Excessive fructose intake causes 1,25-(OH) 2 D 3 -dependent inhibition of intestinal and renal calcium transport in growing rats

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1 Am J Physiol Endocrinol Met 3: E33 E33, 3. First pulished April 9, 3; doi:.5/jpendo.58.. Excessive fructose intke cuses,5-(oh) D 3 -dependent inhiition of intestinl nd renl clcium trnsport in growing rts Veronique Dourd, Yves Sgh, Jcklyn Lee, Chirg Ptel, Frncis W. Kemp, 3 John D. Bogden, 3 Sheldon Lin, nd Ronldo P. Ferrris Deprtment of Phrmcology nd Physiology, University of Medicine nd Dentistry of New Jersey - New Jersey Medicl School, Newrk, New Jersey; Tissue Protection nd Repir, Snofi-Genzyme R&D Center, Genzyme, A Snofi Compny, Frminghm, Msschusetts; 3 Trce Element nd Minerl Reserch Lortory, Deprtment of Preventive Medicine nd Community Helth, University of Medicine nd Dentistry of New Jersey - New Jersey Medicl School, Newrk, New Jersey; Deprtment of Orthopedics, University of Medicine nd Dentistry of New Jersey - New Jersey Medicl School, Newrk, New Jersey Sumitted Novemer ; ccepted in finl form 3 April 3 Dourd V, Sgh Y, Lee J, Ptel C, Kemp FW, Bogden JD, Lin S, Ferrris RP. Excessive fructose intke cuses,5-(oh) D 3 -dependent inhiition of intestinl nd renl clcium trnsport in growing rts. Am J Physiol Endocrinol Met 3: E33 E33, 3. First pulished April 9, 3; doi:.5/jpendo.58.. We recently discovered tht chronic high fructose intke y lctting rts prevented dptive increses in rtes of ctive intestinl C trnsport nd in levels of,5-(oh) D 3, the ctive form of vitmin D. Since sufficient C sorption is essentil for skeletl growth, our discovery my explin findings tht excessive consumption of sweeteners compromises one integrity in children. We tested the hypothesis tht,5-(oh) D 3 medites the inhiitory effect of excessive fructose intke on ctive C trnsport. First, compred with those fed glucose or strch, growing rts fed fructose for wk hd mrked reduction in intestinl C trnsport rte s well s in expression of intestinl nd renl C trnsporters tht ws tightly ssocited with decreses in circulting levels of,5-(oh) D 3, one length, nd totl one sh weight ut not with serum prthyroid hormone (PTH). Dietry fructose incresed the expression of -hydroxylse (CYPA) nd decresed tht of -hydroxylse (CYP7B), suggesting tht fructose might enhnce the renl ctolism nd impir the synthesis, respectively, of,5-(oh) D 3. Serum FGF3, which is secreted y osteocytes nd inhiits CYP7B expression, ws upregulted, suggesting potentil role of one in mediting the fructose effects on,5-(oh) D 3 synthesis. Second,,5-(OH) D 3 tretment rescued the fructose effect nd normlized intestinl nd renl C trnsporter expression. The mechnism underlying the deleterious effect of excessive fructose intke on intestinl nd renl C trnsporters is reduction in serum levels of,5-(oh) D 3. This finding is significnt ecuse of the lrge mounts of fructose now consumed y Americns incresingly vulnerle to C nd vitmin D deficiency. one; growth; intestine; kidney; prthyroid hormone; vitmin D; FGF3 IN CHILDREN AND YOUNG ADULTS, excessive consumption of sugr-sweetened everges nd of sweets my increse the incidence of one frcture, decrese one minerl density (BMD), nd reduce the rte of one minerl ccrul (35, 5). Moreover, chronic consumption of these sweeteners, including fructose, y young rts reduces one mechnicl strength nd minerl content (, 6, 8, 5) These studies suggest tht Address for reprint requests nd other correspondence: R. P. Ferrris, Dept. of Phrmcology & Physiology, UMDNJ - New Jersey Medicl School, 85 S. Ornge Ave., MSB-H6, Newrk, NJ 7. sweeteners, including fructose, compromise one integrity, ut the mechnism remins uncler (9). Sufficient intke, intestinl sorption, nd renl resorption of C s well s incresed levels of,5-(oh) D 3, or clcitriol, the ctive form of vitmin D 3, re essentil in mintining C homeostsis nd one qulity. Totl intestinl C sorption consists of pssive prcellulr nd,5-(oh) D 3 -dependent ctive trnscellulr pthwy (). Active C trnsport involves C entry through the picl trnsient receptor potentil vnilloid C chnnels (TRPV6 for the intestine nd TRPV5 for the kidney), its intrcellulr diffusion vi C -inding proteins (CBP9k for the intestine nd CBP8k for the kidney), nd its extrusion cross the intestinl or renl solterl memrne through the N /C exchnger (NCX) long with the plsm memrne C -ATPse (PMCA) (6). Since the mjor source for C cquisition is intestinl sorption, vertertes disply dptive increses in ctive C trnscellulr trnsport, chieved y ugmenting levels of,5- (OH) D 3, whenever physiologicl demnds for C increse. The precursor of vitmin D, choleclciferol, is hydroxylted in the liver y 5-hydroxylses, producing clcidiol or 5- (OH)D 3, which is further hydroxylted in the kidney y hydroxylse (encoded y CYP7B) to,5-(oh) D 3. Degrdtion of 5-(OH)D 3 nd,5-(oh) D 3 is medited y - hydroxylse (CYPA). Serum levels of,5-(oh) D 3 re tightly regulted y feedck loops controlling renl CYP7B nd CYPA expression (39). We recently discovered in dult rt models of chronic kidney disese, s well s in lctting rts, tht dietry fructose reduced rtes of intestinl C trnsport nd circulting levels of,5-(oh) D 3, in ssocition with decrese in the inding of the vitmin D receptor (VDR) on the promoter of TRPV6 nd CBP9k genes (, 6). This indicted tht high fructose intke, y ltering,5-(oh) D 3 homeostsis, my inhiit the dptive increses in C trnsport during lcttion. U.S. dietry intke dt pooled y Popkin nd Nielsen () show n 83 kcl/dy increse of cloric sweetener consumption in the U.S. etween 96 nd. This upsurge represents % increse in the proportion of dily energy requirements otined from cloric sweeteners, including fructose. Fructose is trnsported pssively cross the picl memrne of the intestinl cell y the fcilittive glucose trnsporter GLUT5 (SlcA5), while the sodium-dependent glucose trnsporter SGLT (Slc5A) is responsile for most glucose trnsport (). Both sugrs re then trnsported cross the solterl mem- Downloded from y on Novemer 3, /3 Copyright 3 the Americn Physiologicl Society E33

