obesity is characterized by a body - mass index of 30  kg / m and excessive body fat accumulation . globally , estimated 502 million adults and 170 million children were classified as overweight or obese in 2008 and the rates of obesity are still increasing . obesity is associated with many chronic disorders such as type 2 diabetes mellitus , coronary heart disease , sleep - breathing disorders , certain cancers , and osteoarthritis . a correlation between obesity and bone metabolism bone forming osteoblasts are derived from stem cells that can also give rise to adipocytes and chondrocytes . stem cells proliferate and differentiate into preosteoblasts and finally into mature osteoblasts that are characterized by cessation of cell division , production of bone matrix , and synthesis of several essential marker enzymes and proteins for bone formation , for example , collagen type 1 , osteocalcin , and alkaline phosphatase ( alp ) . collagen type 1 is with approximately 90% of the main component of the bone matrix . in lamellar bone , its fiber organization allows the highest density per unit volume of tissue . in the biomechanically weaker woven bone collagen fibers crystals of hydroxyapatite are situated on the collagen fibers , within them , and in the matrix around them . alp is the main enzyme for formation of hydroxyapatite crystals and is therefore important for mineralization of bone and bone mineral density ( bmd ) . besides the collagens several non - collagenous proteins are present in the bone matrix . it is produced by osteoblasts , makes up 1% of the matrix , and plays a role in calcium binding and stabilization of hydroxyapatite in the matrix . for example , mechanical loading promotes osteoblastogenesis and inhibits differentiation of stem cells into adipocytes by increasing stable beta - catenin and reducing peroxisome proliferator - activated receptor - gamma ( ppar ) whereas stimulation of ppar decreases osteoblast differentiation and enhances adipogenesis [ 510 ] . however , the loss of bone mass determined in aging , osteoporosis , and after administration of glucocorticoids is associated with an increase in bone marrow adipogenesis ( reviewed in ) . the underling mechanisms for these effects are still unknown . the involvement of adipocyte - derived proinflammatory cytokines and hormones is discussed [ 1012 ] . since several studies focused on obesity associated with low - grade chronic inflammation , there is an increasing amount of reports describing detrimental effects of excessive body fat on bone [ 1315 ] . there is a higher incidence of clinical fractures in obese postmenopausal women   and in overweight adolescents [ 1719 ] . several animal studies supported this negative effect on bone strength [ 20 , 21 ] , bone mineral density , and bone formation . however , the traditional view of obesity is that overweight is beneficial to bone [ 11 , 2325 ] since the femoral neck of obese women especially with osteoporotic bones showed a reduction in fracture risk   and an increase in bmd . the enhanced bmd on the weight - bearing site implies that the mechanical effect of overweight stimulates bone mineralization   in addition to upregulation of bone formation enhancing molecules , for example , adipocytic estrogens . thus , we analyzed in the present study the bone density , the cellular structures , and the molecular bone markers . since cao et al . , 2010 ,   reported detrimental effects on bone in a murine obesity model with high - fat diet ( hfd ) determined by means of ct experiments , we hypothesized that our investigation will also point out negative effects of obesity on bone properties using several cell biological methods for investigation of in vivo bone samples . the expected results will gain new insights into the mechanism underling alterations of bone structure by obesity . seven 4-week - old male c57bl/6j mice ( janvier , france ) were fed for 23 weeks with a high - fat diet ( altromin , lage , germany , 60% kcal from fat ) and six with normal diet ( altromin , 10%  kcal from fat ) . the animals were kept under a 12-hour ( h ) light and dark cycle and had free access to chow and water . the animals were euthanized by inhalation of co2 and directly scanned via dual - x - ray absorptiometry ( dxa , lunar prodigy , ge healthcare , munich , germany ) for determination of bone mineral density ( bmd ) . afterwards bones were extracted and used for conduction of cell and molecular - biological methods . samples of femur and vertebrae l2 were fixed in 4% phosphate buffered paraformaldehyde ( carl roth , karlsruhe , germany ) and demineralized in 10% ethylene diamine tetra acetic acid ( merck , darmstadt , germany ) in 0.281  m tris - buffer . the samples were dehydrated using increasing ethanol concentrations ( 70% , 80% , and 96% each for 2 h , 3x 100% for 3  h ) and incubated in xylol ( carl roth , 3x 1  h ) and afterwards in liquid paraffin . after blocking out , paraffin sections were cut with a thickness of 4 - 5  m at the rotation microtome ( rm 2155 , leica , bensheim , germany ) . sections were stained with hematoxylin and eosin ( he , merck ) or used for enzyme- or immunohistochemistry ( ihc ) . to detect macrophages and osteoclasts the tartrate - resistant acidic phosphatase ( trap ) enzyme histochemistry was performed . therefore paraffin sections were deparaffinized with xylol and a decreasing series of ethanols . after washing in 0.1  m acetate buffer ( ph 5.2 ) sections were incubated in a solution of naphthol - as - tr - phosphate ( sigma - aldrich , steinheim , germany ) , di - sodium - tartrate - dihydrid ( merck ) , and fast red tr salt ( sigma - aldrich ) in acetate buffer at 37c for 30 minutes ( min ) . after washing in aqua dest . the rehydrated paraffin sections were incubated in a solution of 5-bromo-4-chloro - indolyl - phosphate ( bcip ) and nitro blue tetrazolium ( nbt , kpl , gaithersburg , md , usa ) salt for 45  min in a moist chamber at 37c . , sections were counterstained with nuclear fast red , dehydrated , and coverslipped with depex ( serva , heidelberg , germany ) . immunohistochemical incubation with an antibody for detection of collagen-1 was conducted in the present study to evaluate bone architecture . therefore rehydrated paraffin sections were treated with a tris - nacl buffer ( tbs , ph 7.4 ) containing 0.025% triton - x-100 ( merck ) . afterwards after washing in tbs , the sections were incubated with the primary collagen-1 antibody ( biomex , heidelberg , germany ) in a dilution of 1  :  50 in diluting buffer ( dako , hamburg , germany ) overnight at 4c . after washing in tbs , sections were incubated for 30  min with a biotinylated goat anti - rabbit secondary antibody ( dilution 1  :  500 ; vector laboratories inc . , burlingame , ca , usa ) and subsequently with the streptavidin - biotin - peroxidase complex ( vector laboratories inc . ) . to visualize the peroxidase a nova - red substrate kit ( vector laboratories inc . ) was used and according to the manufacturer 's protocol with an incubation time of 5  min . the nuclei were counterstained with hematoxylin and the sections were coverslipped with depex . as negative control all labeled paraffin sections were evaluated light microscopically with a photomicroscope ( axiophot-2 , zeiss , jena , germany ) equipped with a digital camera ( dc 500 , leica , bensheim , germany ) . for quantification of two dimensional trabecular regions in relation to the whole tissue histomorphometrical analyses were performed according to the methods described by dempster et al . , 2013 , . in brief , an area of interest from the tissue was defined ( in mm ) in which the trabeculae were marked and calculated in mm with the image - pro plus software ( media cybernetic , maryland , usa ) . as area of interest the metaphyseal region of the proximal and distal femur and the spongious part of the corpus vertebrae were used . for expression analyses humeri and vertebrae l3 were collected in rna - later ( ambion applied biosystems , foster city , ca , usa ) , homogenized with a mortar , and transferred into 1  ml trizol ( invitrogen , darmstadt , germany ) . after 5  min 200  l chloroform ( sigma - aldrich ) was added , the samples were centrifuged ( 12,000  g , 15  min , 4c ) , and the upper phase containing the rna was transferred into a new cup . isopropanol ( 0.5  ml ) was added and total rna precipitated by centrifugation ( 12,000  g , 10  min , 4c ) . for reverse transcription of total rna the quantitect reverse transcription kit ( 1  g rna was cleaned from genomic dna by incubation with 2  l dna wipeout buffer for 2  min at 42c . afterwards rna was transcribed to cdna with 1  l quantiscript reverse transcriptase , 4  l buffer , and 1  l primer mix containing oligo(dt)s and random - primers at 42c for 30  min . subsequent real - time rt - pcr was performed in the lightcycler ( roche , rotkreuz , schweiz ) . therefore 2  l of cdna was added to 2  l roche reagent ( lightcyclerfaststart dna maser sybr green i , roche , mannheim , germany ) , 0.2  l forward and reverse primer ( eurofins mwg operon , ebersberg , germany , table 1 ) , and 6.8  l rnase free water . the samples were incubated for 10  min at 95c , followed by 40 cycles of 5 seconds ( sec ) heating at 95c , annealing for 5  sec at 5862c , and elongating for 5  sec at 72c . subsequently the pcr product was controlled by melting curve and gel electrophoresis . as