assay [ 19 , 57 ] . with these technological development
, recent studies showed that the ratio of ucocn / ocn usually did not change , while the total amount of ucocn in circulation had the similar changes as the circadian rhythm of ocn [ 8 , 5862 ] . in clinical studies
the data were mostly focused on the effects of ocn on bone formation and metabolism .
children and adolescents , who have the need to increase bone formation , usually have higher levels of ocn and ucocn and this may facilitate the utilization of glucose in bone .
clinical studies also showed that bone loss was associated with high level of ucocn in human , and in older people high ocn level predicted increased fracture risk or lower bone density [ 64 , 65 ] .
the phenomenon can be explained by studies in mice that bone resorption facilitates decarboxylation of ocn and releases ocn from bone matrix to the blood .
however , whether ocn has effects on skeleton or not is still not clear ; the ocn knocked out mice seem quite normal .
the minor differences between the knocked out and wild type mice suggest that ocn may play some roles in regulation of bone mineralization . only in recent years many studies are focused on the hormone functions of ucocn and ocn in energy metabolism .
patients who suffer from type 1 or type 2 diabetes mellitus have lower level of ocn than healthy people [ 6669 ] . patients with obesity also show lower level of ocn [ 70 , 71 ] .
however , studies showed in male patients with type 2 diabetes adiponectin was associated with ucocn / ocn ratio instead of the concentration of ucocn , while in female patients it was correlated with ocn , but not ucocn .
it is now very clear that ocn regulates testicular function and does not have such function on female gonad . whether it has something to do with the differences between male and female diabetic patients is still unknown
anyway , more studies need to be done to reveal the mechanism of this phenomenon .
studies in animal models showed that ucocn targets  cells in the pancreas to directly regulate insulin synthesis and regulates insulin sensitivity through adiponectin .
and experiments in mice showed that ucocn may be a potential therapy for diabetic patients .
one of the regulators of ocn is ost - ptp , which promotes the carboxylation of ocn and reduces the concentration of ucocn or the ratio of ucocn / ocn .
there are also many clinical evidences indicating that ocn or probably ucocn is associated with fasting glucose and insulin sensitivity . however , ost - ptp is not a functional protein in human , and the exact role which ocn plays in human still needs to be studied .
anyway all these studies suggest us skeleton can behave not only as a structure scaffold but also as an endocrine organ , which regulates energy metabolism , even though all the studies are mostly done in mice and only partially confirmed in human .
therefore , more investigations are needed to reveal the total functions of ocn as a hormone in energy metabolism .
 skeleton was considered as a dynamic connective tissue , which was essential for mobility , calcium homeostasis , and hematopoietic niche . 
 however more and more evidences indicate that skeleton works not only as a structural scaffold but also as an endocrine organ , which regulates several metabolic processes . 
 besides osteoprotegerin ( opg ) , sclerostin ( sost ) , and dickopf ( dkk ) which play essential roles in bone formation , modelling , remodelling , and homeostasis , bone can also secret hormones , such as osteocalcin ( ocn ) , which promotes proliferation of  cells , insulin secretion , and insulin sensitivity . 
 additionally ocn can also regulate the fat cells and male gonad endocrine activity and be regulated by insulin and the neural system . in summary , skeleton has endocrine function via ocn and plays an important role in energy metabolism , especially in glucose metabolism . 