2 E3,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE rne y GLUT (SlcA) into the portl vein nd the liver, where fructose is rpidly metolized. Mny recent epidemiologicl studies correlte the rise in fructose consumption with vrious modern-dy helth concerns, including oesity, metolic syndrome, nd osteoporosis (5, 9). Since fructose is nturl component of humn diets, its ssocition with metolic diseses my rise from recently documented, chroniclly excessive intke. Prticulr emphsis is plced on helth concerns of ctively growing children nd dolescents whose intke per unit ody weight of sweeteners, prticulrly fructose, is much greter thn tht of dults (3). Few of the lrge numer of studies on the metolic effects of dietry fructose in humns nd rodent models hve utilized neontl, wening, or postwening ge groups (9,,, 5, 3), which consume the gretest quntity of fructose per kilogrm of ody weight. Since rpid growth in young mmmls requires mrked increses in C intke nd sorption driven y higher level of,5-(oh) D 3 thn older dults (7), we investigted in wening rts the effect of chroniclly high fructose intke on intestinl C trnsport,,5- (OH) D 3 homeostsis, nd one qulity. To test our hypothesis tht the fructose-induced decrese in serum level of,5- (OH) D 3 is the mechnism y which dietry fructose impirs intestinl C sorption, we then treted fructose-fed rts with,5-(oh) D 3 nd determined whether the tretment prevented the deleterious effects of fructose. MATERIALS AND METHODS Animls. All the procedures in this study were pproved y the Institutionl Animl Cre nd Use Committee, UMDNJ-New Jersey Medicl School. Studies were conducted on postwening Sprgue Dwley (Chrles Rivers) mle, virus/ntiody-free rts ( dys old). Rts were kept under stndrd conditions: :-h light-drk cycle nd C in dust-free cellulose edding. Experimentl design. In the first study, postwening rts ( 55 g) were rndomly divided into three groups (n 7 9 rts) nd then fed 63% glucose, fructose, or strch diets sed on stndrd Americn Institute of Nutrition (AIN)-93G formul contining norml C nd phosphte (Pi) levels designed to meet growth requirements of young rodents (, 6). Animls were fed the diets d liitum for wk, from to 5 dys old. The AIN recommends either 73% (AIN-93M diet) or 63% (AIN-93G) crohydrte levels, slightly higher thn the verge for humns (5%) consuming high-crohydrte diet (7). Since this is mong the initil studies evluting the effects of dietry fructose on,5-(oh) D 3 nd C homeostsis, we used n niml model with high demnd for these nutrients nd then chllenged this model with n experimentl diet contining high concentrtion of fructose to llow us to detect ny potentil effect it might hve on selected outcomes. Although humns consume lower concentrtions of fructose tht re often mixed with other crohydrtes, studies hve shown tht mixing with glucose my ccelerte fructose effects nd tht lower fructose concentrtions cuse similr deleterious effects to higher fructose concentrtions if consumed for longer time period (5, ). In second study, sme-ge postwening rts from different tch ( 5 g) were rndomly divided into two groups fed 63% glucose or fructose diet, nd then ech group ws further sudivided into two sugroups (n 6 rts), ech receiving dily intrperitonel injections t noon of either.% (vol/vol) ethnol in sline (vehicle) or g/kg ody wt,5-(oh) D 3 for dys. The nimls were euthnized 8 h fter the lst,5-(oh) D 3 or sline injection. In vitro intestinl nutrient trnsport mesurements. Intestinl segments were everted quickly fter isoltion nd prepred s everted scs or sleeves to determine nutrient trnsport rtes t 37 C with 95% O -5% CO s descried previously (). C uptke. The everted gut scs were mde y using the first cm of proximl duodenum, where ctive trnscellulr trnsport of C is loclized (), nd then incuted in C trnsport uffer s previously descried (). The outer luminl nd inner serosl comprtments hd equl initil concentrtions (.5 mm) of nonrdioctive C ; then, 5 C ws dded to the outer mucosl comprtment. After h, the ctive ccumultion of 5 C in the inner serosl comprtment ws clculted s rtio of the finl concentrtion of ( 5 C serosl/ 5 C mucosl comprtments) nd then normlized to tht of rts fed strch. Fructose nd glucose uptke. Four -cm jejunl segments were mde into everted sleeves, mounted on rods, nd preincuted for 5 min in Kres ringer icronte (KRB) s descried previously (). Two segments ech were then incuted in 5 mm glucose- or fructose-krb solutions contining trcer concentrtions of [ C]glucose or [ C]fructose, respectively. Phosphte uptke. Intestinl Pi trnsport ws determined in two consecutive -cm segments of medil jejunum using the previously descried everted gut sc ssy (). Briefly, the everted intestinl segment for determining totl Pi trnsport ws incuted for h in N -contining Pi trnsport uffer (. mm Pi), while the djoining segment for determining N -independent Pi trnsport ws incuted in N -free trnsport uffer. The totl nd N -independent trnsport of 33 Pi ws expressed s rtio of the finl concentrtion of ( 33 Pi serosl/ 33 Pi mucosl comprtments) nd normlized to the rtio otined from scs of rts fed strch. Mesurements of serum clinicl prmeters. Following erlier work (), serum uric cid concentrtions were determined using QuntiChrom Uric Acid Assy Kit (BioAssy Systems, Hywrd, CA), nd Pi concentrtions were determined using QuntiChrom Pi Assy Kit. The totl serum C concentrtions were determined y previously descried techniques using flme tomic sorption spectrophotometry (model 63; PerkinElmer, Norwlk, CT) ().,5-(OH) D 3, firolst growth fctor 3, nd PTH ssys. Following erlier work (, 53), serum,5-(oh) D 3 levels were mesured y enzyme immunossy [ImmunoDignosticSystems (IDS)]. Briefly, serum smples were delipidted nd,5-(oh) D 3 immunoextrcted efore the ssy. Serum 5-(OH)D 3 levels were mesured directly y IDS. Intct rt PTH (Immutopics, Sn Clemente, CA) nd intct firolst growth fctor 3 (FGF3; Kinos, Tokyo, Jpn) ELISAs were performed ccording to the mnufcturers instructions. Rel-time PCR. Totl RNA from homogenized intestinl mucos or kidney ws isolted nd reverse trnscried, nd rel-time PCR ws performed using Mx3P (Strtgene, L Joll, CA) s previously descried (3). The control group ws the rts fed strch. The reference gene ws -elongtion fctor, Ef, whose expression is independent of ge nd dietry crohydrte (). Previously pulished primer sequences nd nneling tempertures were used () (). Bone nlyses. Briefly, hrvested right femor were clened of soft tissues nd stored t C wrpped in sline-soked guze (.9% NCl) until testing. Prior to testing, the longitudinl length nd the dimeter t the diphysis (the midsection of the shft) were mesured with sliding cliper nd recorded. The dimeters were mesured t the midshft of the diphysis. The mximum ( o) nd minimum ( o) outer dimeters were mesured efore reking the one, wheres the mximum ( i) nd minimum ( i) inner dimeters were mesured fter reking. The corticl thickness ws clculted y tking the difference etween the mximum nd minimum outer nd inner dimeters [mx ( o i)/; min ( o i)/]. Determintion of one clcium, mgnesium, nd phosphorus. Hrvested humer were dried to constnt weight, nd then orgnic mterils in the dried one were removed using methnol-chloroform mixture (: vol/vol). After extrction, ones were shed overnight in muffle furnce t 8 C. Ashed ones were dissolved in hot nitric-perchloric cid mixture (3:) nd diluted to 5 ml with % Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

