A mouse model of vitamin D insufficiency: is there a relationship between 25(OH) vitamin D levels and obesity?

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1 Seldeen et l. Nutrition & Metolism (217) 14:26 DOI /s RESEARCH Open Access A mouse model of vitmin D insufficiency: is there reltionship etween 25(OH) vitmin D levels nd oesity? Kenneth L. Seldeen, Mnhui Png, Mri Rodríguez-Gonzlez, Mirey Hernndez, Zchry Sheridn, Ping Yu nd Bruce R. Troen * Astrct Bckground: Vitmin D insufficiency (serum 25-OH vitmin D > 1 ng/ml nd < 3 ng/ml) is prevlent in the oese (ody mss index (BMI) > 3 kg/m 2 ), yet reltionships etween the two re poorly understood. Ojectives of this study include identifiction of the impct of oesity on reducing serum 25-OH vitmin D concentrtion, prticulrly in response to ltered vitmin D 3 supplementtion, nd to elucidte the longitudinl impct of serum 25-OH vitmin D on ody mss index. Methods: Twenty four-week-old len nd oese mle C57BL/6 J mice were fed low, stndrd, or high levels of choleclciferol supplementtion nd followed for 24 weeks. Longitudinl mesurements include serum 25-OH nd 1,25-(OH) 2 vitmin D, intct PTH, nd clcium concentrtions, s well s BMI, one density nd ody ft/len mss. Results: Bseline serum 25-OH concentrtions were not different in len nd oese mice (len 32.8 ± 4.4 ng/ml versus oese 3.9 ± 1.6 ng/ml p =.9). Len mice receiving low supplementtion exhiited rpid declines in serum 25-OH vitmin D concentrtions, flling from 33.4 ± 5.4 ng/ml to 14.5 ± 3.4 ng/ml fter 2 weeks, while oese mice declined t lower rte, flling from 3.9 ± 1.5 to 19. ±.9 ng/ml within the sme time period. Surprisingly, high vitmin D 3 supplementtion did not sustntilly increse serum vitmin D concentrtions ove stndrd supplementtion, in either len or oese mice. No differences in serum 1,25-(OH) 2 vitmin D, intct prthyroid hormone (PTH) or serum clcium were oserved etween len nd oese mice within the sme vitmin D supplementtion group. Yet oese mice exhiited lower serum clcitriol, higher serum PTH, nd lower one minerl density (BMD) thn did len mice. Additionlly, neither ody mss index nor ody ft % ws significntly correlted with vitmin D concentrtions. Interestingly, len mice with high vitmin D supplementtion consumed significntly more food thn did len mice with stndrd or low supplementtion (14.6 ± 1.7 kcl/mouse/ dy versus 11.8 ± 1.4 nd 12.3 ± 1.7 respectively, p <.1 for oth). Conclusions: Low choleclciferol supplementtion in oth len nd oese mice significntly nd sustinly reduces serum 25-OH vitmin D concentrtions. Interestingly, oesity slowed the rte of decline. Over the period of the study, vitmin D insufficiency ws not susequently correlted with greter BMI/ody ft, lthough len mice with high supplementtion consumed greter clories with no pprent BMI increse. Keywords: Vitmin D, PTH, Insufficiency, Oesity, Weight, BMI, Body Composition, Mice, Ft, High ft diet * Correspondence: troen@ufflo.edu Division of Geritrics nd Pllitive Medicine, Deprtment of Medicine, Jcos School of Medicine nd Biomedicl Sciences, University t Bufflo nd Reserch Service, Veterns Affirs Western New York Helthcre System, Bufflo, NY, USA The Author(s). 217 Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 2 of 9 Bckground Vitmin D insufficiency (serum 25-OH vitmin D >1 ng/ml nd <3 ng/ml) is prevlent condition with n estimted 7% of the popultion considered t risk [1, 2]. Concurrent with widespred vitmin D insufficiency, the Centers for Disese Control lso reports tht lrge percentge of the popultion (>35%) hs ody mss index (BMI) considered oese. Severl humn studies hve explored the possiility tht these two phenomen my e linked [3 6], however, it remins uncler if vitmin D insufficiency promotes weight gin nd/or if oesity modultes serum vitmin D concentrtion. Interestingly, studies seeking to modulte ody weight y correcting vitmin D sttus hve een unsuccessful [7 9], lthough other eneficil effects, including reduced ody ft [7], reduced inflmmtory profile [9], nd improved insulin resistnce [1] were seen. Contrry to wht is seen in humn trils, niml studies pper to show tht vitmin D my promote weight gin. Vitmin D receptor (VDR) knock-out mice were shown to e resistnt to weight gin [11, 12], s were diet-induced vitmin D deficient/insufficient mice to high ft [13] nd western diets [14]. Additionlly, over-expression of VDR in dipocytes induced weight gin in mice [15]. Although interprettion of these dt suggests tht vitmin