Soy protein isolates prevent loss of bone quantity associated with obesity in rats through regulation of insulin signaling in osteoblasts

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1 The FASE Journl Reserh ommunition Soy protein isoltes prevent loss of one quntity ssoited with oesity in rts through regultion of insulin signling in osteolsts JinRn hen,,,, Jin Zhng,,, Oxn P. Lzrenko,, Jy J. o, Mihel L. lkurn,, Thoms M. dger,,, nd Mrtin J. J. Ronis,, Arknss hildren s Nutrition enter, Little Rok, Arknss, USA; Deprtment of Peditris, Deprtment of Physiology nd iophysis, nd Deprtment of Phrmology nd Toxiology, University of Arknss for Medil Sienes, Little Rok, Arknss, USA; nd Grnd Forks Humn Nutrition Reserh enter, Grnd Forks, North Dkot, USA ASTRAT In oth rodents nd humns, exessive onsumption of typil Western diet high in sturted fts nd holesterol is known to result in disruption of energy metolism nd development of oesity nd insulin resistne. However, how these highft, energydense diets ffet one development, morphology, nd modeling is poorly understood. Here we show tht mle wenling rts fed highft (HF) diet ontining 45% ft nd 0.5% holesterol mde with sein (HFs) for 6 wk displyed signifint inrese in one mrrow diposity nd insulin resistne. Sustitution of sein with soy protein isolte (SPI) in the HF diet (HFSPI) prevented these effets. Mintenne of one quntity in the SPIfed rts ws ssoited with inresed underroxylted osteolin seretion nd ltered JNK/ IRS/Akt insulin signling in osteolsts. The HFs group hd signifintly greter serum nonesterified free ftty id (NEFA) onentrtions thn ontrols, wheres the HFSPI prevented this inrese. In vitro tretment of osteolsts or mesenhyml stroml ST ells with NEFAs signifintly deresed insulin signling. An isoflvone mixture similr to tht found in serum of HFSPI rts signifintly inresed in vitro osteolst prolifertion nd loked signifintly redued NEFAindued insulin resistne. Finlly, insulin/igf ws le to inrese oth osteolst tivity nd differentition in set of in vitro studies. These results suggest tht highft feeding my disrupt one development nd modeling; high onentrtions of NEFAs nd insulin resistne ourring with high ft intke re meditors of redued osteolst tivity nd differentition; diets high in soy protein my help prevent high dietry ftindued one impirments; nd the moleulr mehnisms underlying the SPIprotetive effets involve isoflvoneindued normliztion of insulin signling in one. hen, J.R., Zhng, J., Lzrenko, O. P., o, J. J., lkurn, M. L., Arevitions: ALP, lkline phosphtse; s, sein; H&E, hemtoxylin nd eosin; HF, high ft; IGF, insulinlike growth ftor ; IRS, insulin reeptor sustrte; LF, low ft; NEFA, nonesterified free ftty id; OGTT, orl gluose tolerne test; PND, postntl dy; RTPR, reverse trnsriptionpolymerse hin retion; SPI, soy protein isolte dger, T. M., Ronis, M. J. J. Soy protein isoltes prevent loss of one quntity ssoited with oesity in rts through regultion of insulin signling in osteolsts. FASE J. 7, (03). Key Words: highft diet free ftty id isoflvone osteolin Postntl development of oesity is ssoited with exessive onsumption of Western diet (defined s hving high levels of sturted ft nd holesterol). Feeding highft (HF) diet to rodents hs een reported to result in systemi insulin resistne nd metoli syndrome (). Development, mturtion, nd modeling of the skeletl system in the peditri popultion re ffeted y nutritionl sttus, dietry ftors, ody omposition, nd the effets of weight ering (). Inresed one mrrow diposity nd systemi insulin resistne re ommon fetures of impired one qulity/quntity in ptients with vriety of onditions, suh s oesity, hroni lohol use, dietes mellitus, nd ging (3 6). Therefore, it is possile tht insulin signling in one is ritil for mintining one mrrow diposity nd one mss t welllned level. A vriety of hormones nd metolites re elevted in plsm of oese nimls nd humns, inluding insulin, gluose, leptin, insulinlike growth ftor (IGF), nd nonesterified free ftty id (NEFA) onentrtions (7 ). Reent evidene in mie hs shown tht the seretion of osteolin, prtiulrly underroxylted osteolin, n osteolstderived tive form of the hormone, regultes insulin seretion, insulin sensitivity, nd energy expenditure in one (). Insulin reeptor signling in osteolsts hs een suggested s These uthors ontriuted eqully to this work. orrespondene: Arknss hildren s Nutrition enter, 5 hildren s Wy, Slot 50, Little Rok, AR 70, USA. Emil: henjinrn@ums.edu doi: 96/fj.6464 This rtile inludes supplementl dt. Plese visit to otin this informtion /3/ FASE

