Effect of Diets Containing Sucrose vs. D-tagatose in Hypercholesterolemic Mice

Transcription

1 nture pulishing group ARTICLES Effet of Diets Contining Surose vs. D-tgtose in Hyperholesterolemi Mie S r B. Poli e 1, J. C l y Hr r is 2, Roert A. Lodder 1, 2 nd Lis A. Cssis 1 Effets of funtionl sweeteners on the development of the metoli syndrome nd theroslerosis re unknown. The ojetive ws to ompre the effet of dietry rohydrte in the form of surose () to D-tgtose (; n isomer of frutose urrently used s low-lorie sweetener) on ody weight, lood holesterol onentrtions, hyperglyemi, nd theroslerosis in low-density lipoprotein reeptor defiient (LDLr / ) mie. LDLr / mle nd femle mie were fed either stndrd murine diet or diet enrihed with or s rohydrte soures for 16 weeks. nd diets ontined equivlent mounts (g/kg) of protein, ft, nd rohydrte. We mesured food intke, ody weight, dipoyte dimeter, serum holesterol nd lipoprotein onentrtions, nd orti theroslerosis. Mrophge immunostining nd ollgen ontent were exmined in orti root lesions. nd -fed mie exhiited similr energy intke, ody weights nd lood gluose nd insulin onentrtions, ut -fed mie exhiited inresed energy intke nd eme oese nd hyperglyemi. Adipoyte dimeter inresed in femle -fed mie ompred to nd. Mle nd femle -fed mie hd inresed serum holesterol nd triglyeride onentrtions ompred to nd. Atheroslerosis ws inresed in -fed mie of oth genders ompred to nd. Lesions from -fed mie exhiited pronouned mrophge immunostining nd redutions in ollgen ontent ompred to nd mie. These results demonstrte tht in omprison to surose, equivlent sustitution of s dietry rohydrte does not result in the sme extent of oesity, hyperglyemi, hyperlipidemi, nd theroslerosis. Oesity (28) doi:.38/oy INTRODUCTION Oesity is t epidemi proportions in the United Sttes, s two-thirds of the dult popultion is lssified s either overweight or oese (s defined y BMI; >25 kg/m 2 or >3 kg/m 2, respetively) ( 1 ). Numerous studies desrie the link etween oesity nd diseses suh s hypertension, dyslipidemi, nd type 2 dietes ( 2 ). The lustering of dominl oesity with these diseses is defined s the metoli syndrome ( 3 ). Importntly, the primry use of deth in oese ptients with the metoli syndrome is rdiovsulr disese ( 4 ). Sine the mjority of the US popultion is overweight or oese with n inresed risk of rdiovsulr disese, signifint portion of the popultion is striving to lose weight. In 27, out third of dults (36%) report hving tried to lose weight in the pst, nd two in five (41%) were urrently trying ( 5 ). A ommon prtie to redue lori intke is to derese or eliminte tle sugr, or surose, wheres some dieters opt for use of lterntive sweeteners. Alterntive sweeteners provide zero (nonnutritive, or rtifiil sweeteners) or redued mount (nutritive sweeteners) of lories., n isomer of frutose, is ~9% s sweet s surose nd is used s nutritive or low-lorie sweetener ( 6 ). supplies 1.5 kl/g of energy (s ompred to 4 kl/g from surose), in lrge prt euse is inompletely sored y the smll intestine ( 7 ). ws lssified in 21 s Generlly Reognized s Sfe produt y the United Sttes Food nd Drug Administrtion ( 7 ). Some pproved uses of inlude s sweetener in diet everges t onentrtions up to 1%, light ie rems or yogurts t onentrtions up to 3%, nd regulr or dieteti hrd ndies t levels up to 15% ( 8 ). Animls or humns fed high-energy diets from frutose or surose exhiit n elevtion in serum holesterol euse of n imlne of very low-density lipoprotein (VLDL)-triglyeride prodution nd lerne, whih is diretly influened y ftty id vilility. Consumption of high-rohydrte diet, prtiulrly frutose nd surose, hs een demonstrted to elevte irulting holesterol levels in rodents ( 9 ), dogs ( ), nonhumn primtes ( 11 ), nd humns ( 12 ). In low- density lipoprotein reeptor defiient (LDLr / ) mie fed diet enrihed in surose, serum LDL holesterol onentrtions nd theroslerosis were inresed ompred to mie fed 1 [Q1] Grdute Center for Nutritionl Sienes, University of Kentuky, Lexington, Kentuky, USA ; 2 Deprtment of Chemistry, University of Kentuky, Lexington, Kentuky, USA. Correspondene: Lis A. Cssis ( lssis@uky.edu ) Reeived 14 Deemer 27 ; epted 9 Septemer 28 ; dvne online pulition Month 28. doi:.38/oy OBESITY 1