D, through VDR, increses weight gin, other studies hve shown tht dministrtion of 1,25-(OH) 2 vitmin D, the ctive form of vitmin D nd direct lignd to VDR, decreses weight nd hs other eneficil effects [16, 17]. Alterntively, dt from severl studies suggest oesity my impir the effectiveness of vitmin D supplementtion. Supplementtion in the oese hs resulted in less thn expected supplementtion, [18 21] nd these findings re further supported y met-nlysis of greter thn 12, ptients showing tht BMI negtively correltes with the effect of supplementtion [22]. Additionlly, mice given 2, IU vitmin D 3 /kg chow in western diet hd similr serum 25-OH vitmin D concentrtion s their len counterprts given 1, IU vitmin D 3 /kg chow t the conclusion of the study [14]. It hs een speculted tht ft sequestrtion of vitmin D leds to lower serum concentrtions [21], lthough this trditionl view hs recently een chllenged y one study suggesting dilution y greter ody mss nd not ft sequestrtion leds to the dmpening effects [23]. Studies exmining the interreltionships etween serum vitmin D insufficiency nd oesity frequently focus on humn popultions where differing lifestyle nd/or genetic fctors my ofuscte findings. Animl models, on the other hnd, predomintely mke use of receptor knockouts or complete dietry removl to simulte vitmin D deficient conditions, which my not ccurtely reflect the more prevlent condition of insufficiency in humns. To further explore the reltionships etween 25-OH vitmin D sttus nd weight/ody ft, we induced vitmin D insufficiency in len nd oese mice nd exmined food consumption, ody weight, nd ody composition. Our dt indicte tht higher BMI modestly uffers serum 25- OH vitmin D concentrtion in response to lower supplementtion. Further, our findings lso suggest tht neither high or low vitmin D 3 supplementtion modultes ody weight or ody ft. Methods Mice All studies nd experimentl protocols were pproved y nd in complince with guidelines of the Mimi VA Animl Cre nd Use Committee. At ll times mice were provided d liitum ccess to chow/wter nd covered to void exposure to fcility lighting. 8-weekold mle C57BL/6 mice were rndomly ssigned to either high ft (6% FDC, Dyets Inc, Bethlehem, PA, ID# D18988) or low ft (1% FDC, ID# D18989) feeding for 16 weeks. Mice filing to gin more thn 1% ody weight were removed from the study. At 24weeksofge,lenmicewererndomlyssignedto low ft diets contining 125, 1 or 4 IU vitmin D 3 /kg chow nd oese mice to high ft diets contining 162, 1282 or 5169 IU vitmin D 3 /kg chow (Tle 1), nd were followed for 24 dditionl weeks. Numers of nimls were nine for len-low & len-stndrd, eight for len-high, six for oese-low & oese-stndrd, nd Tle 1 Diet compositions Component (g/kg) Low ft Diet High ft diet Vitmin Free Csein L-Cystine 3 4 Dyetrose Sucrose Cornstrch 315 Soyen oil Lrd t-butylhydroquinone (TBHQ).5.6 Cellulose 5 65 Diclcium Phosphte Clcium Cronte Potssium Citrte Choline Bitrtrte Vitmin/Minerl Mix (Vit D free) 2 26 Vitmin D 3 125/1/ 4 IU/kg 3.1/25./1. μg/kg 162/1282/ 5169 IU/kg 4./32.3/129. μg/kg

3 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 3 of 9 seven for oese-high. Mice nd chow from cges were weighed on weekly sis. Additionlly, mouse length ws mesured t the end of the study y nesthetizing mice nd mesuring from nose to the se of the til. BMI ws clculted s g/cm 2 [24]. Serum nlysis Blood ws extrcted through the su-mndiulr vein using mouse lncet (MEDIpoint, inc., Mineol, NY.) nd collected into microcentrifuge tues. Smples were held t room temperture for 1 min to llow cogultion nd then centrifuged t 13, RPM for 1 min to llow seprtion of serum. Anlysis of serum ws performed using specific ELISA kits for 25-OH vitmin D (ImmunoDignostic Systems, Inc., Scottsdle, AZ), 1,25- (OH) 2 vitmin D (MyBioSource, Sn Diego, CA.) nd intct PTH (MyBioSource), while colorimetric ssys were used to ssess clcium (Biovision, Sn Frncisco, CA.) ccording to mnufcturer protocols. Dul-energy X-Ry sorptiometry Anlysis of one minerl density, ody ft % nd len mss ws performed using Lunr PIXImus II (GE Helthcre, United Kingdom). Animls were nesthetized nd then nlyzed with single scn t seline nd every 8 weeks therefter. Sttistics Two-wy ANOVA, followed y post hoc Tukey s Multiple Comprisons test when pplicle, ws used for comprisons etween ll vitmin D nd diet groups t ech time point s well s etween vitmin D groups nd time points, seprtely for oese nd len (In E. Hollidy, 212, Two-Wy ANOVA (v1..3) in