2 positive regultor of postntl one quisition (3). Prtiulrly, reent linil dt hve shown tht eing overweight with insulin resistne nd viserl diposity dversely influene dolesene one mss (4). Effetive pprohes to mnge oesity re extremely limited. Medition, weight loss progrms, nd dietry interventions hve een the most widely used. However, presently there re only meditions pproved in the United Sttes for longterm use, nd they re ssoited with vriety of side effets (5). Weightloss progrms hve een suessful; however, they re often ompnied y signifint one loss, espeilly without pproprite nutritionl supplementtion (6, 7). Furthermore, oese hildren present speil prolem, euse pproprite interventions would need to improve ody omposition while simultneously mintining norml growth. Dietry intervention my e more pproprite hoie. In this regrd, soy protein isolte (SPI) diet hs een investigted s ndidte for the prevention of metoli syndrome in erly development (8). Soy foods hve een reported to hve vriety of helth enefits inluding improved insulin sensitivity, weight mngement, nd improved ody omposition (9). Prevention of one loss in oth dult humns nd niml models of osteoporosis hs lso een reported for soy foods (0 ). Ativtion of AMPkinse in dipose tissue nd skeletl musle hs een implited in the improvement in lipid nd gluose signling fter soy feeding in mie (3). The effets of soy diet on one hve een ttriuted to potentil estrogeni tions relted to its high ontent of phytoestrogens suh s genistein nd didzein, whih re isoflvones struturlly similr to 7 estrdiol (4 6). Dietry ftors other thn isoflvone omponents, inluding unhrterized peptides ppering in serum fter feeding soy diets, my hve effets on one nd other orgn systems. We hve reently demonstrted positive tions of soy diet on systemi insulin resistne (8). The urrent study ws designed to explore the potentil link etween NEFA, insulin sensitivity, nd osteolsti ell differentition nd tivity in the young HF fed rt s model of hildhood oesity. Our dt suggest tht NEFA, one insulin resistne, nd redued osteolsti ell differentition nd tivity pper to e entrl meditors of HFindued impirment of one development nd modeling. We further found tht SPIontining diets loked the impirment of one formtion nd modeling during erly development, nd this ours with redued one diposity nd improved one insulin sensitivity. MATERIALS AND METHODS Animls nd diets Mle SprgueDwley rts were purhsed from Hrln Industries (Indinpolis, IN, USA), rrived on postntl dy (PND) 0, nd on PDN4 were rndomly ssigned to one of 4 groups (n 0). Rts were fed one of 4 diets: stndrd lowft (LF; 5%) AIN93G diet formulted with sein (s) s the sole protein (LFs); n LF diet mde with the AIN93G diet formul, exept tht s ws repled with SPI (LFSPI); n HF/highholesterol diet ontining 8.8 MJ/g energy, 95 g/kg s protein, 483 g/kg rohydrte, 0 g/kg nhydrous milk ft, 5 g/kg holesterol, nd 50 g/kg ellulose fier (HFs); nd the sme HF diet s the preeding, exept thts ws repled y SPI (HFSPI) plus supplementl mino ids. Thus, there were LF diets, one with s (LFs) nd one with SPI (LFSPI), nd HF diets, one with s (HFs) nd one with SPI (HFSPI). Rts were fed their respetive diets from PND4 to PND68. The HFSPI group hd d liitum ess to food nd wter, ut the other 3 groups were pir fed to the HFSPI sed on totl lories. Sine pirfed rts onsumed ll their food, onsumption ws tightly ontrolled mong the 4 groups. Rts were housed in n Assoition for Assessment nd Aredittion of Lortory Animl repproved niml fility t the Arknss hildren s Hospitl Reserh Institute with onstnt humidity nd lights on from 06:00 to 8:00 t. Physil tivity mong groups of nimls ws expeted to e similr. All niml proedures were pproved y the Institutionl Animl re nd Use ommittee t University of Arknss for Medil Sienes. Rt ody weights were monitored /wk. At 7 d prior to killing, rts were given stndrd orl gluose tolerne test (OGTT), nd til lood ws tken s desried previously (8). At the ompletion of the experiment, rts were nesthetized y injetion with 00 mg nemutl/kg ody weight (Avnt Lortories, Avnt, OK, USA), followed y depittion, nd legs nd serum were olleted. Gondl nd dominl ft tissues were lso tken, nd their weights were reorded. Serum insulin onentrtions were mesured using n ELISA (EZRMI3K) for rt serum (Lino Reserh, St. hrles, MO, USA). Serum gluose ws mesured using the gluose oxidse method (IR070; Synermed, Westfield, IN, USA). one nlyses Miroomputed