controls rt in addition negative controls were made where the template was omitted and water was used instead ( water control ) . using the lightcycler software cp - values were measured , normalized to the reference gene -actin and the cp , and relative expression was calculated according the cp method . vertebrae l4 were fixed in yellow fix ( 2% paraformaldehyde , 2% glutardialdehyde and 0.02% picrinic acid in 0.01  m phosphate buffer , ph 7.2 ) for 6  h. after washing in 0.1  m cacodylate buffer samples were incubated for 2  h in 1% osmium tetroxide solution . afterwards they were dehydrated through an increasing ethanol series , washed 3x in xylol , and incubated in a solution of xylol and epon ( serva ) . finally the samples were polymerized in epon and cut into semi - thin ( 500  nm ) and ultra - thin sections ( 6080  nm ) . the semi - thin sections were stained with toluidine blue and safranin - o and evaluated with a light microscope . the ultra - thin sections were contrasted with uranyl acetate and lead citrate and analyzed with a tem ( leo em 912 , zeiss , oberkochen , germany ) equipped with a ccd - camera ( olympus , mnster , germany ) . the spss software ( version 21.0 ; spss institute inc , chicago , il , usa ) was used for statistical analysis . the weight of mice receiving a hfd ( 60%  kcal of energy from fat ) was 11% higher ( p = 0.001 ) than that of control animals ( 10% kcal of energy from fat ) ( figure 1 ) . dxa - scan did not reveal significant differences in bone mineral density between both experimental groups ( figure 2 ) . the trabecular structure was evaluated using epon embedded calcified semi - thin sections ( figure 3 ) and demineralized paraffin sections ( figure 4 ) that were stained routinely with hematoxylin and eosin ( he ) ( figures 4(a ) , 4(b ) , 4(e ) , and 4(f ) ) or immunohistochemically labeled with an antibody against collagen-1 ( figures 4(c ) and 4(g ) ) . semi - thin and he sections demonstrated that the trabeculae of animals fed with hfd were smaller ( figures 3(a ) and 3(b ) ) , peaked at the ends ( rod - like shape , figures 4(b ) and 4(f ) ) , and less linked with other trabeculae ( figures 3(a ) and 3(b ) ) . the lamellar structure was often dissolved inside the trabeculae so that there was space in between the different lamellae ( figure 4(f ) ) . in the hfd group the trabeculae contained more woven bone and less lamellar bone than in the control group as shown by collagen-1 ihc ( figures 4(c ) and 4(g ) ) . furthermore the animals with high - fat diet exhibited less osteoid and more megakaryocytes in the bone marrow of the same samples ( figure 3(b ) ) . however , no changes could be observed in the amount , distribution , and size of osteoclasts identified with the trap enzyme histochemical staining ( figure 4(h ) ) . the alp staining could not point out differences between both animal groups . no alterations were shown in the labeling intensity , amount , and distribution of alp ( figure 4(c ) ) . comparing the average of the values a distinct decrease in bone was observed for all measured areas ( proximal , distal femur , and vertebrae , table 2 ) . however , because of the high standard deviation ( sd ) only the values of the distal femur showed significant changes ( p = 0.026 , table 2 ) . the expression of collagen 11 was significantly downregulated in humeri ( p = 0.002 , relative expression 0.34  0.19 ) and vertebrae ( p = 0.004 , relative expression 0.4  0.14 ) whereas osteocalcin and cathepsin k were not significantly changed due to hfd ( figure 5 ) . tem analysis further confirmed the presence of less osteoid in hfd mice compared to controls ( figure 6 ) . even if collagen fibrillae were found the striation was less distinct than in control mice . besides , we observed a dissolving of the cell - cell and cell - matrix connections ( figure 6(d ) ) . the membrane of osteocytes and osteoblasts was sometimes leaky and folded ( figures 6(e ) and 6(f ) ) and more apoptotic cells were found in the hfd group . the presented study was conducted to analyze the bones of male mice ( strain c57bl/6j ) after feeding with a hfd ( 60%  kcal from fat ) for 23 weeks . this animal procedure is a well - known model for induction of obesity that is used in a number of studies [ 15 , 2931 ] . it remains still unclear through which mechanism the adipose tissue affects the musculoskeletal system . it is known that additional load leads to functional and structural limitation of the soft structures such as tendons . furthermore overweight results in less physical activity   and that causes a loss of bone mass . on the other hand adipose tissue secretes adipose - derived hormones and cytokines ( e.g , tnf , il-1 , and