Free Sttistics Softwre (v r7), Office for Reserch Development nd Eduction, 2ANOVA.wsp). An * indictes sttisticlly significnt results corresponding to * for p <.5, ** for p <.1, *** for p <.1 nd **** for p <.1. All dt were screened for potentil outliers using GrphPd online softwre (L Joll, CA.), using significnce cut-off of.5. All dt re presented s men ± stndrd devition. Results nd Discussion Serum 25-OH vitmin D concentrtions fll rpidly with low supplementtion to constnt insufficient level, while greter supplementtion leds to little increse; oesity tempers decline in response to low vitmin D chow In order to understnd the vried interreltionships etween vitmin D nd ody weight/composition, we set out to estlish rnge of serum 25-OH vitmin D concentrtions etween insufficiency (>1 ng/ml) nd ove sufficiency (>3 ng/ml) in len nd oese mice. To do so, we provided len mice vitmin D 3 supplementtion t low (125 IU/kg chow), stndrd for most niml fcilities (1, IU/kg chow) nd high (4, IU/kg chow) mounts nd in oese mice y providing low (162 IU/ kg chow), stndrd (1,282 IU/kg chow) nd high (5,169 IU/kg chow) mounts (Tle 1). We incresed vitmin D 3 supplementtion for oese groups to ccount for the lower food consumption rte tht occurs during high ft feeding [25, 26]. As expected, we found no sttisticlly significnt difference in the mount of vitmin D 3 eing consumed etween len nd oese mousegroups(tle2).toconfirmestlishmentof vitmin D insufficient nd sufficient mice we mesured serum 25-OH vitmin D concentrtions t seline nd every two weeks therefter (Fig. 1). There ws no sttisticlly significnt difference in serum 25-OH vitmin D etween len nd oese mice (len n = 26, 32.8 ± 4.4 ng/ml versus oese n = 19, 3.9 ± 1.6 ng/ml, p =.9). Similrly, Prk et l. did not find differences in serum 25-OH vitmin D in oese nd len 22 week old mice, suggesting ody composition does not modulte serum vitmin D levels during consistent supplementtion [25]. We then modified choleclciferol given in chow to elucidte whether ody composition ffects response to ltered supplementtion. Mice given low supplementtion experienced rpid decline evident t two weeks, with stiliztion etween ng/ml y 8 weeks, independent of eing len or oese. The rte of decline in serum 25-OH Tle 2 Men food nd vitmin D 3 consumption in mice. Len nd oese mice were given d liitum ccess to chow contining vrying mounts of vitmin D 3 nd food ws weighed weekly to determine men (± stndrd devition) consumption Supplementtion Group Food Consumption (g/mouse/dy) Vitmin D 3 Consumption (IU/mouse/dy) Comprison Len nd Oese Low Len 3.38 ± ±.6 p = 1. Oese 2.82 ± ±.3 Stndrd Len 3.24 ± ±.39 p =.86 Oese 2.77 ± ±.25 High Len 4. ± ± 1.87 p =.57 Oese 2.94 ± ±.85

4 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 4 of 9 Fig. 1 Anlysis of serum 25-OH vitmin D 3 concentrtions of len nd oese mice in response to ltered vitmin D 3 supplementtion. Serum 25-OH vitmin D 3 concentrtion ws mesured every two weeks using ELISA in len (L) nd oese (O) mice given low (125 IU/kg chow), stndrd (STD, 1 IU/kg chow) or high (4 IU/kg chow) mounts of vitmin D 3 Decline from seline serum 25-OH vitmin D concentrtion ws further clculted for L-LOW nd O-LOW. Symols indicte p <.5 etween L-HIGH nd L-STD (*), L-HIGH nd O-HIGH ( ),ndl-lowndo-low( ) vitmin D for len mice is consistent with findings in rts, gots nd cows [27 29], ut more rpid thn the reported hlf-life of pproximtely 2 dys in humns [3]. However, the rte of decline ws steeper in len compred to oese mice, with the decline from seline over two weeks eing 18.8 ± 5.7 ng/ml versus 11.9 ± 1.1 ng/ml (**p =.84), respectively (Fig. 1). This slient oservtion supports the notion tht ft sequestrtion my uffer ginst the loss of serum 25-OH vitmin D during low supplement intke. Furthermore, serum 25-OH vitmin D ws significntly greter t severl time points in low supplemented oese mice compred to len (week 8: *p =.247, week 2: *p =.41, nd week 24: ***p =.3, Fig. 1), which would not e consistent with the Drincic et l. dilution model [23]. Yet, the overll mgnitude of the differences (~1 2 ng/ml) does not strongly support role of ft sequestrtion in controlling serum 25-OH vitmin D. Interestingly, higher supplementtion filed to significntly increse serum 25-OH vitmin D concentrtions compred to stndrd mounts fter 24 weeks in oth len nd oese mice. Non-liner increses during supplementtion to rise serum vitmin D ove 3 ng/ml hve een oserved in rts [31], nd similr 