2 n energy-mthed diet enrihed in sturted ftty ids ( 13 ). However, it hs not een determined whether nutritive or funtionl sweetener, suh s, would indue similr levels of therosleroti disese. The purpose of this study ws to define the effets of diet enrihed with s the rohydrte soure on ody weight, serum gluose nd lipids, nd theroslerosis in LDLr / mie. In ddition, we ompred n equivlent grm sis of dietry to surose s rohydrte soure. We hypothesized tht in omprison to surose, dietry -enrihment would result in miniml hnges in ody weight, lipid metolism, gluose, or theroslerosis formtion in theroslerosissuseptile LDLr / mie. METHODS AND PROCEDURES Mie nd diets LDLr / (krossed times on C57BL/6J kground) mle nd femle mie were red in-house. All mie were mintined in rrier fility nd 12-h light/drk yle. Diets were designed (Hrln Tekld) with D-tgtose (; TD5579) or surose (; TD97111) s the primry rohydrte soure, nd ontined n equivlent grm mss (g/kg of diet) of nd surose ( Tle 1 ). In ddition, we mthed (g/kg of diet) ll other ingredients (oonut, olive oil, orn oil, sein, mltodextrin) etween the nd diets. Due to differenes in energy ontent etween (~1.5 kl/g) nd surose (4. kl/g), the -ontining diet provided more lories from ft nd protein ( Tle 1 ). Both diets ontined equivlent mounts of the following (in g/kg):. Vitmin Mix Tekld (46), 2.3 Choline Bitrtrte,.4 Ethoxyquin (ntioxidnt), 36.8 Minerl Mix AIN-93G-MX (TD 9446), 4.2 Clium Phosphte disi CHPO 4. At ~8 weeks of ge, groups of mle nd femle mie were pled on diets enrihed with ( N = 6/gender) or surose (; N = 6/gender) for 16 weeks. We inluded groups of LDLr / mie fed stndrd murine diet (Tekld, TD218, N = 5/gender) for omprison ( Tle 1 ). Before the 16-week feeding period, nd diets were introdued into the diet in grdul mnner over 3 weeks, using n inresing rtio of powdered - or - ontining diet to stndrd murine lortory diet (TD218). Food intke ws mesured for the first 4 weeks nd ody weight ws monitored weekly. Food intke is expressed s verge kl/dy, given the differenes in energy density etween the diets. Fsting (4 h) lood gluose onentrtions were determined t the time of killing through til vein punture (Freestyle Flsh gluometer; Aott Dietes Cre, Almed, CA). At the study end point, mie were nesthetized (ketmine/xylzine, / mg/kg, IP.) nd the left ventrile ws puntured to otin lood. After the right trium ws ut, mie were exsnguinted y perfusion through the left ventrile nd tissues (liver, kidneys, spleen, epididyml ft, retroperitonel ft, tiilis nterior musle ( 14 ), nd orti tissue from the hert to ilel ifurtion) were disseted. Tissues were snpfrozen in liquid nitrogen nd then stored t 8 C; the hert nd ort were fixed overnight in 4% prformldehyde mde with phosphte uffered solution, nd then trnsferred to phosphte uffered solution for storge. Retroperitonel dipose tissue, viserl dipose depot, ws stored in formlin nd susequently emedded in prffin for exmintion of dipose morphology nd mrophge immunostining (see elow). The wet weight of dipose tissues (epididyml ft, retroperitonel ft) ws normlized to ody weight (%) s n index of diposity. Blood nlyses Totl serum holesterol nd plsm triglyeride onentrtions were determined on individul mie using enzymti ssy kits (Wko