tomogrphy (T) mesurements of treulr of the tiil one were evluted using Sno mirot snner ( T40; Sno Medil AG, ssersdorf, Switzerlnd) t 6 m isotropi voxel size with n Xry soure power of 55 kv nd 45 A nd integrtion time of 300 ms. The grysle imges were proessed y using lowpss gussin filter (, support ) to remove noise, nd fixed threshold of 0 ws used to extrt the minerlized one from the soft tissue nd mrrow phse. nellous one ws seprted from the ortil regions y semiutomtilly drwn ontours. A totl of 0 slies strting from mm distl to growth plte, onstituting 0.70 mm length, ws evluted for treulr one struture y using softwre provided y Sno, s desried in detil previously (7). one hemtoxylin nd eosin (H&E) stining on delified tii setions ws rried out using the stndrd protool from VetStin A kit (Vetor Lortory, urlingme, A, USA). Serum one turnover mrkers nd NEFAs The serum one formtion mrker lkline phophtse (ALP) nd the serum one resorption mrker proollgen rosslinks RtLps were mesured y RtMID ALP ELISA nd RtLps ELISA, respetively, from Nordi iosienes Dignosti (Herlev, Denmrk). Serum underroxylted osteolin nd totl osteolin (inluding in ulture medium) levels were mesured y n ELISAsed kit from Tkr io (Otsu, Jpn). An ELISA test for roxyterminl peptide frgment of type ollgen levels ws performed using enzyme immunossy kits from TSZ ELISA (TSZ Sientifi, Frminghm, MA, USA). Totl NEFAs (Wko Dignostis; Wko hemils, Rihmond, VA, USA) were mesured using stndrd ELISA method ording to proe SOY IMPROVES ONE QUALITY 355

3 dures provided y the mnufturer. NEFA omposition in serum ws hrterized nd quntified using Shimdzu QP00 GMS (Shimdzu orp., Kyoto, Jpn) fter TL seprtion. ell ulture one mrrow stroml ell line ST or osteolsti ell line O6 ws ultured in MEM supplemented with 0% FS (Hylone, Logn, UT, USA), peniillin (00 U/ml), streptomyin (00 g/ml), nd glutmine (4 mm). For different ssy purposes (mrna, proteins), different sizes of ell ulture pltes were used, nd ells were treted in the presene or sene of NEFAs, isoflvone, insulin, nd IGF for different durtions (see Results). Free ids were dissolved in 95% ethnol t 60 nd then mixed with prewrmed SA (0%) to yield stok onentrtion of 8 mm. Working onentrtions of NEFAs nd isoflvone were djusted to rtios similr to their pperne in serum from HFs nd SPI diet rts respetively. Individul isoflvones were purhsed from Plnte, Reding, UK, nd NEFAs were purhsed from SigmAldrih (St. Louis, MO, USA). Western lotting nd oimmunopreipittion Right tiil one tissue nd in vitro ellulr proteins were extrted for Western immunolot nlysis using ell lysis uffer s desried previously (0). riefly, fter lening of surrounding onnetive tissues nd spirtion of one mrrow ells, tii ws smshed to smll piees using surgil pliers. After dding 400 ml of ell lysis uffer, one tissues were homogenized using tissue homogenizer. Western lot nd oimmunopreipittion nlyses were performed using stndrd protools. The following primry ntiodies were used: pirs(ser307); rit, polylonl (tlog no. 0747; Millipore, illeri, MA, USA); pirs(tyr6), rit, polylonl (6653; Am, mridge, MA, USA); TIRS, rit, polylonl (38; ell Signling; Dnvers, MA, USA); pakt, rit, polylonl (s35650; Snt ruz iotehnology, Snt ruz, A, USA); TAkt, rit, polylonl (97; ell Signling); tin, mouse, monolonl (978; Sigm Aldrih); pjnk, mouse, monolonl (J4750; SigmAldrih); nd TJNK, mouse monolonl (SA40076; SigmAldrih). Seondry ntiodies were purhsed from Snt ruz iotehnology (got ntirit IgG, s004; got ntimouse IgG, s005). lots were developed using hemiluminesene (Piere iotehnology, Rokford, IL, USA) ording to the mnufturer s reommendtions. RNA isoltion, reltime reverse trnsriptionpolymerse hin retion (RTPR) RNA from one tissues or ultured ells ws isolted nd extrted using TRI Regent (MR In., ininnti, OH, USA) ording to the mnufturer s reommendtion, followed y DNse digestion nd olumn lenup using Qigen miniolumns (Qigen, Vleni, A, USA; ref. 8). Reverse trnsription ws rried out using n isript DNA synthesis kit from iord (Herules, A, USA). All primers for reltime PR nlysis used in this report were designed using Primer Express. softwre (Applied iosystems, Foster ity, A, USA) nd re listed in Supplementl Tle S. Sttistil nlyses Numeril vriles re expressed s mens sem. omprisons etween groups were performed with the nonprmetri KruskllWllis test, followed with y omprisons performed with MnnWhitney U test djusted for multiple omprisons. For pired mesures, the nonprmetri Wiloxon mthedpirs signedrnks test ws used. The ritil P vlue for sttistil signifine ws P 5. RESULTS SPI prevents HFindued oesity, one mrrow diposity, nd insulin resistne in one Rts were fed LF or HF diets mde with either s or SPI. Rts were pirfed the HFSPI diet. HFsfed rts were hevier thn LFsfed rts: vs g, respetively (P 6). LFSPIfed rts hd the lowest ody weight (P 5), nd HFSPIfed rts hd ody weights similr to the LFs group (Fig. A), suggesting tht SPIontining diets proteted ginst HFindued weight gins. When regionl ft pds were expressed s perentge of ody weight, the HFsfed rts hd lrger dominl ft pds (.6 0.