25-OH concentrtions were detected in len mice given 1 IU/kg chow compred to oese mice given just over 2 IU/kg chow [14]. Tken together, these reports nd our dt reflecting non-liner increse in supplementtion etween 1 4 IU/kg chow re inconsistent with those of Fleet et l. [32] demonstrting liner increses from 4 IU/kg chow through 2, IU/kg chow. It is importnt to note tht Fleet et l. used wenling mice in nd did not specificlly interrogte supplementtion with 2 or 4 IU/kg chow. Furthermore, our dt suggest tht the oese/len sttus of the mouse does not modulte the effects of higher supplementtion. These findings run contrry to numerous humn clinicl trils showing diminished elevtions with supplementtion due to oesity [2, 21, 33, 34], ut not ll [19, 35]. A possile explntion for the lck of effect in our study ws the filure of higher supplementtion to increse 25-OH vitmin D levels ove stndrd supplementtion in either len or oese mice, ceiling effect tht my e msking potentil oesity impcts. Our dt show tht high supplemented len mice hd significntly greter 25-OH vitmin D concentrtion t week 16 over oese mice (week 16, **p =.4 versus high supplemented oese mice), however, s this difference did not persist we were unle to confidently conclude tht oesity modultes the effects of higher supplementtion. Oese mice, independent of serum vitmin D sttus, exhiit differences in serum 1,25-(OH) 2 vitmin D nd intct PTH concentrtion reltive to len mice, with no effect on serum clcium To further identify the impcts of vried 25-OH vitmin D levels, we nlyzed the serum concentrtion of 1,25-(OH) 2 vitmin D, intct prthyroid hormone (PTH) nd clcium (Fig. 2-c) in len nd oese mice fter 24 weeks of ltered vitmin D 3 supplementtion. Our findings revel there were no significnt differences in ny serum 25-OH vitmin D levels etween vitmin D groups in either len or oese mice. However, oese mice exhiited lower 1,25-(OH) 2 vitmin D nd higher intct PTH compred to len mice (****p <.1 for oth), nd these findings in our mouse model re consistent with dt from study in len nd oese humns [36]. We did not oserve sttisticlly significnt differences in serum clcium due to oesity (p =.27). Our findings for intct PTH nd serum clcium in oese nd len 48-week-old mice re in greement with the reported levels in 22-week-old oese nd len mice [25]. However, we do report significntly lower, nd Prk et l. [25] significntly higher, serum 1,25-(OH) 2 vitmin D in oese mice reltive to len. As expected, we detected positive ssocition etween serum levels of 1,25-(OH) 2 vitmin D nd intct PTH (r 2 =.24, **p =.14, Fig. 2d).

5 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 5 of 9 Serum 1,25-(OH) 2 Vit-D (pmol/l) c Serum Clcium (mmol/l) Len Oese Whether this reltionship exists in Prk et l. is not reported. The nture of these conflicting dt my e due to the greter length of time of exposure to high ft diet (48 weeks in our study versus 18 weeks for Prk et l.) tht hs llowed greter time for n equilirium etween PTH nd serum 1,25-(OH) 2 vitmin D consistent with oservtions in humn studies [36]. To our surprise, differing supplementtion nd the susequent impcts on serum 25-OH vitmin D concentrtions, hd no significnt correltion with 1,25-(OH) 2 vitmin D or ipth serum mrkers in these mice, finding tht is comprle to oservtions from study in rts [31]. Although, this study my hve een underpowered to detect effects on 1,25-(OH) 2 vitmin D, intct PTH nd clcium, this phenomenon my lso reflect the nrrow dosing rnge designed to induce insufficiency. Indeed effects on these serum mrkers were reported in other studies when supplementing outside the normo-physiologicl rnge used in our study of roughly 125 5, IU/kg chow [32, 37]. Len mice receiving higher mounts of choleclciferol hve higher cloric intke yet mintin similr ody weight Numerous humn studies hve identified negtive ssocitions etween ody mss index (BMI) nd serum 25- OH vitmin D concentrtions [2, 4, 6, 38], leding to Serum intct PTH (nmol/ml) Len Oese Len Oese d Intct PTH (nmol/l) R² = ,25(OH) 2 vitmin D (pmol/l) Fig. 2 Serum profiles of len nd oese mice following supplementtion with vried mounts of Vitmin D 3. Serum ws collected following 24 weeks of either low, stndrd or high vitmin D 3 supplementtion, nd nlyzed for serum 1,25-(OH) 2 vitmin D 3,intctPTH nd clcium c concentrtion using ELISA or colorimetric ssy. Correltive nlysis ws further performed etween serum intct PTH nd serum 1,25-(OH) 2 vitmin D d. Different