Pure Chemil, Rihmond, VA). Lipoprotein distriutions were evluted in individul serum smples (5 μl) from four mie of eh group fter frtiontion y size exlusion hromtogrphy on Superose six olumn. Ser smples hosen for lipoprotein nlysis were within 5% of the men ser holesterol onentrtion for eh group. Frtions were olleted nd holesterol onentrtions were determined using n enzymti kit (Wko Pure Chemil, Rihmond, VA). Plsm insulin levels were mesured on individul mie using n ELISA kit (Merodi Insulin ELISA; Merodi AB, Sweden). Quntifition of theroslerosis Atheroslerosis ws quntified y en fe nlysis of lesion surfe re ording to previously desried methods ( 15 ). Briefly, ort were lened of dventitil tissue, ut longitudinlly long the entire length, nd then pinned out on wx surfe. Digitl imges of the orti rh nd thori surfe were ptured with Q Color 3 Olympus mer (Center Vlley, PA). Atherosleroti lesions were quntified y two independent oservers using Imge Pro Plus 5.1 (Medi Cyernetis, Silver Spring, MD). Dt re expressed s the perentge of the orti rh overed with grossly disernle therosleroti lesions. Immunoytohemil nd morphologil hrteriztion of orti root omponents nd dipose tissue Seril setions (5 μm) were generted from the orti sinus to the sending rh (7 μm) of the orti root t the entry into the hert nd mounted onto hrged nd prelened mirosope slides. In ddition, setions from speifi regions of the vessel (orti sinus, orifies of oronry rteries, nd the sending ort) were pled on rium fluoride windows for infrred mirospetrosopy. Setions (5 μm) were lso generted from prffin-emedded viserl retroperitonel dipose tissue. Slides ontining orti root nd dipose tissue setions were nlyzed y immunohistohemistry for the detetion of mrophges using CD68 (primry ntiody: 1:2 dilution; rt ntimouse; Serote, Kidlington, Oxford, UK) nd F4/8 (primry ntiody: 1:2 dilution; rt ntimouse; Serote), respetively. From eh experimentl group, four orti root (5 μm prt) nd four dipose tissue setions were nlyzed for mrophge immunostining. For oth tissues, primry ntiser were inuted t 4 C for 15 min, followed y inution with seondry iotinylted ntiody under the sme onditions (1:5 dilution; rit-ntirt; Vetor Lortories, Burlingme, CA). Prffin-emedded dipose tissue setions underwent ntigen retrievl (stem; -min inution in Antigen Unmsking Solution; Vetor Lortories, Burlingme, CA) efore ntiody inutions. A peroxidse-sed ABC system nd red hromgen AEC (oth Tle 1 Composition of ontrol nd experimentl diets Energy (kl/g) Energy soures (% kl) Protein Ft Crohydrte Components (g/kg) Surose D-tgtose Corn strh Csein Cellulose Coonut oil Olive oil Corn oil

3 from Vetor Lortories) were used to identify the ntigen-ntiody retion. A nonimmune serum ws used s ontrol; nulei were visulized y ounterstining with hemtoxylin. Aorti root setions (two slides ontining nine setions eh nd representing distne of 8 μm) were lso stined using Gomori Trihrome. For hrteriztion of dipoyte morphology, dipose tissue setions (two setions/gender/diet group) were deprffinized nd stined using hemtoxylin nd eosin. In four fields from eh setion, dipoyte numer, dimeter, nd re were determined using Imge Pro Plus 5.1. Infrred mirosopy The infrred mirospetrometer used t emline U2 of the vuum UV storge ring of the Ntionl Synhrotron Light Soure t Brookhven Ntionl Lortory, Upton NY onsisted of Ni PLAN infrred mirosope interfed to Niolet Mgn 86 infrred spetrometer (Thermo Eletron, Mdison, WI). A liquid nitrogen ooled 25 m MCT detetor with frequeny rnge of 5 4, m 1 ws used. Shwrtzhild 32 _nd _ll-refleting mirror lenses were used for the ojetive nd ondenser, respetively. A remote projeted imge plne msk efore the ojetive produed the pertures used for single point spetr or rster