il-6 ) leading to a low - grade systemic inflammation [ 12 , 13 , 15 ] . . however , relation between obesity and bone is still controversially discussed , and therefore the aim of the present study was to investigate the bone structure of obese mice in comparison to normal animals by means of dxa , histological methods , histomorphometrical measurement , real - time rt - pcr , and transmission electron microscopy . dxa - scan allows the calculation of bmd which in our study showed no differences between obese mice and controls getting normal chow ( 10%  kcal from fat ) . a close correlation of body weight and bmd , however , was found in humans where an approximate increase in 10  kg body weight causes a 1% increase in bmd . this positive correlation is stronger in women than in men and in postmenopausal than in premenopausal women . this effect could be explained by the conversion of androgens into estrogen in adipocytes and by the protective role of estrogen against osteoporosis and bone loss ( reviewed in ) . besides , ehrlich and lanyon described that the biomechanical loading of the additive weight stimulates bone formation and therefore increases bone mass and bmd . contrasting studies demonstrated that the bone strengthening effects of heavy bodies were not only due to adipose tissue but also due to elevated muscle mass . changes in bone mineral density are often induced by an imbalance in the amount and activity of bone forming osteoblasts and bone resorbing osteoclasts . enzyme histochemistry of alkaline phosphatase is a common method for testing the activity of osteoblasts . alp activity showed no alteration in bone sections from mice receiving hfd compared to control diet . 2009 , found an upregulation of alp positive colony forming units after culturing bone marrow stromal / osteoblastic cells of mice fed with hfd . the direct comparison between these results is not possible because of the different methods and the different hfds . in the present study we used a hfd where 60  kcal% energy as fat was given whereas the mice in cao et al hence , there could be a metabolic window were an increase in fat has positive effects on bone formation . such a window would also explain the controversial discussion in the literature and this would be in line with the report ofnez et al . furthermore , bone stimulating effects are only found when the energy upregulation is not correlated with an increase of insulin . in patients with type ii diabetes mellitus osteoblasts increased their cell division and proliferation in presence of insulin ( 1.2- to 1.7-fold ) but the alp activity and the production of mineralized matrix was reduced to 55% in comparison to control . it is linked to the membrane of matrix vesicles via a glycosylphosphatidylinositol ( gpi ) anchor by means of posttranslational modification . alp is involved in the formation of hydroxyapatite crystals within the vesicles by hydrolyzing monophosphate esters at a ph of 810 . after the matrix vesicles are budding from the osteoblast , the hydroxyapatite crystals penetrate the vesicle membrane and fill the space between the collagen fibrils ( reviewed in ) . besides alp , osteocalcin and collagen-1 are very prominent in bone . with an amount of 1% of the bone matrix , osteocalcin is the most important member of the group of noncollagenous matrix proteins . in our study we analyzed the possible regulation of osteocalcin on mrna level where we did not find any alteration . on the protein level cao et al .   could measure a significant down - regulation and therefore supposed a delay in bone formation . besides , collagen-1 is the main component of the non - mineralized bone matrix . using real - time rt - pcr we observed a significant down - regulation of collagen 11 mrna in both bone types , long bone ( tibia ) as well as irregular bone ( vertebrae ) of hfd mice . thus , on mrna level , the formation of non - mineralized matrix is changed in our obesity model . since the collagen builds up the skeletal structure of bone , we measured the cancellous area in relation to the whole tissue by means of histomorphometry . our results showed a distinct downregulation in the relative trabecular area of hfd mice in comparison to the control mice . a decrease in cancellous bone mass has also been reported by other studies using hfd in mice as model for obesity [ 29 , 30 , 40 ] . in addition to histomorphometry used in our study patsch et al . also used microcomputed tomography ( ct )   and fujita et al . and cao et al . focused on ct and serum levels of bone markers ct analysis possesses the advantage of analyzing the bone structures 3-dimensionally whereas histomorphometry is restricted to 2 dimensions . all three reports [ 29 , 30 , 40 ] measured a decrease in the ratio of bone volume to tissue volume and trabecular number . in addition , patsch et al . and cao et al . also described an increase in trabecular separation , connectivity density , and structure model index ( smi ) [ 29 , 40 ] . in contrast to our study the reports of fujita et al .   