letters denote significnt difference etween len nd oese (****p <.1) specultion tht low 25-OH vitmin D my promote weight gin nd correction of vitmin D sttus my promote weight loss in overweight individuls. To investigte the modulting effects of serum vitmin D on ody mss index, we followed ody weight chnges in len nd oese mice supplemented with vrying mounts of vitmin D for 24 weeks (Fig. 3). Our findings suggest vitmin D insufficiency does not potentite dditionl weight gin in len or oese mice compred with sufficiency. Furthermore, no effect on BMI ws oserved in highly supplemented len or oese mice, which is consistent with humn studies tht hve ttempted to promote weight loss through vitmin D supplementtion [7 9, 39]. However, in our study, mice with high supplementtion hd serum 25-OH nd 1,25-(OH) 2 vitmin D concentrtions similr to mice given stndrd mounts, which my indicte tht these mice did not receive high enough supplementtion to ffect BMI. Indeed, evidence tht greter supplementtion might ffect BMI ws recently reported y Mrcotorchino et l. [4], where BMI (g/cm 2 ).1-2 Mouse Length (cm) L-LOW L-STD L-HIGH O-LOW O-STD O-HIGH Time (weeks) Len Oese c Food Consumption (kcl/mouse/dy) Len Oese Fig. 3 Food consumption nd BMI in mice fed high or low ft diet contining vrying levels of vitmin D 3. Twenty-four week old mle mice were rndomly sorted into low, stndrd (STD) or high vitmin D 3 supplementtion groups nd followed for 24 weeks. Body mss index for ech mouse ws determined weekly using mouse length mesurements from week 24. Mouse length endpoint mesurements reflect the distnce from nose to the se of the til. Food consumption ws mesured weekly nd group mens were verged c. Different letters denote significnt differences (****p <.1)

6 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 6 of 9 15, IU/kg chow given to mice prevented weight gin during high ft diet. Mice in tht study were only 6 weeks of ge t the strt of the experiment nd not oese t seline, yet these findings my indicte necessry level of supplementtion to see n effect. Despite the lck of n effect of higher supplementtion on BMI or serum vitmin D metolites, our len mice supplemented with 4 IU/kg chow hd greter cloric consumption over the length of the study (14.6 ± 1.7 kcl/ mouse/dy versus 11.8 ± 1.4 for 1 IU nd 12.3 ± 1.7 for 125 IU, ****p <.1 for oth, Fig. 3). The inference from these dt is tht the greter food consumption ws offset y higher metolism, perhps suggesting weight loss strtegy involving cloric restriction might e improved y vitmin D supplementtion. Interestingly, Shpses et l. were unle to discern n effect on weight loss in humns with dul vitmin D supplementtion nd cloric restriction protocol [41]. But it is possile tht the 25 IU vitmin D 3 /dy supplementtion strtegy ws not equivlent to the higher level of supplementtion in our mice. Overll, our findings tht vitmin D insufficiency hd no effect on BMI in our mice run contrry to findings y Bstie et l. [14] nd to lesser extent Liu et l. [13], where diet induced vitmin D deficient mice were resistnt to western nd high ft diet induced weight gin, respectfully. However these studies induced vitmin D deficiency concurrently with ltertions in diet. Wheres in our study the mice hd een fed HFD for four months prior to initition of the vitmin D intervention. Additionlly we elieve the ge t which mice were mde insufficient/deficient my lso e n importnt fctor. We oserved tht the verge length of our oese mice t 48 weeks of ge ws Fig. 4 Impcts on one minerl density nd reltionship with serum iomrkers. The effects of high ft nd low ft diet contining vrying levels of vitmin D on one minerl density were nlyzed using DEXA No sttisticl differences oserved etween vitmin D groups. Differences etween ll len nd oese mice were significnt for one minerl density ( p =.4; p <.1; c p <.8). Bone minerl density ws not correlted with serum 25-OH or 1,25-(OH) 2 vitmin D c, ut ws correlted with serum intct PTH (R 2 =.32,****p <.1)d