3 ,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE E35 nitric cid. For C determintions, the smples were diluted with.5 M K-% nitric cid. Then C contents were determined y previously descried techniques using flme tomic sorption spectrophotometry (Anlyst ; PerkinElmer Norwlk, CT) (7). For phosphorus, the smples were diluted in % trichlorocetic cid nd iron molydte solution to produce colortion within the rnge of stndrd concentrtions prepred with KH PO regent. Smples re then red on visile spectrum spectrophotometer t 66 nm. Western lot nlysis. Western lot nlyses were performed using 5 g of intestinl or renl protein extrcts, following erlier work (). Memrnes were proed with polyclonl ntiodies ginst CBP9k (Swnt Swiss Antiodies) nd CYP7B (Snt Cruz Bioctechnologies) nd then stripped nd reproed with -ctin ntiody (Chemicon Interntionl). Histology nd immunohistochemistry. Immunohistochemistry ws performed on kidney prffin slides using rit nti-mouse NPi [donted y Y. Sgh (), :, in 5% got serum, in Piuffered sline (PBS)], incuted overnight t C, nd then wshed in PBS. The secondry ntiody ws got nti-rit IgG leled with Cy3 (:, Chemicon Interntionl), diluted in % BSA in PBS, nd pplied for h t C. The stined sections were exmined t or mgnifiction with lser scnning confocl microscope (Bio-Rd, Rdince ). All imges of sections eing compred were otined with the sme settings of the microscope. Nonspecific stining with secondry ntiodies only ws consistently negligile. Sttisticl nlyses. Dt re presented s mens SE. For the first experiment (-wk feeding), one-wy ANOVA ws used to determine the differences mong groups with different diets. If there ws significnt difference, Fisher s pired lest significnt difference (LSD) test ws used (STATVIEW, Acus Concepts) to determine differences mong mens. Differences were considered significnt if P.5. For the,5-(oh) D 3 rescue experiment, if n initil two-wy ANOVA indicted significnt effect of,5-(oh) D 3 tretment nd/or diet, one-wy ANOVA followed y LSD test ws used to determine differences mong mens. Simple liner regressions were used to exmine the reltionship etween circulting levels of,5-(oh) D 3 nd FGF3 s well s etween levels of,5-(oh) D 3 nd PTH. A trnsport Reltive B Reltive to EFα C RESULTS 3 CBP9k βctin glucose fructose strch 7 C + Fructose Glucose totl Pi Glucose Fructose Strch pssive Pi TRPV6 CBP9k PMCA NCX GLUT5 SGLT NPi CBP9k/βctin.5.5 Fructose feeding inhiits C trnsport. Chronic consumption of high-fructose diet contining norml C levels more thn douled intestinl fructose uptke ut reduced ctive trnsepithelil C trnsport y lmost one-hlf compred with strch nd glucose diets (Fig. A). The dietry fructose effect on C nd fructose trnsport is specific, ecuse ctive glucose uptke, nd totl Pi trnsport, s well s N -independent pssive Pi trnsport, remined similr mong the three diet groups. To determine whether chnges in trnsport rtes prlleled chnges in mrna levels, we next nlyzed expression of the vrious trnsporters tht ply significnt roles in fructose nd C trnsport (Fig. B). mrna levels of TRPV6 nd CBP9k ech decresed more thn threefold in the duodenum of fructose-fed rts, prlleling mrked reductions in ctive C trnsport. In contrst, the mrna expression of GLUT5 ws upregulted more thn tenfold y dietry fructose. In the distl jejunum (dt not shown), fructose lso mrkedly downregulted TRPV6 nd CBP9k, except tht mrna expression levels were lredy orders of mgnitude less thn in the duodenum. These fructose effects on TRPV6, CBP9k, nd GLUT5 levels were specific, s expression of the solterl C trnsporters PMCA nd NCX were not ffected y the diet. Likewise, the mrna expression of the N /glucose cotrnsporter SGLT nd the intestinl N -dependent phosphte cotrnsporter NPi remined independent of diet. Chnges in protein levels of CBP9k followed the chnges oserved for mrna nd showed lower level of proteins in the intestine of fructose-fed rts (Fig. C). Blood chemistry nd hormone levels. We then investigted the plsm level of,5-(oh) D 3, since it is one of the key hormones controlling intestinl ctive C trnsport, minly y regulting TRPV6 nd CBP9k expression (Tle ). We oserved significnt 3 % decrese in,5-(oh) D 3 plsm concentrtion in rts fed fructose. It is well estlished Fig.. Intestinl C, fructose, glucose, nd Pi trnsport nd trnsporter expression in 5-dy-old rt fed glucose, fructose, or strch fter wening. A:C nd Pi trnsport were mesured using everted intestinl scs formed from the duodenum nd proximl jejunum, respectively. Reltive trnsepithelil C nd Pi trnsport from the luminl to the solterl comprtment were expressed s rtio of ( 5 C inside/ 5 C outside or 33 Pi inside/ 33 Pi outside, respectively) of the everted scs. Reltive fructose nd glucose uptkes into enterocytes were mesured using everted intestinl sleeves from the jejunum. B: mrna expression of C (TRPV6, CBP9k, PMCA, NCX) fructose (GLUT5), glucose (SGLT), nd phosphte (NPi) trnsporters ws nlyzed y rel-time PCR using EF s reference. All dt were normlized reltive to levels seen in rt fed strch. Dt re mens SE (n 8 per group). Mens with different superscript letters re significntly different t P.5. C: protein undnce of CBP9k using -ctin s reference. C trnsport nd trnsporter expression decresed with chronic consumption of fructose. Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