%) nd gondl ft pds (.39 0.%) thn LF ontrols (5 8 nd %, respetively; P 5). The LFSPI diet group hd smller gondl ft pds (P 5), ut similr dominl ft pds ompred with LFsfed rts (Fig. ). Thus, SPI ws le to prevent HFindued inreses in rt ody ft. We performed n OGTT, nd til lood insulin onentrtion mesurement t PND6. In HFsfed nimls, greter serum gluose nd insulin onentrtions were found t 60, 90, nd 50 min fter gluose hllenge ompred with the other groups (P 5; Fig. ). Insulin sensitivity ws the gretest in the LFSPI group nd similr etween the LFs nd HFSPI groups (Fig. D). This ws ompnied y prevention of hyperglyemi in the HFSPI group ompred with the HFs group (Fig. ). To determine whether SPI ould lok or prevent one mrrow diposity, nlyses of tii one/one mrrow histology were onduted. H&E stining reveled the HFsfed rts hd exessive ft deposition in the one mrrow, while one mrrow diposity did not differ signifintly etween LFs, LFSPI, nd HFSPI groups (Fig. A). We exmined insulin signling in one tissue nd found tht phosphoryltion t the inhiitory site Ser307 on insulin reeptor sustrte (IRS) ws sustntilly inresed while phosphoryltion of Tyr6 ws deresed on IRS in the one of HFsfed rts (Fig. ) despite totl IRS ppering to e lower in the HFs group. No effets were found in the other groups, suggesting tht SPI loked HFindued phosphoryltion of IRS in one. In ordne with these dt, Akt phosphoryltion ws mrkedly downregulted in one from the HFs group ompred to the LFs group (Fig. ). The LFSPI diet group inresed Akt (Thr308) phosphoryltion in one ompred with LFs (Fig. ). onsistent with previous dt in mouse liver (9), rts fed HFs lso exhiited sustntil elevtion of JNK phosphoryltion in one (Fig. ). There were no signifint differenes in JNK phosphoryltion sttus mong the other groups (Fig. ). Phosphorylted IRS (Ser Vol. 7 Septemer 03 The FASE Journl HEN ET AL.

4 A ody Wt (g) lood gluose (mg/dl) PND Minutes After Gluose Injetion LFs LFSPI HFs HFSPI } % A Ft Wt / wt.0.6. LFs HFs HFSPI LFSPI D Serum insulin (ng/ml).5 LFs HFs.0 HFSPI LFSPI LFs HFs HFSPI LFSPI % Gon Ft Wt / wt Min. fter gluose injetion.6. Figure. ofeeding SPI diet prevents HFindued oesity. A) Growth urves from the 4 diet groups. ) Viserl (gondl ft) nd dominl ft perentges of ody weight of rts from the 4 diet groups t euthnsi. Dt re expressed s mens sem (n 0/group). Mens with different letters differ signifintly (P 5; ), s determined y wy ANOVA followed y StudentNewmnKeuls post ho nlysis for multiple pirwise omprisons. ) Serum gluose onentrtions fter OGTT in PND6 rts fed 4 different diets from PND4. Dt re mens sem, n 0 rts. Animls were denied ess to food overnight, nd til lood ws smpled over 50min period following.5 g/kg orl gluose hllenge. D) Serum insulin onentrtion fter n OGTT in PND6 rts fed 4 different diets. P 5 vs. LFs nd HFs groups. nd phosphorylted JNK were oimmunopreipitted with JNK when one proteins from HFsfed rts were sujeted to oimmunopreipittion experiment (Fig. ). SPI diet prevented this HFindued inrese of pjnk nd pirs ssoition in one (Fig. ). SPI prevents HFindued impirment of one qulity MiroT nlysis of tiil ones reveled their phenotypes represented in imges presented in Fig. 3A. Speifilly, one volume, treulr numer, nd treulr thikness were ll deresed in HFsfed rts ompred to LFsfed nimls (Fig. 3). These prmeters were highest in the LFSPI group (Fig. 3). However, treulr sping ws greter in HFsfed rts ompred with the other groups. Impired one quisition in HFsfed rts ws onsistent with our previous oservtions in whih we utilized rt totl enterl nutrition model of HF feeding, while keeping the ody weights equl etween groups, to demonstrte tht low one qulity ws ssoited with development of oesity (0). Serum one turnover mrker mesurements reveled tht osteolin levels were lower in HFsfed rts ompred to the LFs diet group (Tle ). Underroxylted osteolin, n endorine tive form of osteolin sereted only y