7 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 7 of 9 significntly longer thn len mice (15.5 ± 1.3 cm versus 1.6 ± 2.3 cm, ****p <.1, Fig. 3c). We found no length differences etween vitmin D groups, which would e expected s mice hve likely stopped growing y the time supplementtion ws djusted. However, oth Bstie et l. nd Liu et l. vried vitmin D supplementtion efore 3 5 weeks of ge which my hve led to numerous impcts on the development of the mice not seen in our study since vritions in vitmin D supplementtion occurred fter mturity. Interestingly, lthough our study is the first report of high ft feeding leding to incresed length in mice, severl humn studies hve found trends etween higher BMI nd height in dolescents [42 44]. Serum 25-OH vitmin D insufficiency does not modulte ody ft, len mss or one minerl density in either len or oese mice Vitmin D is cnoniclly recognized s eing importnt for one helth, nd more recently for impcts on ody ft nd len mss, yet the impcts of chronic insufficiency on these prmeters hve not een elucidted. Therefore, we performed dul X-Ry sorptiometry (DEXA) nlysis t seline nd every two months therefter on our mouse cohorts (Figs. 4 nd 5). Surprisingly, despite 6 months of vitmin D insufficiency, neither len nor oese insufficient mice exhiited lower BMD compred to sufficient mice (Fig. 4). These findings re in generl greement with Fleet et l. [32], with the exception of lower BMD seen in group receiving 25 IU vitmin D 3 /kg chow group, for which supplementtion ws elow our lowest tested group. Interestingly, high supplementtion filed to increse BMD in mice s well, lthough we note this my e due to the inility of this level of supplementtion to sustntilly increse serum levels of 25-OH vitmin D or the possiility tht we were underpowered to oserve this effect. Furthermore, we did not identify ssocition etween BMD with either serum 25-OH vitmin D (Fig. 4) or 1,25-(OH) 2 vitmin D (Fig. 4c), ut there ws negtive correltion etween BMD nd serum intct PTH (r 2 =.33, ****p <.1 Fig. 4d). This difference remined significnt when controlling for BMI of the mice (**p =.14, dt not shown) nd reflects the key role of PTH in one homeostsis [45]. However, oese mice exhiited lower BMD t ll time points except t seline ( weeks: p =.17; 8 weeks: **p =.4; 16 weeks: ****p <.1; nd 24 weeks: **p =.8, Fig. 4). Interestingly, in other mouse studies such differences in totl BMD etween oese nd len were not oserved, yet differences in one density were oserved when utilizing μct [46 48]. One key distinction etween our study nd the other reports is tht the length of nerly 48 weeks in our study tht my hve provided enough time for oesity relted-effects on BMD to ecome mnifest. It is lso noteworthy tht higher supplementtion ws not le to counterct the effects of oesity. However, s higher supplementtion did not significntly ffect 25-OH or 1,25-(OH) 2 vitmin D concentrtion, the possiility remins tht supplementtion eyond 4, IU/kg chow my rescue oesity relted one deficits. Our dt revel tht vitmin D insufficiency does not modulte ody ft percentge (Fig. 5) or len tissue mss (Fig. 5) in len or oese mice. Yet, we did detect lrger gin of len mss in oese mice versus len mice over the 48-week period (**p =.2, Fig. 5). Interestingly, no effects were seen in mice receiving higher mounts of vitmin D 3 in chow contrry to oservtions in severl humn Body Ft (%) L-STD LFD1 L-HIGH LFD4 O-LOW HFD125 L-LOW LFD125 O-STD HFD1 HFD4 O-HIGH Gin in len mss (g) Time (weeks) Len Oese Fig. 5 Anlysis of ody composition of len nd oese mice given low, stndrd nd high vitmin D supplementtion. The effects of high ft nd low ft diet contining vrying levels of vitmin D on ody ft % nd the gin in len ody mss over 48 weeks were nlyzed using DEXA. No sttisticl differences oserved etween vitmin D groups. Differences etween ll len nd oese mice were significnt for ody ft % (t ll time points, ****p <.1) nd gin in len ody mss ( versus, **p =.2)

8 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 8 of 9 studies [7, 49, 5], ut not ll [33]. This discrepncy my e due to differing rnge of supplementtion levels, or tht vitmin D my hve roles unique to humn physiology. Conclusions We hve estlished model of vitmin D insufficiency in len nd oese mice y reducing vitmin D 3 supplementtion in chow. Oese mice exhiited slower rte of decline in serum 25-OH vitmin D nd modestly higher levels t equilirium. No differences in serum 1,25-(OH) 2 vitmin D, intct prthyroid hormone (PTH) or serum clcium were oserved etween len nd oese mice within the sme vitmin D supplementtion group. Yet oese mice exhiited lower serum clcitriol, higher serum PTH, nd lower one minerl density (BMD) thn did len mice. It lso ppers tht 25-OH vitmin D concentrtions etween 1 to 4 ng/ml did not correlte with ody weight or ody ft in mice, lthough higher supplementtion in mice fed stndrd diet correlted with incresed food