4 E36,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE Tle. Serum iochemistry in 5-dy-old rts fed glucose, fructose, or strch diet for wk Glucose Fructose Strch Significnce,5-(OH) D 3, pmol/l PTH, pg/ml Clcium, mg/dl Phosphte, mg/dl Uric cid, mg/dl Dt re mens SE; n 7 9 per group. Superscript letters refer to results of lest significnt difference (LSD) post hoc tests fter one-wy ANOVA (P.5). Mens with different superscript letters re significntly different. tht low plsm level of C promotes synthesis of PTH, which in turn increses,5-(oh) D 3 levels. Despite the mrked decrese in rtes of intestinl C trnsport in the fructose group, plsm levels of C, Pi, nd PTH remined similr mong the three diet groups. Since fructose my cuse hyperuricemi rising from fructose-induced renl dmge (37) nd heptic uric cid production (3), concentrtions of uric cid were determined ut were lso found to e independent of diet. Fructose lters CYP7B nd CYPA expression. We exmined the effects of fructose on the kidney, since it is the mjor orgn system regulting metolism of,5-(oh) D 3, the hormone regulting intestinl C trnsport. mrna expression of CYP7B, which synthesizes,5-(oh) D 3, ws significntly downregulted threefold in the kidney of rts fed fructose compred with those fed glucose (Fig. A). In contrst, expression of CYPA, which ctolizes,5-(oh) D 3, ws two- to threefold higher in fructose-fed thn in oth glucose- nd strch-fed rts (Fig. B). The protein levels of CYP7B decresed in the kidney of fructose-fed rts (Fig. C). Thus, the decrese in circulting levels of,5-(oh) D 3 might hve resulted from reduced synthesis nd incresed degrdtion. Fig.. Expression of,5-(oh) D 3 metolic enzymes nd C trnsporters in kidney of 5- dy-old rts fed glucose, fructose, or strch fter wening. mrna expression of CYP7B (A) nd CYPA (B) nlyzed y rel-time PCR using EF s reference. C: protein undnce of CYP7B using -ctin s reference. D: mrna expression of TRPV5, CBP8k, CBP9k, PMCA, NCX, nd GLUT5. All dt were normlized reltive to levels seen in rts fed strch. Dt re mens SE (n 8 per group). Mens with different superscript letters re significntly different t P.5. CYP7B, TRPV5, nd CBP8k mrna expression ech decresed with dietry fructose; CYPA nd GLUT5 expression incresed with fructose. A Reltive to EFα 3 We lso performed gene expression nlyses of,5- (OH) D 3 trget genes in the kidney involved in C resorption from glomerulr filtrtes (Fig. D). Expression levels of TRPV5 nd CBP8k decresed in the fructose-fed compred with the glucose- nd strch-fed rts, n expression pttern similr to tht oserved for C -trnsporting proteins in the smll intestine. The mrna expression of CBP9k (the sole CBP in the smll intestine ut lso expressed in the kidney, where it serves significnt ut supportive role to CBP8k), PMCA nd NCX remined unchnged; thus, the inhiitory effect of fructose on TRPV5 nd CBP8k is specific. As in the smll intestine, renl GLUT5 mrna levels incresed mrkedly, sixfold.,5-(oh) D 3 tretment rescues rts from the hrmful effects of fructose. To prove tht the deleterious effect of dietry fructose on intestinl C trnsport ws directly medited y fructose-induced reductions in,5-(oh) D 3, fructose- nd glucose (control)-fed rts were treted with,5-(oh) D 3. After wk, fructose feeding cused twofold decrese in duodenl C trnsport in vehicle-treted rts (Fig. 3A). However,,5-(OH) D 3 tretment clerly prevented this deleterious effect of fructose. A similr, modest rescue effect of,5-(oh) D 3 ws lso oserved on the mrna expression level of TRPV6 nd CBP9k (Fig. 3B). In vehicle-treted rts, dietry fructose clerly reduced TRPV6 nd CBP9k mrna expression more thn twofold. This reduction ws not oserved in fructose-fed,5-(oh) D 3 -treted rts. In glucose-fed rts, tretment with,5-(oh) D 3 did not induce ny chnge in C trnsport rte or in TRPV6 s well s CBP9k mrna expression ut mrkedly incresed the protein levels of CBP9k (Fig. 3C). The protein level of CBP9k lso responded roustly to,5-(oh) D 3 tretment in fructose-fed rts. Exogenous,5-(OH) D 3 lso produced no toxic effects, since PMCA, NCX, nd SGLT expression, which were not ffected y diet, were lso not ffected y tretment with,5-(oh) D 3. In fct,,5-(oh) D 3 tretment did not inter- B D Reltive to EFα 6 5 Reltive to EFα 5 3 Glucose Fructose Strch C CYP7B βctin CYP7B/βctin TRPV5 CBP8k CBP9k PMCA NCX glucose fructose strch.5 5. GLUT5 Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

5 ,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE E37 A Reltive trnsport B Reltive to EFα G + veh F + veh G +,5D 3 F +,5D 3 C CBP9k βctin TRPV6 CBP9k PMCA NCX GLUT5 SGLT fere with regultion of vitmin D-independent genes, s clerly shown y the drmtic fructose-induced increse in GLUT5 expression in the presence or sence of,5-(oh) D 3. Wht is the effect of exogenous,5-(oh) D 3 tretment on expression of CYP7B nd CYPA? After wk of feeding, the mrna levels of CYP7B decresed threefold in fructose vehicle rts (Fig. A). Tretment with exogenous,5-(oh) D 3 inhiited expression of renl CYP7B in oth glucose- nd fructose-fed rts, s would e expected since CYP7B is inhiited y its product (39).,5- (OH) D 3 tretment lso decresed the protein levels of CYP7B in the glucose-fed rts (Fig. C). Since CYP7B A B C Reltive to EFα D Reltive to EFα.6 Reltive to EFα 3 CBP9k/βctin 3 CYP7B βctin G + veh F+ veh G+,5D 3 F +,5D c CYP7B/βctin TRPV5 CBP8k CBP9k PMCA NCX c c NPi Fig. 3. Tretment in vivo with,5-(oh) D 3 reverses fructose-dependent reductions in intestinl C uptke nd C trnsporter expression. A: reltive trnsepithelil C trnsport from the luminl to the solterl comprtment expressed s rtio of ( 5 C inside/ 5 C outside) of everted gut scs. B: mrna expression of C (TRPV6, CBP9k, PMCA, NCX), fructose (GLUT5), glucose (SGLT), nd phosphte (NPi) trnsporters ws nlyzed y rel-time PCR using EF s reference. All dt were normlized reltive to levels seen in rts fed glucose nd injected with vehicle. Dt re mens SE (n 6 per group). Mens with different superscript letters re significntly different (P.5). Tretment with,5-(oh) D 3 prevented the fructose effect on the smll intestine. C: protein undnce of CBP9k using -ctin s reference. protein levels were lredy very low in fructose-fed rts,,5-(oh) D 3 tretment resulted in no further decreses. In contrst, wk of dietry fructose ws not sufficient to ffect CYPA expression (Fig. B). However, levels of CYPA were lredy very low in this cohort of rts fed norml levels of C, nd further decreses would likely not hve een detectle. Tretment with exogenous,5-(oh) D 3 stimulted expression of renl CYPA in oth glucose- nd fructose-fed rts, s would e expected since CYPA is stimulted y excess,5-(oh) D 3. In the fructose vehicle rts, serum,5-(oh) D 3 levels decresed y % compred with those in the glucose vehicle c GLUT5 c Fig.. Effect of,5-(oh) D 3 tretment on renl expression of CYP7B, CYPA, nd C trnsporters in postwening rts fed glucose or fructose. mrna expression of CYP7B (A), CYPA (B), nd protein undnce of CYP7B (C) using -ctin s reference. D: TRPV5, CBP8k, CBP9k, PMCA, NCX, nd GLUT5 were nlyzed y rel-time PCR using EF s reference. All dt were normlized reltive to levels seen in rt fed glucose nd injected with vehicle. Dt re mens SE (n 6 per group). Mens with different superscript letters re significntly different (P.5). Tretment with,5-(oh) D 3 prevented the fructose effect on the kidney. Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