and patsch et al . fujita et al . analyzed animals after 4 , 8 , and 12 weeks of hfd . we used only a long - term hfd ( 23 weeks ) where we could confirm the results of patsch et al .   and fujita et al . . although cortical bone formation was slower in obese mice compared to controls , the periostal bone formation increased with age . thus , they assumed that the underlying mechanism of bone loss was different at these two sites of bone . confirmed these results in a study with young animals receiving a hfd . when the hfd was given to adults they measured a decrease in femoral diameter , bone strength , fracture toughness , and alignment of osteocytes . our results also depict a lamellar disorganization that affects the osteocyte microarchitecture . beyond the bone forming osteoblast lineage , osteoclasts are multinucleated cells with a specific endowment of enzymes and transporters that facilitate them to resorb bone . in the present study osteoclasts were analyzed on mrna level by measuring cathepsin k expression and histologically by trap enzyme histochemistry . no differences were observed in the osteoclast population between hfd mice and controls using both methods . multinucleated osteoclasts with a ruffled border , sealing zone , and several vesicles in their cytoplasm were found to be located at the surface of the mineralized bone . no structural aberration or obvious difference in the amount was found in the hfd mice in comparison to the controls . despite this osteoblasts are usually situated on the surface of growing bone as a closely packed layer of cells   that are connected to each other , to osteocytes , and to osteoid via gap junctions , connexons , and hemi - channels . an unusual big space was observed between the osteoblasts among themselves and between osteoblasts and the bone surface . the tem observations did not give information about the connections from osteoblast to the osteocytes . however the cell - to - cell and cell - to - matrix communications are important for maintaining bone homeostasis . the expression of connexin43 , which is the main component of gap junctions and hemi - channels is needed for functioning of mature osteoblasts and osteocytes . this report indicates that gap junctions and hemi - channels play an important role for bone cell survival . in hfd mice several apoptotic formations were observed for osteocytes and osteoblasts , and additionally , tem analysis showed a reduced osteoid production and striation and a decrease in osteoblast endoplasmatic reticulum in hfd mice . thus , we suspect that the reduction of cell - to - cell contacts is followed by an increase in apoptosis of bone forming cells and therefore the assembling of new bone is delayed . in summary , the presented study demonstrated a miss - arrangement of cell - cell and cell - matrix contacts , osteoblast and trabecular structure , and collagen-1 and osteoid synthesis that altogether outlines a negative effect of obesity on bone microstructure .
a correlation between obesity and bone metabolism is strongly assumed because adipocytes and osteoblasts originate from the same precursor cells and their differentiation is conversely regulated by the same factors . 
 it is controversially discussed if obesity protects bone or leads to loss of bone mass . 
 thus , the aim of the present study was to investigate the influence of diet - induced mild obesity ( 11% increased body weight compared to control ) on bone microstructure in mice . four - week - old male c57bl/6j mice received a high - fat diet ( hfd , 60% kcal from fat ) and were analyzed by means of dual x - ray absorptiometry , histological methods , real - time rt - pcr , and transmission electron microscopy in comparison to control animals ( 10% kcal from fat ) . 
 the cancellous bone mass , collagen 11 expression , amount of osteoid , and cohesion of cells via cell - to - cell contacts decreased in hfd mice whereas the bone mineral density and the amount of osteoblasts and osteoclasts were not modified . 
 the amount of apoptotic osteocytes was increased in hfd mice in comparison to controls . 
 we conclude that moderately increased body weight does not protect bone architecture from age - dependent degeneration . 
 by contrast , bone microstructure is negatively affected and reduced maintenance of cell - cell contacts may be one of the underlying mechanisms .