consumption weight or len/ft mss. The increse in cloric consumption without concurrent increse in energy storge implies heightened metolism, which, if coupled to cloriclly restricted diet my prove to e n effective strtegy for weight loss. Arevitions BMI: Body mss index; DEXA: Dul Emission X-Ry Asorptiometry; ipth: Intct prthyroid hormone; μct: Micro computed tomogrphy; VDR: Vitmin D receptor Acknowledgements The uthors thnk Guy Howrd of the University of Mimi nd Jessic Reynolds of the University t Bufflo for their suggestions nd insights during the preprtion of this mnuscript. The uthors lso wish to thnk the University t Bufflo, University of Mimi, Mimi VA, nd the reserch service of the VA Western New York Helthcre System. Funding This work ws supported y grnts from VA RR&D RX166 nd VA BLR&D BX758 s well s from the Indin Tril Chritle Foundtion, Inc. Avilility of dt nd mterils Plese contct uthor for dt requests. Authors contriutions KLS nd BRT conceived of the study nd drfted the mnuscript. KLS nd MP performed dt nlysis. MP nd PY performed DEXA nlysis of mice. KLS, MP, MRG, MH, PY nd ZWS performed longitudinl lood collections, hrvested mouse tissues nd performed ELISA nd colorimetric nlyses. KLS, MRG nd MH monitored niml weight, chow nd helth. All uthors red nd pproved the finl mnuscript. Competing interest The uthors declre tht they hve no competing interests. Consent for puliction Not pplicle. Ethics pprovl nd consent to prticipte Not pplicle. Received: 27 Octoer 216 Accepted: 13 Ferury 217 References 1. Wei MY, Giovnnucci EL. Vitmin D nd multiple helth outcomes in the Hrvrd cohorts. Mol Nutr Food Res. 21;54(8): Dong Y, et l. Low 25-hydroxyvitmin D levels in dolescents: rce, seson, diposity, physicl ctivity, nd fitness. Peditrics. 21;125(6): Mithl A, et l. Glol vitmin D sttus nd determinnts of hypovitminosis D. Osteoporos Int. 29;2(11): Mi XM, et l. Cross-sectionl nd prospective cohort study of serum 25- hydroxyvitmin D level nd oesity in dults: the HUNT study. Am J Epidemiol. 212;175(1): Goshyeshi L, et l. Assocition etween metolic syndrome, BMI, nd serum vitmin D concentrtions in rheumtoid rthritis. Clin Rheumtol. 212;31(8): Frrell SW, Willis BL. Crdiorespirtory fitness, diposity, nd serum 25- dihydroxyvitmin D levels in women: the Cooper Center Longitudinl Study. J Womens Helth (Lrchmt). 212;21(1): Slehpour A, et l. A 12-week doule-lind rndomized clinicl tril of vitmin D(3) supplementtion on ody ft mss in helthy overweight nd oese women. Nutr J. 212;11: Sneve M, Figenschu Y, Jorde R. Supplementtion with choleclciferol does not result in weight reduction in overweight nd oese sujects. Eur J Endocrinol. 28;159(6): Zittermnn A, et l. Vitmin D supplementtion enhnces the eneficil effects of weight loss on crdiovsculr disese risk mrkers. Am J Clin Nutr. 29;89(5): Kmychev E, Berg V, Jorde R. Insulin-like growth fctor I, growth hormone, nd insulin sensitivity: the effects of one-yer choleclciferol supplementtion in middle-ged overweight nd oese sujects. Endocrine. 213;43(2): Nrvez CJ, et l. Len phenotype nd resistnce to diet-induced oesity in vitmin D receptor knockout mice correltes with induction of uncoupling protein-1 in white dipose tissue. Endocrinology. 29;15(2): Weer K, Eren RG. Differences in triglyceride nd cholesterol metolism nd resistnce to oesity in mle nd femle vitmin D receptor knockout mice. J Anim Physiol Anim Nutr (Berl). 213;97(4): Liu XJ, et l. Vitmin d deficiency ttenutes high-ft diet-induced hyperinsulinemi nd heptic lipid ccumultion in mle mice. Endocrinology. 215;156(6): Bstie CC, et l. Dietry choleclciferol nd clcium levels in Western-style defined rodent diet lter energy metolism nd inflmmtory responses in mice. J Nutr. 212;142(5): Wong KE, et l. Trgeted expression of humn vitmin D receptor in dipocytes decreses energy expenditure nd induces oesity in mice. J Biol Chem. 211;286(39): Alkhrfy KM, et l. Effects of vitmin D tretment on skeletl muscle histology nd ultrstructurl chnges in rodent model. Molecules. 212; 17(8): Yin Y, et l. Vitmin D ttenutes high ft diet-induced heptic stetosis in rts y modulting lipid metolism. Eur J Clin Invest. 212;42(11): Aguirre Cstned R, et l. Response to vitmin D3 supplementtion in oese nd non-oese Cucsin dolescents. Horm Res Peditr. 