6 E38,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE Tle. Serum iochemistry in 5-dy-old rts fed glucose or fructose nd then treted with,5-(oh) D 3 or vehicle Vehicle,5(OH)D3 Two-Wy Significnce Glucose Fructose Glucose Fructose Diet Rescue Diet Rescue,5-(OH) D 3, pmol/l Clcium, mg/dl Dt re mens SE; n 6 per group. Superscript letters refer to results of LSD post hoc tests fter one-wy ANOVA (P.5). Mens with different superscript letters re significntly different. rts (Tle ), trend similr to tht oserved in Tle. It is not cler why there re differences in levels of hormones etween experiments s indicted in Tles nd, ut the second feeding experiment ws conducted over yer fter the first, so different tches of ssy kits were used for nlysis. Tretment with exogenous,5-(oh) D 3 further decresed serum,5-(oh) D 3, to 5% lower in oth glucose- nd fructose-fed rts compred with the glucose vehicle group, for resons mentioned in DISCUS- SION. As expected,,5-(oh) D 3 tretment incresed serum levels of C in oth glucose- nd fructose-fed rts. Thus, s in the previous experiment, dietry fructose hd no significnt effect on C levels. The mgnitude of the rescue effect of,5-(oh) D 3 ws less mrked in the kidney (Fig. D). TRPV5 expression decresed y over 5% in fructose vehicle rts compred with tht in glucose vehicle rts. This inhiitory effect of fructose ws clerly leit modestly rescued y,5-(oh) D 3 tretment, s indicted y compring the fructose,5-(oh) D 3 group to the glucose vehicle nd fructose vehicle groups.,5- (OH) D 3 lso hyperstimulted TRPV5 expression, s indicted y the 5% increse in the glucose,5-(oh) D 3 rts. Fructose tended to reduce CBP8k mrna expression, nd,5-(oh) D 3 tretment did not prevent this fructose-induced reduction in expression of CBP8k in the kidney. Renl CBP9k expression ws similr etween the glucose vehicle nd fructose vehicle groups. Thus CBP9k expression ws not ffected y dietry fructose, s ws lso oserved in experiment (Fig. ). Renl CBP9k expression, however, ws hyperstimulted y,5-(oh) D 3 tretment.,5-(oh) D 3 did not ffect seline PMCA nd NCX expression. Moreover,,5-(OH) D 3 tretment did not interfere with regultion of GLUT5 expression, which incresed mrkedly with dietry fructose in vehicle- nd,5-(oh) D 3 - A [FGF3] pg/ml 6 B ] pmol/l [,5-(OH) D 3 injected rts. Thus,,5-(OH) D 3 tretment likely hd no cute toxic effects on renl function, s in the smll intestine. Fructose incresed FGF3 serum levels. Among the key regultors of,5-(oh) D 3 synthesis, FGF3 is known to reduce,5-(oh) D 3 levels y inhiiting CYP7B expression. We found tht wk of fructose feeding significntly incresed circulting levels of FGF3 y more thn 6% (Fig. 5A) nd tht there ws significnt negtive correltion etween the serum levels of,5-(oh) D 3 nd of FGF3 (Fig. 5B). PTH is nother key fctor regulting the circulting levels of,5- (OH) D 3. However, there ws no significnt correltion etween the plsm levels of,5-(oh) D 3 nd those of PTH (Fig. 5C), suggesting tht the fructose-induced reduction in,5-(oh) D 3 levels my not involve PTH. To support this new finding nd demonstrte tht fructoseinduced increses in FGF3 ffected the kidney, where,5- (OH) D 3 is synthesized, we investigted whether fructose feeding ws ssocited with the well-estlished inhiitory effects of FGF3 on renl Pi resorption, s FGF3 inhiits the trnsloction of NPi to the proximl tuulr memrnes (8,, ). Fructose-fed rts (Fig. 6) clerly displyed lower level of NPi in the picl memrne of cells of the proximl tuules (white rrow) thn those of rts fed glucose or strch. Fructose feeding compromised growth rte. Despite similr rtes of food intke mong diets (dt not shown) nd similr rtes of initil growth in the first week, rts fed fructose hd slightly lower increses in ody weight out wk fter the eginning of feeding compred with littermtes fed glucose or strch (Fig. 7). After wk more of feeding, t the time of deth, the fructose group weighed 7% less thn the glucose or the strch group. This modest effect on ody weight hs een oserved previously in other studies involving fructose feeding of postwening rodents (5, 3). Glucose Fructose Strch 8 C 3 ] pmol/l 5 -(OH) D y = -.536x y =.57x R² =.9 R² =. P<.33 P < [FGF3] pg/ml [PTH] pg/ml Fig. 5. FGF3 nd,5-(oh) D 3. A: circulting levels of FGF3 in rts fed glucose, fructose, or strch for wk fter wening. B: vritions in circulting levels of,5-(oh) D 3 re strongly negtively correlted with vritions in circulting levels of FGF3. C: vritions in circulting levels of,5-(oh) D 3 re independent of PTH levels. Mens with different superscript letters re significntly different.,5-(oh) D 3 levels seem more strongly dependent on FGF3 ut not PTH levels. [, 8 Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

7 ,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE E39 NPi in kidney Glucose Fructose m m cytosol p cytosol p Strch cytosol m The modest effect of fructose on ody weight ws lso oserved in experiment. Feeding rtes nd ody weights were likewise similr t the strt of the experiment when rts were dys of ge, ut t the midwy point of the experiment fter 7 dys of feeding, fructose vehicle rts exhiited significntly (P.3) lower ody weight ( g) thn glucose vehicle rts (78..5 g). Tretment with,5- (OH) D 3 prevented the fructose-induced, significnt decrese in ody weight (glucose-fed, ; fructose-fed, g; P.). At the end of the rescue experiment, dys fter initition of feeding, fructose vehicle rts ( 5 g) hd lower ody weights thn glucose vehicle rts ( g). However, the men ody weight of fructose,5- (OH) D 3 rts ( g) ws not significntly different from tht of glucose,5-(oh) D 3 rts ( ). Thus,,5- Body weight (g) 5 Glucose Fructose Strch * 7 * * * 3 9 # p Negtive control Fig. 6. Effect of dietry