sn mpping vi digitlly ontrolled motorized mirosope stge. Spetr were reorded in trnsmission mode. A ler lotion on the infrred mirosope slide (Delmr Ventures, Sn Diego, CA) ws used to otin kground spetrum. Mpping ws lso omplished from glor soure fol plne rry instrument. The Perkin-Elmer Spotlight model 3 ws used to otin retngulr mps of selet regions of the setions eing exmined. For fol plne rry imges, the μm pixel size ws used. Clultions nd sttistis Dt re presented s the men ± s.e.m. Dt were nlyzed using 1-wy ANOVA, nd tested for use of prmetri or nonprmetri post ho nlysis. If sttistil differenes existed etween experimentl groups, Tukey s test ws utilized for post ho nlyses. Vlues of P <.5 were onsidered to e sttistilly signifint. All sttistil nlyses were performed using GrphPd Prism 4. (Sn Diego, CA) of SigmStt (SPSS, Chigo, IL), with exeption of sttistil nlyses of the infrred mirospetrosopy dt. Detiled sttistil methods for nlysis of infrred mirosopy dt n e found in Supplementry Mteril online. RESULTS Surose, ut not, inreses ody weight, diposity, lood gluose, nd insulin onentrtions In oth genders, mie fed diet enrihed with surose exhiited mrked inrese in ody weight ompred to nd ( P <.1 ompred to or ; Figure 1,, Tle 2 ). In ontrst, mle nd femle mie fed -enrihed diet exhiited ody weight similr to ( Figure 1, ). Mle nd femle mie exhiited elevtions in energy intke (14.4 ±.6 nd 12.8 ±.4), ompred to (9.4 ±.7 nd.8 ±.4) nd -fed mie (.9 ±.6 nd 9.8 ±.4 kl/dy, Figure 1,d ; P <.1). Totl dipose tissue mss (epidydiml + retroperitonel dipose weight) ws inresed in oth mle nd femle -fed mie ompred to nd ( P <.1 ompred to or ; Supplementry Figure S1e,f online). When normlized for totl ody weight, gondl dipose tissue mss remined lrger in ll -fed mie ompred to nd ( P <.1); this trend ws lso oserved for retroperitonel dipose djusted to ody weight in mles ut not femles ( Supplementry Figure S1 d online). Representtive setions of retroperitonel dipose tissue illustrte inresed dipoyte size in femle -fed mie ( Figure 2 ) ompred to ( Figure 2 ) or ( Figure 2 ), whih ws onfirmed in quntittive mesurements of dipoyte dimeter (, 3 ± 1;, 5 ± 1;, 31 ± 1 μm, P <.5; Figure 2g nd Tle 2 ). Adipose tissue from mle mie Body Weight (g) Averge energy intke (kl/dy) Weeks Mle Body Weight (g) Averge energy intke (kl/dy) Weeks.. d Femle Figure 1 A diet ontining surose, ut not, promotes the development of oesity in mle nd femle LDLr / mie. ( ) Mle nd ( ) femle -fed mie exhiit inresed ody weight ompred to nd mie. Beginning on week 7 in mles nd week 11 in femles, ody weight ws greter in -fed mie ompred to nd. Dt re men ± s.e.m. from N = 5 6 mie/ gender. Aordingly, ( ) mle nd ( d ) femle -fed mie exhiit inresed energy intke ompred to oth nd mie ( P <.1). LDLr /, low-density lipoprotein reeptor defiient. Tle 2 Metoli prmeters of ontrol nd experimentl mie Gender Diet Body weight (g) Plsm insulin (uiu/ml) Adipoyte dimeter (µm) Totl plsm triglyerides (mg/dl) Tiilis nterior (mg) Mle 3 ± 17 ± 3 74 ± 3 1 ± 2 58 ± 4 4 ± 2 28 ± 6 83 ± ± ± 3 27 ± 13 ± 1 79 ± ± ± 2 Femle 24 ± 1 15 ± 1 3 ± 1 79 ± ± 7 31 ± 1 17 ± 2 5 ± ± ± 2 24 ± 1 14 ± 1 31 ± 1 54 ± 8 42 ± 3 Dt re men ± s.e.m. from N = 5/6 mie/group. P <.1 ompred to. P <.1 ompred to nd. OBESITY 3