212;78(4): Cnto-Cost MH, Kunii I, Huche OM. Body ft nd choleclciferol supplementtion in elderly homeound individuls. Brz J Med Biol Res. 26;39(1): Lee P, et l. Adequcy of vitmin D replcement in severe deficiency is dependent on ody mss index. Am J Med. 29;122(11): Wortsmn J, et l. Decresed iovilility of vitmin D in oesity. Am J Clin Nutr. 2;72(3): Sli W, Brnett-Griness O, Rennert G. The reltionship etween oesity nd the increse in serum 25(OH)D levels in response to vitmin D supplementtion. Osteoporos Int. 213;24(4): Drincic AT, et l. Volumetric dilution, rther thn sequestrtion est explins the low vitmin D sttus of oesity. Oesity (Silver Spring). 212;2(7): Mffei M, et l. Leptin levels in humn nd rodent: mesurement of plsm leptin nd o RNA in oese nd weight-reduced sujects. Nt Med. 1995; 1(11): Prk JM, Prk CY, Hn SN. High ft diet-induced oesity lters vitmin D metolizing enzyme expression in mice. Biofctors. 215;41(3):

9 Seldeen et l. Nutrition & Metolism (217) 14:26 Pge 9 of Fenton PF, Crr CJ. The nutrition of the mouse. XI. Response of four strins to diets differing in ft content. J Nutr. 1951;45(2): Brouwer DA, et l. Rt dipose tissue rpidly ccumultes nd slowly releses n orlly-dministered high vitmin D dose. Br J Nutr. 1998;79(6): Hidiroglou M. Kinetics of intrvenously dministered 25-hydroxyvitmin D3 in sheep nd the effect of exposure to ultrviolet rdition. J Anim Sci. 1987;65(3): Wilkens MR, et l. Is the metolism of 25-hydroxyvitmin D3 ge-dependent in diry cows? J Steroid Biochem Mol Biol. 213;136: Smith JE, Goodmn DS. The turnover nd trnsport of vitmin D nd of polr metolite with the properties of 25-hydroxycholeclciferol in humn plsm. J Clin Invest. 1971;5(1): Anderson PH, et l. Vitmin D depletion induces RANKL-medited osteoclstogenesis nd one loss in rodent model. J Bone Miner Res. 28;23(11): Fleet JC, et l. Serum metolite profiles nd trget tissue gene expression define the effect of choleclciferol intke on clcium metolism in rts nd mice. J Nutr. 28;138(6): Gllgher JC, Ylmnchili V, Smith LM. The effect of vitmin D supplementtion on serum 25(OH)D in thin nd oese women. J Steroid Biochem Mol Biol. 213; 136: Hrel Z, et l. Low vitmin D sttus mong oese dolescents: prevlence nd response to tretment. J Adolesc Helth. 211;48(5): Gllgher JC, et l. Dose response to vitmin D supplementtion in postmenopusl women: rndomized tril. Ann Intern Med. 212;156(6): Prikh SJ, et l. The reltionship etween oesity nd serum 1,25-dihydroxy vitmin D concentrtions in helthy dults. J Clin Endocrinol Met. 24; 89(3): Rowling MJ, et l. High dietry vitmin D prevents hypoclcemi nd osteomlci in CYP27B1 knockout mice. J Nutr. 27;137(12): Forsythe LK, et l. Effect of diposity on vitmin D sttus nd the 25- hydroxycholeclciferol response to supplementtion in helthy young nd older Irish dults. Br J Nutr. 212;17(1): Belenchi AM, et l. Correcting vitmin D insufficiency improves insulin sensitivity in oese dolescents: rndomized controlled tril. Am J Clin Nutr. 213;97(4): Mrcotorchino J, et l. Vitmin D protects ginst diet-induced oesity y enhncing ftty cid oxidtion. J Nutr Biochem. 214;25(1): Shpses SA, et l. Vitmin D supplementtion nd clcium sorption during cloric restriction: rndomized doule-lind tril. Am J Clin Nutr. 213;97(3): Krzyznowsk M, Umlwsk W. The reltionship of Polish students height, weight nd BMI with some socioeconomic vriles. J Biosoc Sci. 21; 42(5): Stovitz SD, et l. Growing into oesity: ptterns of height growth in those who ecome norml weight, overweight, or oese s young dults. Am J Hum Biol. 211;23(5): Stovitz SD, et l. The interction of childhood height nd childhood BMI in the prediction of young dult BMI. Oesity (Silver Spring). 28;16(1): Sneve M, et l. The ssocition etween serum prthyroid hormone nd one minerl density, nd the impct of smoking: the Tromso Study. Eur J Endocrinol. 28;158(3): Co JJ, Sun L, Go H. Diet-induced oesity lters one remodeling leding to decresed femorl treculr one mss in mice. Ann N Y Acd Sci. 21;1192: Ionov-Mrtin SS, et l. Chnges in corticl one response to high-ft diet from dolescence to dulthood in mice. Osteoporos Int. 211;22(8): Lu XM, Zho H, Wng EH. A high-ft diet induces oesity nd impirs one cquisition in young mle mice. Mol Med Rep. 213;7(4): Cron-Join M, et l. Elevted serum 25(OH)D concentrtions, vitmin D, nd clcium intkes re ssocited with reduced dipocyte size in women. Oesity (Silver Spring). 211;19(7): Crrillo AE, et l. Impct of vitmin D supplementtion during resistnce trining intervention on ody composition, muscle function, nd glucose tolernce in overweight nd oese dults. Clin Nutr. 213;32(3): Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t