osteolsts, ws found to e the highest in the LFSPI group, nd deresed in the HFsfed rts ompred to LFs group (Tle ). There were no differenes etween HFSPI nd LFs groups, nd this is refleted in the one mss dt presented in Fig. 3. Serum NEFA onentrtions in HFsfed rts were lmost 3 times higher thn those in LFsfed rts (Tle ), while these levels did not differ mong the other groups ompred to LFs. Serum NEFA omposition of HFsfed rts ws lso hrterized nd quntified using Shimdzu QP00 GMS fter TL seprtion. We found tht there were 5 mjor ftty ids with rtios similr to results previously reported in our lortory (0), with plmitte ( 00 M) hving the highest onentrtion of ftty ids in serum of HFsdiet rts (dt not shown). Quntittive reltime PR nlysis of osteolin mrna expression in one ws very similr to its pttern in serum (Fig. 3 nd Tle ). Gene expression of nother osteolsti ell tivity mrker, ollgen type (ol), ws lso lower in HFsfed rts ompred to LFsfed rts (Fig. 3). There were no differenes in ol mrna expression etween the LFSPI groups nd HFSPI ontrols (Fig. 3). Role of NEFAs NEFAs from inresed ft stores my e one of the most importnt ftors ffeting one in oesity. SPI onsumption results in pperne of mny iotive ompounds in lood. To dte, isoflvones re the most studied omponents ssoited with dietry soy nd hve een found to hve enefiil effets on one (30). Therefore, we ssessed the serum isoflvone levels in rts fed SPI diets using method previously pulished in our lortory (3, 3). Totl isoflvone onentrtions were in the rnge of M, nd the mjor isoflvone ws the estrogeni didzein metolite equol (3, 33). Next, osteolsti O6 ells were treted with NEFAs in the presene or sene of isoflvones. onentrtion nd rtios of individul SOY IMPROVES ONE QUALITY 357

5 A LFs HFs IP: JNK LFs HFs HFSPI LFSPI HFSPI LFSPI I: pirs (Ser307) I: pjnk I: TJNK pirs (Ser307) pirs (Tyr6) TIRS pakt (Thr 308) TAkt pjnk TJNK βtin LFs HFs HFSPI LFSPI Figure. ofeeding SPI diet prevents HFindued one mrrow diposity nd insulin resistne in one. A) Representtive H&E stining imges of inresed one mrrow diposity in tiil one setion from HFindued oese nimls ( 0). White rrows re pointing to treulr one spiule; lk rrows re pointing to diposity. ) Western lots of phosphorylted IRS (pirs; Ser307 nd Tyr6), totl IRS (TIRS), phosphorylted Akt (pakt, Thr308), totl Akt (TAkt), tin, phosphorylted JNK nd (pjnk, ), nd totl JNK nd (TJNK, ) re depited for 3 smples from the 4 diet groups. ) Western lots showing oimmunopreipittion of endogenous pirs, p JNK, nd JNK in triplite from pooled proteins isolted from ones from the 4 diet groups. Proteins were isolted from long one fter spirtion of one mrrow ells; 0 smples from eh group were pooled to 3 smples/group. NEFAs nd isoflvones were similr to their pperne in the serum of rts from HF nd SPI, respetively. As to NEFAs, 5 mjor ftty ids, plmiti, steri, olei, linolei, nd rhidoni id, were mixed in the rtio of 5:::3: t onentrtions ppering in the serum of HFsfed rts. Similrly, isoflvones is the mixture of glyetein, genistein, didzein, nd equol with onentrtion of eh individul ompound similr to their level in niml serum fter SPI diet. Tretment of ells with this NEFA mixture for 3 d signifintly deresed, wheres isoflvones inresed, osteolst prolifertion, refleted y the hnges in osteolst numers (Fig. 4A). However, isoflvones did not lok NEFAindued downregultion of osteolst prolifertion (Fig. 4A); suggesting tht NEFAs nd isoflvones hve independent effets on osteolsti ell prolifertion. To further otin mehnisti explntions for our in vivo oservtions, we next studied insulin signling in osteolsts fter 4 h tretment with NEFAs in the presene or sene of isoflvones. We found tht NEFAs not only phosphorylted IRS t its inhiitory site Ser307, it lso inhiited totl IRS expression (Fig. 4). More strikingly, NEFAs downregulted oth phosphorylted nd totl Akt expression (Fig. 4) nd sustntilly phosphorylted JNK (Fig. 4). Isoflvone tretment hd opposite effets on osteolsts, inhiiting IRS phosphoryltion nd upregulting totl IRS expression (Fig. 4). Moreover, isoflvones inresed Akt phosphoryltion nd totl Akt expression nd deresed JNK phosphoryltion (Fig. 4). All the ove oserved effets of NEFAs were ompletely rogted y otretment of osteolsts with isoflvones (Fig. 4). In the presene of insulin in the ell ulture, the sttus of pakt nd pirs were similr etween ontrol nd isoflvone tretment (Supplementl Fig. S). ST ells were similrly treted with NEFAs in the presene or sene of isoflvone. NEFAs