fructose on NPi expression in renl proximl tuules. Immunolocliztion of FGF3-regulted N -dependent phosphte trnsporter NPi in renl proximl tuules (p) of 5-dy-old rts fed glucose, fructose, or strch fter wening. Brs, m. White rrows indicte presence of NPi in the picl or rushorder memrne (m) or its reltively low undnce in the cytosol of cells lining the proximl tuules. The negtive control ws processed without the primry ntiody. Excessive fructose consumption clerly incresed FGF3 to physiologicl levels high enough to reduce trnsloction of NPi to the picl memrne of renl proximl tuule cells. (OH) D 3 tretments prtilly rescued the rts from the deleterious effects of fructose on ody weight. Consequences of chronic fructose intke on one. Fructosefed rts hd femur lengths significntly shorter thn those of glucose- nd strch-fed ones (Tle 3), suggesting tht the fructose-induced decrese in totl ody weight my hve een due, in prt, to retrdtion of liner skeletl growth. Although diet hd no effect on outer one dimeters, glucose-fed rts tended to hve greter inner one dimeters thn fructose-fed nd strch-fed rts. Thus, the net effect is trend for glucose-fed rts to hve less corticl thickness thn fructose-fed nd strch-fed rts. Fructose-fed rts lso displyed lower ( %) dry weight of the humeri thn glucose nd strch groups (Tle ). Susequently, their one sh weight ws lso significntly lower y %; the percentge of one sh to dry weight, however, ws similr mong the tretment groups. Bone C ws lso higher in the glucose- nd strch-fed rts, lthough the difference from tht of the fructose-fed rts ws not sttisticlly significnt. Glucose-fed rts contined significntly higher mount of P thn those fed fructose. The [C P] product ccounts for much of the hydroxyptite tht mkes up the one mtrix. [C P] tends to e lower in humeri of fructose-fed rts thn in those fed glucose or strch, explining their reduced length nd lower weight. Downloded from y on Novemer 3, Age of post-wened rt (d) Fig. 7. Body weight of wenling rts fed glucose, fructose, or strch diet for wk. *Significnt differences in ody weight mong the diets. Excessive intke of fructose grdully slowed the growth of rt pups. DISCUSSION The present study demonstrtes tht decrese in circulting levels of,5-(oh) D 3 is the key mechnism y which fructose inhiits ctive trnsport of C in the smll intestine nd likely in the kidney s well. This finding is highly significnt, AJP-Endocrinol Met doi:.5/jpendo.58.

8 E3,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE Tle 3. Femor length nd dimeters Glucose Fructose Strch Significnce Bone Length, mm Mximum outer dimeter, o Minimum outer dimeter, o Mximum inner dimeter, i Minimum inner dimeter, i Mximum corticl thickness, o i Minimum corticl thickness, o i Dt re mens SE in mm; n 6 8 per group. Superscript letters refer to results of LSD post hoc tests fter one-wy ANOVA (P.5). Mens with different superscript letters re significntly different. ecuse totl fructose intke now constitutes lmost % of totl energy intke of verge Americns, nd % of totl energy intke of the highest 5% of fructose consumers (3).,5-(OH) D 3 tretment prevents the fructose-induced decrese in intestinl C trnsport. When C sttus is deficient ecuse dietry supply is limiting or physiologicl demnd is high, levels of the iologiclly ctive,5-(oh) D 3 hormone increse drmticlly to restore C sufficiency y inducing ctive intestinl sorption nd renl resorption of C. We recently discovered tht dietry fructose prevents these compenstory increses in C trnsport in rts whose C requirements re incresed ecuse of lcttion (6). But the signl mediting the inhiitory fructose effect hd not een clerly identified until the present study. The fructose-induced decrese in intestinl C trnsport ws linked to reductions in duodenl expression of TRPV6 nd CBP9k, oth known to e trnscriptionlly regulted y,5-(oh) D 3 (5). We focused on the duodenum ecuse contriutions of the more distl regions to dptive,,5- (OH) D 3 -medited increses in ctive trnscellulr C trnsport re likely to e smll. Moreover, since fructose lso decreses TRPV6 nd CBP9k expression levels in the distl jejunum, there likely cn e no compenstion of C trnsport in these distl regions. CBP9k-null mice re still le to trnsport C ; thus, CBP9k deletion lone will not stop ctive trnsport (). In TRPV6 knockout mice, C sorption cn still e stimulted y low-c diet nd,5- (OH) D 3 injections (, 8); hence, TRPV6 deletion lone is insufficient to fully inhiit trnsport ctivity. However, simultneous deletion of TRPV6 nd CBP9k impirs the ility to respond to C insufficiency or,5-(oh) D 3 () tretment, indicting tht oth CBP9k nd TRPV6 re required for dptive increses in C trnsport. In our postwening rt Tle. Humerus weight nd minerl composition mesurements Glucose Fructose Strch Significnce Dry weight Totl sh weight %Ash/one Totl weight of C %C /sh weight Totl weight of P %P/sh weight [C ] [P] Dt re mens SE, weights in mg; n 6 8 per group. Superscript letters refer to results of LSD post hoc tests fter one-wy ANOVA (P.5). Mens with different superscript letters re significntly different. model requiring C levels sufficient to support rpid growth, TRPV6 nd CBP9k expression ws compromised y dietry fructose, providing clue tht,5-(oh) D 3 ws involved. Since serum levels of,5-(oh) D 3 decrese when it is injected (Tle ), how cn,5-(oh) D 3 tretments rescue intestinl nd renl C trnsporters? The low serum,5- (OH) D 3 concentrtion is likely due to reductions in CYP7B expression nd increses in CYPA expression, potentilly preventing the synthesis of endogenous,5-(oh) D 3 nd ensuring the rpid clernce of injected,5-(oh) D 3. Similr findings hd een previously oserved in hypoclcemic CYP7B-deficient mice treted dily with,5-(oh) D 3 for 5 wk tht eventully rescued the hypoclcemi despite drmtic increse in CYPA expression nd the undetectle mounts of,5-(oh) D 3 in the plsm (8). Although,5-(OH) D 3 tretment clerly rescued intestinl C trnsport, its cute effects on mrna expression of intestinl trget genes were modest. The cute effects of,5(oh) D 3 my e difficult to demonstrte due to the short hlf-life of intestinl CBP9k [ 6 h ()] nd TRPV6 [ 6 h (6)] reltive to the time gp etween the lst,5(oh) D 3 injection nd deth ( h). Thus,,5(OH) D 3 -induced chnges in levels of intestinl CBP9k mrna were smll nd contrsted with those of CBP9k protein, which hs longer hlf-life nd thus would e more