4 d e f g % Of ells Distriution of dipoyte size Cell dimeter (µm) Figure 2 ( ) Adipoyte dimeter nd ( d f ) mrophge immunostining re inresed in retroperitonel dipose tissue setions from -fed ompred to nd LDLr / mie. Representtive dipose setions from ( ), ( ), nd ( ) -fed mie, demonstrting lrger dipoytes in ompred to nd mie. F4/8 mrophge immunostining ws not evident in setions from ( d ) or ( f ) -fed mie (ll imges re 2 mgnifition). In ontrst, dipose tissue mrophges were oserved in setions from -fed mie ( e ; rrows denote positively stined ells; inset is 4). ( g ) Distriution of dipoyte size. LDLr /, low-density lipoprotein reeptor defiient. exhiited similr results ( Figure 2g nd Tle 2 ). Mrophge positive F4/8 immunostining ws deteted in dipose setions from mie ( Figure 2e ), ut not in setions from ( Figure 2d ) or mie ( Figure 2f ). At the study end point, mle -fed mie exhiited inresed fsting lood gluose onentrtions (267 ± 13 mg/dl) ompred to (27 ± 23 mg/dl) nd -fed mie (168 ± 5 mg/dl; P <.5). Femle -fed mie exhiited inresed fsting lood gluose onentrtions (177 ± 6 mg/dl) ompred to (145 ± 11 mg/dl; P <.5), ut not mie (151 ± 8 mg/dl). Interestingly, fsting lood gluose onentrtions were greter in mle ompred to femle -fed mie. Plsm insulin onentrtions were inresed in mle mie ompred to ( P <.1), wheres plsm insulin onentrtions of femle diet groups were similr ( Tle 2 ). Surose inreses serum lipids nd theroslerosis to greter extent thn Mle nd femle -fed mie exhiited mrkedly inresed totl serum holesterol onentrtions (1,368 ± 98 nd 912 ± 71, respetively) ompred to (519 ± 32 nd 464 ± 4) nd mie (25 ± 6 nd 184 ± 16 mg/dl; P <.1) of eh gender ( Figure 3, ). In ddition, totl serum holesterol onentrtions in mle nd femle -fed mie were inresed ompred to ( Figure 3, ; P <.5). Chrteriztion of lipoprotein-holesterol distriutions Serum holesterol (mg/dl) Asorne (6 nm) 1,5 1,25 1, Mle VLDL HDL Frtion numer (mg/dl) Serum holesterol (mg/dl) reveled signifintly elevted onentrtions of VLDL- nd LDL-holesterol in mle nd femle -fed ompred to or mie, s quntified y re-under-the urve nlysis ( Figure 3,d ; P <.1 vs., ). In ddition, -fed mie exhiited inresed VLDL- nd LDL-holesterol onentrtions ompred to ( Figure 3,d ; P <.5 vs. ). Elevtions in serum holesterol onentrtions were ssoited with striking inrese in therosleroti lesion surfe re in mle nd femle -fed mie ompred to nd mie of eh gender ( Figure 4, ; P <.1). In mles, -feeding did not signifintly inrese the extent of theroslerosis. However, in femles, -fed mie exhiited inresed theroslerosis ompred to ( Figure 4 ; P <.5). Immunohistohemistry ws used to ssess the degree of mrophge infiltrtion in the orti root of eh group. LDLr / mie did not exhiit positive mrophge immunostining, s genetilly modified hyperlipidemi mie do not redily develop theroslerosis unless hllenged with high-energy diet ( 16 ). However, therosleroti lesions in orti roots of -fed mie stined positive for mrophges ( Figure 4e ). Furthermore, the lrger lesions in orti roots of -fed mie were ssoited with more pronouned mrophge immunostining ompred to reltively smller therosleroti lesions in -fed mie. d 1,5 1, LDL VLDL LDL HDL.7 Asorne (6 nm) Femle Frtion numer (mg/dl) Figure 3 Totl serum holesterol onentrtions nd lipoprotein holesterol onentrtions re mrkedly inresed in -fed ompred to nd LDLr / mie. Totl serum holesterol onentrtions re inresed in ( ) mle nd ( ) femle -fed mie ompred to nd. ( ) Mle nd ( ) femle -fed mie exhiit inresed totl serum holesterol onentrtions ompred to. Lipoprotein holesterol onentrtions in ( ) mle nd ( d ) femle mie demonstrte mrked inrese in VLDL/LDL holesterol in - ompred to nd. However, VLDL nd LDL holesterol onentrtions were inresed in ( ) mle nd ( d ) femle -fed mie ompred to. Dt re men ± s.e.m. from N = 5 6 mie/gender/diet group., Denotes signifintly different from nd ;, denotes signifintly different from, P <.5. HDL, high-density lipoprotein; LDL, low-density lipoprotein; LDLr /, LDL reeptor defiient; VLDL, very LDL. 4