signifintly downregulted mrna expression of wellknown osteolsti ell differentition mrkers Runx, osterix, nd ALP in ST ells (Fig. 4), inditing tht NEFAs lso suppress osteolst differentition, whih is onsistent with our previously pulished dt (0, 34). Isoflvones did not signifintly hnge Runx nd osterix mrna expression, ut signifintly upregulted ALP mrna expression (Fig. 4). However, in the presene of osteolsti ell differentition medium (o medium), isoflvones signifintly inresed Runx nd osterix gene expression (Supplementl Fig. S). Interestingly, isoflvones loked NEFAindued downregultion of osteolst differentition mrkers, with the exeption of osterix (Fig. 4). Similr to results shown in osteolsts, isoflvones ompletely inhiited the effet of NEFAs on insulin signling indited in Western lot results on phosphoryltion of IRS nd Akt in ST ells (Fig. 4D). The 4 free ftty ids tht minly ompose NEFAs were lso individully tested t the onentrtion tht ppered in rt serum to determine their effets on JNK phosphoryltion in osteolsts. Sustined JNK phosphoryltion ws found in ells treted with plmiti, olei, nd linolei id (Fig. 5A). Plmiti id not only tivted JNK, ut lso inresed the phosphoryltion of IRS (Fig. 5). JNK inhiitor SP6005 olished the effets of plmiti id on phosphoryltion of oth IRS nd JNK in osteolsts (Fig. 5). Most importnt, when osteolsts were treted with NEFAs, phosphorylted IRS nd phosphorylted JNK were oimmunopreipitted with JNK (Fig. 5). Isoflvones prevented this NEFAindued inresed ssoition of pjnk nd pirs in osteolsts (Fig. 5). The intertion etween phosphorylted IRS nd JNK ssoited with NEFA tretment in osteolsts ws similrly 358 Vol. 7 Septemer 03 The FASE Journl HEN ET AL.

6 A LFs HFs HFSPI LFSPI O mrna ol mrna.6 O mrna. ol mrna V / TV (%) T. Th (mm) T. N (# / mm) T. Sp (mm) thepsin mrna LFs HFSPI HFs Figure 3. SPI diet prevents HFindued impirment of rt one quisition. A, ) Quntittive mirot nlysis of the proximl tiil in rts fed 4 different diets (n 4 rts). A) Representtive imges. ) one volume/ 6 tissue volume (V/TV; %); treulr numer (T. N; 0. 4 no./mm); treulr thikness (T. Th; mm); treulr sping (T. Sp; mm). ) Totl RNA isolted from 0. one nd quntittive reltime PR nlyses onfirmed 0 the signifint downregultion of osteolin gene, s well s ol mrna, from HFsfed rts ompred to those from LFsfed rts. thepsin K is upregulted in HFsfed rts. Dt re expressed s mens sem (n 0/group). Mens with different letters differ signifintly (P 5). LFSPI TRAP 5 mrna reveled in one smples (Fig. ). These dt suggest tht the effet of HF on insulin signling in one in vivo nd in osteolsts in vitro is, t lest in prt, due to elevted serum NEFAs. Isoflvones pper to medite the effets of SPI y ttenuting insulin resistne in one fter HF feeding. Effets of insulin in osteolsti ells We hve demonstrted tht HF or NEFA exposure results in insulin resistne. To ddress whether insulin hs n effet in osteolsti ells, we treted O6 nd ST ells with insulin nd IGF. In O6 ells, insulin nd IGF were le to signifintly stimulte seretion of osteolin nd P, de novo ollgen synthesis mrker mesured in ell ulture medium (Fig. 6A, ). With insulin or IGF tretment, O6 ells lerly showed inresed Akt nd IRS phophoryltion (Fig. 6D). In ST ells, following tretment with insulin or IGF, mrna expression of ll 3 osteolst differentition mrkers, ALP, osterix, nd Runx, ws signifintly inresed ompred with untreted ells (Fig. 6). Similr to the results we found in O6 ells, phosphoryltion of Akt nd IRS ws higher in ST ells following insulin or IGF tretment ompred to those without tretment (Fig. 6E). These dt suggest tht insulin signifintly stimulted osteolst tivity nd differentition in vitro. DISUSSION The urrent study suggests tht insulin resistne oserved in the one of rts fed HF diet my e due to TALE. Effets of HF nd SPI diet on serum one turnover mrkers PND68 mle, 6 wk diet Mrker LFs HFs HFSPI LFSPI Osteolin (ng/ml) Underroxylted osteolin (ng/ml) ALP ( U/min) RtLps (ng/m) NEFAs (mm) Serum one formtion mrkers totl osteolin, underroxylted osteolin, nd ALP; resorption mrker RtLps; nd NEFA levels were mesured using stndrd ELISA methods. P 5 vs. LFs; t test. SOY IMPROVES ONE QUALITY 359