reflective of cute,5(oh) D 3 effects. In vitmin D-deficient young rts,,5-(oh) D 3 tretment lso cused grdul increses in CBP9k mrna nd protein, ut h fter injection, mrna levels hd returned to preinjection levels s protein levels remined high (). Although,5-(OH) D 3 tretment ffects the prcellulr component of C trnsport, its effects re primrily on the ctive component, s indicted y the strikingly similr chnges etween ctive trnsepithelil trnsport nd levels of intestinl TRPV6 nd of CBP9k expression (Figs. nd 3). Nonetheless, mesurements of totl nd prcellulr C trnsport nd of C digestiility re very interesting nd cn e the suject of future work. Wht is the mechnism underlying the fructose-induced decrese in,5-(oh) D 3? The proximte mechnism underlying the regultion of,5-(oh) D 3 y fructose my e the drmtic fructose-induced decreses in CYP7B expression nd the increses in CYPA expression s shown in the first experiment involving 5 wk of fructose feeding. In the second experiment, the renl expression of CYP7B ws lso reduced in fructose-fed compred with glucose-fed rts; however, the stimultory fructose effect on CYPA mrna expression ws not yet pprent, likely ecuse fructose feeding lsted only wk. When serum levels of C, Pi, nd Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

9 ,5-(OH) D 3 re norml,,5-(oh) D 3 inhiits its own synthesis y reducing CYP7B expression so tht low levels of,5-(oh) D 3 re often ssocited with n increse in CYP7B expression to restore homeostsis. Since,5- (OH) D 3 levels nd CYP7B expression re oth low, the,5-(oh) D 3 -CYP7B feedck loop is not responding ppropritely in fructose-fed rts. This feedck loop ws lso shown y us to e disrupted in fructose-fed lctting rts (6), chllenging our understnding of the regultion of,5- (OH) D 3 synthesis under conditions of chroniclly high fructose intke. If this feedck loop is disrupted during chronic fructose feeding, wht else my e mediting the fructose-induced reduction in,5-(oh) D 3? The two other min hormones potentilly regulting,5-(oh) D 3 re PTH, which increses, nd FGF3, which decreses, serum,5-(oh) D 3 concentrtions (3). If the fructose-induced reduction in,5-(oh) D 3 concentrtions is vi PTH, fructose should reduce PTH levels; if vi FGF3, fructose should increse FGF3 levels. In this study, PTH did not vry with diet, nd there ws no correltion etween the circulting levels of PTH nd,5-(oh) D 3, finding similr to tht in our previous work (6). In contrst, fructose incresed FGF3 circulting levels, so tht FGF3 nd,5-(oh) D 3 were significntly negtively correlted. These findings suggest potentil role of FGF3 in the downregultion of,5-(oh) D 3 y fructose. FGF3, which is highly expressed in one tissue mtrixforming cells such s osteolsts nd osteocytes, is phosphturic hormone tht decreses renl Pi resorption (3). We confirmed tht the fructose-induced increse in FGF3 circulting levels is physiologicl, s it led to reduced trnsloction of NPi protein expression to the picl memrne of proximl tuulr cells, s previously shown (). In fct, the mrked effect of fructose vi FGF3 on NPi levels in the PTH 6 7 CYP7B CYPA 8c FGF3 8,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE Serum levels Known from recent studies Hypotheticl mechnism,5-(oh) D 3 5 Fructose 8 Fructose diet 3 C + trnsport one growth E3 proximl tuulr picl memrne (Fig. 6) my result in reduced Pi resorption s well s incresed Pi excretion nd my explin the incresed loss of Pi from the one (Tle ) to keep lood Pi levels from decresing. In ddition, FGF3 is known to regulte the renl synthesis of,5-(oh) D 3 y inhiiting CYP7B nd stimulting CYPA mrna expression (3), findings replicted in this study. Future work using FGF3 / nd klotho / (the oligte renl coreceptor of FGF3) mice should confirm whether the effect of fructose is truly medited through FGF3, nd enle us to determine the mechnism underlying the fructose-induced increse in FGF3 concentrtions. It is not cler how fructose increses FGF3 levels, ut 6-deoxy-6-[ 8 F]fluoro-D-fructose did ccumulte in the one more thn in ny other orgn system (5). Interestingly, we hve immunocytochemicl evidence tht GLUT5 my e expressed in one (V. Dourd nd P. Ferrris, unpulished oservtions), suggesting tht some one cells my e le to trnsport nd metolize fructose. FGF3 is n erly-onset iomrker of renl insufficiency (3), ut it is lso not cler whether this increse in FGF3 is cuse or consequence of kidney dmge (9,, 5). Interestingly, excessive fructose consumption hs een shown to dmge the kidney, s evidenced y mrked renl hypertrophy, ccelerted progression of chronic kidney disese, nd pprent ggrvtion of endstge renl disese symptoms (7,, 6, 9). It lso increses plsm concentrtion of uric cid (3), shown to e positively ssocited with incresed FGF3 in dults nd children with norml kidney function (3, ). However, in this study, the rts fed fructose did not yet exhiit ny increse in plsm levels of uric cid, lthough fructose did induce renl hypertrophy. A recent study showed tht FGF3 my directly lter,5- (OH) D 3 -medited intestinl C trnsport in mice (6), suggesting tht FGF3 my not ct indirectly vi the kidney. Fig. 8. Schemtic digrm of fructose-induced disruptions in,5-(oh) D 3 homeostsis. Under norml conditions such s in rts fed strch or glucose, postwening growth stimultes increses in circulting levels of,5-(oh) D 3, which in turn enhnce rtes of intestinl C trnsport, therey ddressing the incresed C demnd for one growth. Under fructose feeding, serum fructose increses (solid line with rrowhed, step ). Fructose intke is ssocited with decrese in levels of,5-(oh) D 3 (), resulting in reduced intestinl C trnsport (3), decresing the supply of C for pproprite growth (; solid line with end point). Reduction in,5-(oh) D 3 levels results from increses in CYPA nd decreses in CYP7B renl expression, suggesting tht fructose my directly enhnce renl rekdown nd impir synthesis of,5- (OH) D 3 (5, dshed rrow). These fructose effects were independent of ny chnge in PTH (6) nd the negtive feedck loop y which,5-(oh) D 3 controls its own metolism (7). Alterntively, fructose feeding is ssocited with n increse in circulting levels of FGF3, which my result from direct effect of fructose on one cells (8). This fructose-induced increse in FGF3 (8) my e the mechnism y which fructose indirectly ffects renl CYP7B nd CYPA expression (8c). Downloded from y on Novemer 3, 7 AJP-Endocrinol Met doi:.5/jpendo.58.