5 % Lesion re Mle Femle d % Lesion re e Infrred mirospetrometry of lesions in the mie showed the lowest onentrtion of lipid ( Figure 5, doulet etween 2,8 nd 3, m 1 ). Atherosleroti lesions of -fed mie hd the lrgest onentrtion of lipids, while lesions in -fed mie showed n intermedite lipid ontent. The rod pek t 1, m 1 ssoited with ollgen, whih my strengthen firous ps in lipid-filled theroms, is missing in the spetrum of lesions from -fed mie ( Figure 5 ). Gomori Trihrome stining for ollgen illustrtes n inrese in ollgen deposition (tel lue olortion in Figure 5, right olumn) within therosleroti lesions of ompred to mie. Figure 4 A diet ontining surose results in mrked inreses in the extent of (, ) theroslerosis nd ( e ) mrophge immunostining in orti roots from LDLr / mie. ( ) Mle nd ( ) femle -fed nimls exhiited mrked extent of theroslerosis in the orti rh ompred to nd. Femle -fed mie exhiited elevted theroslerosis development ompred to. Dt re men ± s.e.m. from N = 5 6 mie/gender/diet group., Denotes signifintly different from nd -fed mie, P <.5., denotes signifintly different from, P <.5. e, Representtive setions revel pronouned mrophge immunostining in the orti root from ( e ) surose-fed mie ompred to ( d ) nd ( ). LDLr /, low-density lipoprotein reeptor defiient. DISCUSSION This study ws designed to ompre diets ontining nd (s the primry rohydrte soure) to investigte their effets on spets of the metoli syndrome nd theroslerosis in LDLr / mie. A diet enrihed in promoted inresed energy intke, oesity, elevted lood gluose nd insulin, inresed totl serum holesterol onentrtions, nd mrkedly ugmented theroslerosis in LDLr / mie. In ontrst, diet ontining n equivlent mount of hd no effet on ody weight or the insulin levels. Although -fed mie exhiited inresed serum holesterol nd theroslerosis ompred to, the extent of these hnges were , 3,5 3, 2,5 2, 1,5 1, Asorne , 3,5 3, 2,5 2, 1,5 1, , 3,5 3, 2,5 2, 1,5 1, Wve numer (m 1 ) Figure 5 Infrred mirospetrometry of lesions in the mie. Left, verge infrred spetrum of lesions from eh group. Middle, infrred spetrl imge, with the intim oriented to the left, nd the lumen in lue. The spetrl imge shows the reltive lipid onentrtion in shdes of red sed on the pek doulet etween 2,8 nd 3, m 1 (imge size = 3 9 µm). Right, Gomori trihrome stining in orti root setions illustrtes inreses in ollgen deposition (tel lue olor) in ompred to -fed mie. Inset is higher mgnifition ( 4) of the lesion re. ( ) mie showed the lowest onentrtion of vsulr wll lipid (pek doulet etween 2,8 nd 3, m 1 ). ( ) Atherosleroti lesions of -fed mie hd y fr the lrgest onentrtion of lipids, while lesions in the ( ) -fed mie showed n intermedite lipid ontent. The rod pek t 1, m 1, ssoited with ollgen, is missing in the spetrum of lesions from -fed mie. OBESITY 5

6 fr less thn those oserved in surose-fed mie. Finlly, orti therosleroti lesions deteted in -fed mie were omprised of inresed ollgen, suggestive of more stle plque phenotype ompred to -fed mie. Results from this study demonstrte surose-enrihed diet promotes the development of oesity in LDLr / mie, while did not promote sustntil weight gin or enhned diposity., urrently used s nutritive or low-lorie sweetener, is inompletely sored from the lower digestive trt nd remins in the olon to e fermented y resident teri. Inomplete sorption of from the gstrointestinl trt my explin the prevention of weight gin y -fed mie. Studies investigting the effet of on food intke hve yielded inonsistent results ( 17 ); however, our dt suggest onsumption does not ffet food intke. Pltle diets (in the form of high sugr or high ft) hve een demonstrted to lter stiety signls nd lend to hyperphgi in rodents ( 18 ). Aordingly, mle nd femle mie exhiit inresed energy intke per dy ompred to oth nd mie. The diet ws more energy dense (4. kl/g) ompred to the (3.3 kl/g) or diet (3.4 kl/g). The omintion of elevted food intke nd inresed energy density of