7 A ells/wellx O6 ell prolifertion pirs TIRS pakt TAkt ontrol NEFA Isof NEFA Isof 0.5 pjnk TJNK Runx mrna ý ALP mrna O differentition mrkers in ST ells ALP mrna OSX mrna ontrol NEFA Isoflvone NEFAIsoflvone OSX mrna D pirs TIRS pakt TAkt ontrol NEFA Isof NEFA Isof Figure 4. NEFAs ply ritil role in inhiiting insulin signling in osteolsti ells, while isoflvone prevents it. A) O6 osteolsti ell prolifertion nlysis fter the tretments of 4 different regimes for 3 d. ontrol, vehile tretment. onentrtions of NEFAs, isoflvone, nd their omintion were sed on their ppernes in niml serum (see Mterils nd Methods). ) Western lots for phosphorylted nd totl IRS, Akt, nd JNK in O6 ells in triplite fter 4 h tretment. ) ST ells were treted with NEFAs, isoflvone, nd their omintion for 3 d; ell RNA ws isolted, nd reltime PR nlyses showed hnges in mrna expression of 3 different osteolst (O) differentition mrkers, ALP, Runx, nd OSX (osterix). Dt re expressed s mens sem (triplite). Mens with different letters differ signifintly (P 5). D) Western lots for phosphorylted nd totl IRSnd Akt in ST ells in triplite fter 4 h tretment. Isof, isoflvone. elevted NEFAs. In ddition, the impired one quntity in rts fed n HF diet my e due to deresed seretion of underroxylted osteolin, nd this is onsistent with reently pulished studies using n osteolsti ellspeifi insulin reeptorknokout niml model in mie (, 3). SPI inorported into the HF diet prevents inresed one mrrow diposity, skeletl insulin resistne, nd impired gluose tolerne. This protetive effet on one y SPI ppers to e, t lest in prt, due to the pperne in serum of isoflvones derived from SPI. Although the hnges in one struture my differ etween oesity (35) nd sex steroid defiieny (6), our results re onsistent with the protetive effets of isoflvone on one oserved in those previous studies. It is well known tht homeostti ontrol of lood gluose is determined y mjor ftors: the onentrtion of insulin in the irultion nd insulin sensitivity t trget orgns. Progression of insulin resistne towrd gluose intolerne ours in oesity due to inresed demnd for insulin nd filure of pnreti ells to meet this demnd (36). While this hs een doumented for vrious orgns, surprisingly, it hs not een estlished in one. oneforming ells highly express insulin reeptor, nd reent rodent study suggested tht insulin reeptor signling in osteolsts is signifint determinnt of wholeody gluose homeostsis (37). This is onsistent with the results desried in the present report. For exmple, IRS/ Akt/JNK insulin signling ws signifintly downregulted in one of HF rts. Also, seretion of osteolin, espeilly the underroxylted tive form, nd osteolin mrna expression were signifintly lower in one from HF nimls. Similr to JNK tivtion in heptoytes, pnreti ells nd other peripherl sites y free ftty ids (38), the mehnisms leding to JNK tivtion in one y NEFAs re unknown. However, the onsequenes my e the sme, i.e., inhiition of insulin signling. Reently, JNK tivtion ws not only shown to e involved in inhiition of insulin signling y elimintion of Akt tivtion, ut lso involved in p53medited ell senesene pthwys (39). It hs een reported tht p53 is JNK sustrte (40) nd pretretment with 350 Vol. 7 Septemer 03 The FASE Journl HEN ET AL.

8 A Time (min): plmiti olei p JNK TJNK p JNK TJNK PA SP6005 pirs(ser307) TIRS pjnk TJNK linolei p JNK TJNK βtin IP: JNK rhidoni SA p JNK TJNK p JNK TJNK I: pirs(ser307) I: pjnk I: TJNK NEFA ontrol NEFA Isof Isof Figure 5. NEFAs tivte JNK nd IRS, while isoflvone dissoites them in osteolsts. A) Western lots showing time ourse of JNK tivtion (phosphoryltion) fter O6 ells were treted with olei, plmiti, linolei, rhidoni id, nd SA s ontrol. ) Western lots showing phosphoryltion of IRS nd JNK in O6 ells fter ells were treted with plmiti id (PA) nd JNK inhiitor SP6005 in duplite for h. ) Western lots showing oimmunopreipittion of endogenous pirs, pjnk, nd TJNK fter O6 ells were treted with NEFAs, isoflvone, nd their omintion in duplite for h. the speifi JNK inhiitor not only dereses senesene, ut lso indues poptosis (4). In shrp ontrst, JNK defiieny hs lso een reported to use p53dependent senesene in primry murine firolsts isolted from emryos (39). Nonetheless, we hve shown tht inresed phosphoryltion of JNK long with IRS in NEFAtreted osteolsts resulted in insulin resistne in this prtiulr ell type. Olei nd plmiti id my e more potent thn other NEFAs, nd they my e ritil in vivo in HFindued oesity A O (ng/ml).0.6. ontrol O Insulin IGF ALP mrna ontrol Insulin IGF Runx mrna ontrol Insulin IGF OSX mrna ontrol Insulin IGF P (ng/ml) ontrol P Insulin IGF D insulin IGF pakt (Thr 308) TAkt pirs(ser307) TIRS βtin E insulin IGF pakt (Thr 308) TAkt pirs(ser307) βtin Figure 6. Insulin tivtes osteolst nd stimultes ell differentition. A, ) Sereted osteolin (O; A) nd