10 E3,5(OH) D 3 PREVENTS DELETERIOUS FRUCTOSE EFFECTS ON C UPTAKE However, klotho is n oligte coreceptor of FGF receptors for FGF3 tht is expressed t high levels only in the kidney nd is poorly expressed in the smll intestine (38); thus, it is not cler how FGF3 would exert its direct effects on the gut. Fructose nd growth. Although serum C concentrtions were not pertured, preserving its essentil electrochemicl grdients nd cell signling functions, fructose-induced reductions in C trnsport my ultimtely hve ffected growth rte, s indicted y reductions in one length, one dry weight, nd ody weight. The cuse cn only e scried to excessive fructose intke, s the diets were isocloric nd isonitrogenous. The effect of chronic fructose feeding on ody weight oserved in oth experiments my e specific for this rpidly growing ge group, s we did not oserve such fructose effects on ody weight in our previous studies involving dult mle nd femle rts (, 6). Two other studies focusing on fructose effects in postwening rts lso oserved reductions in ody weight (5, 3) etween nd 8 wk of ge. Continued fructose feeding in one study eyond 8 wk of ge eventully led to the frequently oserved fructose-induced increses in ody weight nd oesity in older ge groups. Modest differences in food intke were oserved only fter ody weight ws reduced, nd even then, food consumption per kilogrm of ody weight remined the sme. Thus, oth C nd,5-(oh) D 3 re criticl for one development, nd herein we confirm, using fructose model, previous work demonstrting tht the ction of,5-(oh) D 3 on the contriutions of C to one growth nd remodeling is medited y regulting intestinl C sorption (, 33). In contrst, fructose seems to influence Pi levels nd metolism in one without ffecting totl nd N -independent Pi trnsport in the smll intestine. However, fructose, vi n increse in serum FGF3 level, decresed expression of NPi in the picl memrne of the proximl tuule, which likely impired renl resorption of Pi. Interestingly, consumption y humns of high-fructose diet (% of totl clories) for 5 wk mrkedly incresed renl Pi excretion, modestly incresed renl C excretion, nd compromised intestinl C sorption, therey decresing C nd Pi lnce (36). Our findings provide mechnism underlying these interesting oservtions. Perspectives. In conclusion, our studies demonstrte tht chroniclly high fructose intke during postwening hs dverse impcts on the intestine, kidney, nd one (Fig. 8). More studies re required in order to etter understnd the exct signling pthwys involved in the different orgns impcted y excess fructose intke nd to demonstrte tht mixed diet with lower fructose content will hve similr effects s ws demonstrted previously (5, ). Here, we used the sme high fructose concentrtion s in n erlier work (6) to demonstrte tht our previous oservtion in the rt lcttion model could e repeted in growing rts tht were not hyperphgic, to demonstrte roust effect of fructose so tht we could unequivoclly show whether or not,5-(oh) D 3 medited those effects, nd to induce the fructose effect quickly nd demonstrte,5-(oh) D 3 medition while growth rte ws still rpid. ACKNOWLEDGMENTS We re grteful to Dr. Dvid Pgli for technicl dvice, Dr. Peddrick Weis for the use of the muffle furnce nd lortory, nd Humerto Cruz, Ashkn Hmzelou, nd Frnk Portugl for technicl help. GRANTS This work ws supported in prt y NSF Grnts IOS-9 nd Support from Ntionl Institutes of Helth Grnt RC-AI-7858, Foundtion of UMDNJ, nd Benjmin Delessert Foundtion (to V. Dourd) re lso cknowledged. DISCLOSURES No conflicts of interest, finncil or otherwise, re declred y the uthor(s). AUTHOR CONTRIBUTIONS Author contriutions: V.D. nd R.P.F. conception nd design of reserch; V.D., Y.S., J.L., C.P., nd F.W.K. performed experiments; V.D., Y.S., J.L., C.P., F.W.K., J.D.B., S.L., nd R.P.F. nlyzed dt; V.D., Y.S., C.P., J.D.B., S.L., nd R.P.F. interpreted results of experiments; V.D., J.L., nd R.P.F. prepred figures; V.D. drfted mnuscript; V.D., Y.S., J.D.B., nd R.P.F. edited nd revised mnuscript; R.P.F. pproved finl version of mnuscript. REFERENCES. Amling M, Priemel M, Holzmnn T, Chpin K, Rueger JM, Bron R, Demy MB. Rescue of the skeletl phenotype of Vitmin D receptorlted mice in the setting of norml minerl ion homeostsis: forml histomorphometric nd iomechnicl nlyses. Endocrinology : , Armrecht HJ, Boltz MA, Christkos S, Bruns ME. Cpcity of,5-dihydroxyvitmin D to stimulte expression of clindin D chnges with ge in the rt. Arch Biochem Biophys 35: 59 6, Bcchett J, Cocht P, Slusky IB, Wesseling-Perry K. 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