the diet likely ontriuted to the development of oesity in mie. Numerous studies hve demonstrted the ntihyperglyemi effets of (reviewed in ( 6 )). Speifilly, Donner et l. (19 ) exmined effets of on the glyemi response of dieti nd nondieti sujets to surose lod. When dieti sujets were dministered (75 g) efore gluose tolerne test, they exhiited signifintly lunted inrese in lood gluose ompred to ontrol dieti sujets; insulin levels were not ffeted y the tretment. Our results re onsistent with nd extend previous findings y demonstrting tht hroni onsumption of diet enrihed in, in ontrst to surose, does not promote inresed gluose or insulin levels. Colletively, these results suggest tht would ssist with glyemi ontrol. However, -fed mie exhiited elevted energy intke, ody weight, nd diposity ompred to nd ; thus, the diret effet of on gluose regultion, independent of its effet to suppress weight gin, is diffiult to distinguish in the urrent study. Importntly, is urrently in pleo-ontrolled phse 3 linil tril designed to evlute its effiy s n ntidieti gent ( 6 ). Orl dministrtion of (45 g/dy over 3 divided doses) hs een demonstrted to signifintly elevte high-density lipoprotein holesterol ( mg/dl) in type 2 dietis not tking meditions for lipids ( 2 ). While we did not oserve n effet of to signifintly inrese high-density lipoprotein holesterol in LDLr / mie, totl serum holesterol levels were deresed in oth mle nd femle ompred to mie. However, it should e noted tht fed mie were not totlly proteted ginst hyperholesterolemi or theroslerosis, suggesting tht metoli use of my ontriute to some degree of elevted holesterol preursor. Alterntively, modest inreses in ft s n energy soure etween the nd diet my hve ontriuted to inresed serum holesterol nd theroslerosis in -fed mie ompred to. Moreover, the deresed fier ontent of oth experimentl diets, ompred to the diet, my hve further inresed suseptiility to theroslerosis in or mie. One it hs een sored into the lood strem, is metolized in the liver long the sme pthwy used in frutose metolism, through phosphoryltion y frutokinse to -1-P. This metolite, like frutose-1-p, is leved y ldolse to yield glyerldehyde nd dihydroxyetone phosphte. While ldolse ts on oth frutose-1-p nd -1-P s sustrtes, the rte of levge of -1-P is 5% of tht of frutose-1-p ( 21 ). Thus, -1-P onentrtions would inrese, resulting in stimultion of gluokinse tivity nd inresed phosphoryltion of gluose to gluose-6-p to tivte glyogen synthse ( 22 ). The literture lso suggests tht -1-P (similrly to frutose-1-p) inhiits glyogen phosphorylse ( 23 ), therey limiting glyogen utiliztion. The net effets from metolism would e to enhne glyogen synthesis, nd to diminish glyogen utiliztion. Thus, pyruvte genertion from glyolysis would e limited, reduing etyl CoA through the Kres yle s preursor to holesterol. These effets of my hve ontriuted to the oserved redutions in serum holesterol in - ompred to -fed mie. Previous results demonstrte tht irrespetive of the mode of induing oesity, dipoyte hypertrophy with oesity is ssoited with infiltrtion of mrophges into dipose tissue ( 24 ). Further, dipoyte size hs een shown to orrelte positively with the relese of proinflmmtory ftors leptin, monoyte hemottrtnt protein-1, nd interleukin-6, mong others ( 25 ). Indeed, oesity-ssoited inflmmtion is link etween oesity nd inresed risks for the development of type 2 dietes, hypertension, nd rdiovsulr disese ( 24, 26 ). Our results demonstrte tht in - fed mie exhiiting hyperholesterolemi, oesity, hyperglyemi nd theroslerosis, mrophges were deteted in dipose tissue. Lolized nd/ or systemi inflmmtion in mie my hve ontriuted to gluose dysregultion or the development of theroslerosis. In ontrst, smller dipoyte size