rosslinked telopeptide of type ollgen (P; ) in ulture medium were mesured using ELISA fter O6 ells were treted with either insulin or IGF (0 nm) for 3 d. ) ST ells were treted with either insulin or IGF (0 nm) for 3 d, nd osteolsti ell differentition mrkers ALP, Runx, nd OSX (osterix) mrna expression were determined using reltime PR. D) O6 ells were treted with either 0 nm insulin or 0 nm IGF for h (duplited). Western lots for phosphorylted Akt nd IRS, totl Akt, IRS, nd tin re shown. E) ST ells were treted with either 0 nm insulin or 0 nm IGF for h (duplited). Western lots for phosphorylted Akt nd IRS, totl Akt, nd tin re shown. P 5 vs. ontrol. SOY IMPROVES ONE QUALITY 35

9 leding to JNK tivtion, IRS Ser/Thr phosphoryltion, nd downregultion of insulin reeptor signling in osteolsti ells. In ddition, previous study hs shown tht plmitte n exert lipotoxi effets in osteolsts due to mehnisms relting to ftty id synthesis nd Runx inding (4); this my explin the suppressive effets of NEFA on osteolst differentition reported in the urrent study. Insulin indues prodution of the insulin seretgogue osteolin (Fig. 6A), whih my influene gluose utiliztion (37). Although we did not provide diret evidene of ssoition etween insulin resistne nd osteolin in osteolsts in HF rts, insulin resistne ws ompnied y deresed underroxylted osteolin seretion in vivo. Osteolsti ells were further shown to hve inresed osteolin seretion fter insulin nd IGF stimultion. We elieve tht our present report demonstrtes for the first time tht insulin resistne is ssoited with HF feeding in one nd osteolsts. Interestingly, inresed one mrrow diposity ws reported in norexi nervos, nd this ws lso ssoited with insulin resistne (43). Suh insulin resistne ourring in osteolsts my diretly lter the funtion of ells leding to one deteriortion. Using genetilly modified niml models, it my e possile to untngle this reltionship. Signifint dereses in oth gondl nd dominl ft pd weights in the HF group with odministrtion of SPI re onsistent with reent studies demonstrting tht soyontining diets or soy dietderived phytoestrogens, suh s genistein, hve ntilipogeni effets in ovrietomized rodents (44) nd effets on osteolsti ell differentition (45). Moreover, previous studies hve lso shown tht dietry soy protein redues rdi lipid umultion nd ermide onentrtion in HF dietfed rts nd o/o mie (46). These findings greed with our reently pulished dt, whih reveled tht feeding SPIontining diets loked HFindued systemi insulin resistne nd mintined lipid homeostsis (8). Our urrent results were onsistent with our previous oservtion in whih oth soy infnt formulfed infnts nd piglets showed lower totl ody ft ut inresed one quisition rte ompred to ontrols (47). ompred with HF lone, serum NEFA levels in the HFSPI group were signifintly deresed in the present study, refleting dipose tissue mss s the primry soure for NEFA rther thn HF diet itself. Although the positive effet of SPI on treulr one is onsistent with our previous oservtions, the urrent dt lso indited tht one mss ws signifintly proteted from the effets of HF y SPI. We elieve tht this my e linilly pplile. In oth humns nd experimentl nimls, the most reent studies on dietry intervention for oesity use lori restritive diet, ut this diet is linked to onomitnt elerted one loss nd inresed risk of frture (48, 49). The protetive effet of SPI diet on deresed one quisition ssoited with HF feeding hs een suggested to e due t lest in prt to isoflvones. Sine these SPIssoited isoflvones re struturlly similr to 7 estrdiol, mjority of the reserh hs foused on their estrogeni effets on one (45); they my lso exert nonestrogeni effets on one (30, 33). We hve reently ddressed suh differentil effets etween SPI nd estrogen on one (33). However, to definitively eluidte isoflvones nti NEFA effet on one, n in vivo isoflvone nd NEFA odministrtion experiment my e neessry in future studies. In summry, feeding young prepuertl rts diet omposed of s protein nd high in sturted ft nd holesterol produed diposity hrterized y inresed one mrrow ft stores, one insulin resistne, nd ompromised one qulity. High levels of NEFAs ting through underroxylted osteolin seretion nd IRS/Akt insulin signling in osteolsts were shown to impir one qulity in vitro. Repling s protein in the HF diet with SPI prevented one diposity nd one insulin resistne, nd this ws ssoited with lower serum NEFA onentrtions nd improved one qulity. 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