in -fed mie my hve limited mrophge infiltrtion nd inflmmtion, therey deresing spets of the metoli syndrome in ompred to -fed mie. An importnt spet of these studies is the mesurement of theroslerosis in mie with differing holesterol onentrtions from dietry rohydrte mnipultion. Interestingly, we found tht mle nd femle mie differed in their responsiveness to the development of theroslerosis from nd -ontining diets. In mle mie did not result in inresed theroslerosis ompred to ; however, mle mie fed diet exhiited more extensive theroslerosis ompred to femles. In ontrst, femle mie fed the -ontining diet exhiited n inrese in theroslerosis, ut hd less pronouned therosleroti response to ompred to mles. Gender differenes in effets of on theroslerosis my result from differenes in serum holesterol or the lipoprotein holesterol profile. For exmple, femle 6

7 mie fed the -diet exhiited n inresed VLDL/LDL rtio ompred to mles. Interestingly, the elevtion in VLDL holesterol ourred in femle -fed mie despite generlly lower serum holesterol onentrtion thn tht oserved in mle -fed mie. A novel finding in this study ws redution in the ollgen omposition of therosleroti lesions mong the diet groups. The inrese in ollgen ontent of therosleroti lesions from -fed mie ompred to my ontriute to more stle plque phenotype (27,28 ). The mehnism of this effet is unler. However, these results suggest tht in ddition to minimizing the extent of theroslerosis from rohydrte onsumption, diet enrihed in my fvor more stle plque phenotype. A drwk to use of, like tht of other poorly sored sugrs, is tht their onsumption is ssoited with gstrointestinl distress. For instne, 75 g dose of ( 19 ) led to gstri distress in the form of dirrhe, nuse nd/or fltulene in % of sujets. However, when lower doses of ( 3 g) were dministered, only 3 of reported gstrointestinl symptoms. This side effet results from the poor sorption of in the smll intestines, resulting in longer resident time nd teril fermenttion in the lrge intestine, with inresed fltulene nd fluid retention. Asorption studies involving hve shown phse-in period for dministrtion improves overll sorption of, s indited y inresed fel exretion of tgtose (25.7 ompred to 1.7%) in undpted vs. dpted rts (exposed to g/kg of in the diet for 4 weeks) ( 29 ). It will e importnt to determine whether gstrointestinl effets of limit its tolerility in urrent phse 3 linil studies. In onlusion, in omprison to surose, diet enrihed in s rohydrte soure did not promote oesity or hyperglyemi, dipoyte hypertrophy, nd resulted in lesser extent of hyperholesterolemi nd theroslerosis. Future studies should investigte whether D-tgtose ould e used in the mngement of oesity nd its ssoited disorders of dietes nd oronry rtery disese. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t ACKNOWLEDGMENTS Use of the Ntionl Synhrotron Light Soure, Brookhven Ntionl Lortory, ws supported y the US Deprtment of Energy, Offie of Siene, Offie of Bsi Energy Sienes, under Contrt no. DE-AC2-98CH886. We thnk Spherix for the gift of D-tgtose. DISCLOSURE R.A.L. serves s memer of the ord of diretors of Spherix. 28 The Oesity Soiety REFERENCES 1. Egger G, Swinurn B. An eologil pproh to the oesity pndemi. BMJ 1997 ; 315 : Rohini AP. Oesity hypertension. Am J Hypertens 22 ; 15 ( 2 Pt 2 ) S5 S Despres JP, Lemieux I. Adominl oesity nd metoli syndrome. Nture 26 ; 444 : Grundy SM, Brewer HB Jr, Cleemn JI, Smith SC Jr, Lenfnt C. Definition of metoli syndrome: report of the Ntionl Hert, Lung, nd Blood Institute/Amerin Hert Assoition onferene on sientifi issues relted to definition. Arteriosler Throm Vs Biol 24 ; 24 : e13 e Serdul MK, Mokdd AH, Willimson DF et l. 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