The Journal of Physiology

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1 J Physiol (2017) pp Impct of perintl exposure to sucrose or high fructose corn syrup (HFCS-55) on diposity nd heptic lipid composition in rt offspring Crl R. Toop 1, Beverly S. Muhlhusler 2,KerinO De 3 nd Sheridn Gentili 1 1 School of Phrmcy nd Medicl Sciences, Snsom Institute for Helth Reserch, University of South Austrli, Adelide, Austrli 2 FOODplus Reserch Centre, School of Agriculture, Food nd Wine, The University of Adelide, Adelide, Austrli 3 School of Popultion Helth, Snsom Institute for Helth Reserch, University of South Austrli, Adelide, Austrli The Journl of Physiology Key points Fructose-contining sugrs, including sucrose nd high fructose corn syrup (HFCS), hve een implicted in the epidemics of oesity nd type 2 dietes. Few studies hve evluted the impct of perintl exposure to these sugrs on metolic nd physiologicl outcomes in the offspring. Using rt model, offspring exposed to mternl sucrose or HFCS diet during the prentl nd/or suckling periods were found to hve ltered diposity nd liver ft content nd composition t wening. Plsm levels of free ftty cids remined elevted in young dulthood, ut consumption of control diet following wening ppered to meliorte most other effects of perintl exposure to mternl high-sugr diet. Guidelines for mternl nutrition should dvise limiting consumption of fructose-contining sugrs, nd it is prticulrly importnt tht these recommendtions include mternl nutrition during lcttion. Astrct Perintl exposure to excess mternl intke of dded sugrs, including fructose nd sucrose, is ssocited with n incresed risk of oesity nd type 2 dietes in dult life. However, it is unknown to wht extent the type of sugr nd the timing of exposure ffect these outcomes. The im of this study ws to determine the impct of exposure to mternl consumption of 10% (w/v) everge contining sucrose or high fructose corn syrup-55 (HFCS-55) during the prentl nd/or suckling periods on offspring t 3 nd 12 weeks, utilising cross-fostering pproch in rodent model. Perintl sucrose exposure decresed plsm glucose concentrtions in offspring t 3 weeks, ut did not lter glucose tolernce. Incresed diposity ws oserved in 3-week-old offspring exposed to sucrose or HFCS-55 during suckling, with incresed heptic ft content in HFCS-55-exposed offspring. In terms of specific ftty cids, heptic monounsturted (omeg-7 nd -9) ftty cid content ws elevted t wening, nd ws most pronounced in sucrose offspring exposed during oth the prentl nd suckling periods, nd HFCS-55 offspring exposed during suckling only. By 12 weeks, the effects on diposity nd heptic lipid composition were lrgely normlised. However, exposure to either sucrose or HFCS-55 during the prentl period only ws ssocited with elevted plsm free ftty cids t wening, nd this effect persisted until 12 weeks. This study suggests tht the type of sugr nd the timing of exposure (prentl or suckling periods) re oth importnt for determining the impct on metolic helth outcomes in the offspring. (Received 20 Jnury 2017; ccepted fter revision 30 Mrch 2017; first pulished online 26 April 2017) Corresponding uthor S. Gentili: School of Phrmcy nd Medicl Sciences, Snsom Institute for Helth Reserch, University of South Austrli, Adelide, Austrli. Emil: sheridn.gentili@unis.edu.u DOI: /JP274066

2 4380 C. R. Toop nd others J Physiol Arevitions AUC, re under the curve; FFA, free ftty cid; HDL, high-density lipoprotein; HFCS, high fructose corn syrup; IPGTT, intrperitonel glucose tolernce test; LDL, low-density lipoprotein; MUFA, monounsturted ftty cid; NAFLD, non-lcoholic ftty liver disese; PUFA, polyunsturted ftty cid; SCD, steroyl-coa desturse; SFA, sturted ftty cid; SSB, sugr-sweetened everge; T2DM, type 2 dietes mellitus; TG, triglyceride; WHO, World Helth Orgniztion. Introduction The glol epidemic of oesity nd type 2 dietes mellitus (T2DM) remins mjor pulic helth issue. In 2014, lmost 2 illion dults were clssified s overweight or oese (39% nd 13% of the world s popultion, respectively), nd 9% of dults world-wide hd een dignosed with T2DM (WHO, 2014). Numerous epidemiologicl, clinicl nd experimentl niml studies hve demonstrted tht the nutritionl environment tht n individul is exposed to efore irth nd/or in erly infncy is criticl determinnt of their risk of developing oesity nd metolic diseses over their life course (Hles & Brker, 1992; Mcmillen & Roinson, 2005). Exposure to mternl oesity or over-nutrition during criticl windows of development is ssocited with ltered development of key physiologicl systems, resulting in n incresedriskofoesityndpoormetolichelthindult life (Tylor & Poston, 2007; Alfrdhi & Oznne, 2011). While the negtive impcts of erly life over-nutrition hve een well descried, the role of specific nutritionl components nd the criticl periods during which exposure is most detrimentl remin poorly understood. Consumption of excess dded sugrs, i.e. sugrs/syrups ddedtofoodsndevergesssweeteners,hseen identified s mjor fctor contriuting to the epidemic of oesity nd T2DM (Johnson et l. 2009). This prompted the World Helth Orgniztion (WHO) in 2015 to relese recommendtions tht dded sugrs should contriute less thn 10%, nd preferly less thn 5%, to totl energy intke (WHO, 2015). Despite this, significnt proportion of the popultion continue to consume considerly over this mount, with sugr-sweetened everges (SSBs) representing mjor contriutor to sugr intke (Johnson et l. 2009). The two dded sugrs most commonly used in SSBs re sucrose (fructose/glucose discchride 50:50) nd high fructose corn syrup (HFCS; most commonly HFCS-55; fructose/glucose monoscchrides 55:42). Both of these sweeteners contin similr mounts of fructose, nutrient tht hs een implicted in the development of numer of metolic normlities, including insulin resistnce, inflmmtion, dyslipidemi nd oesity, in oth humns nd nimls (Lustig, 2013; Regnult et l. 2013; Stnhope et l. 2013; Toop & Gentili, 2016), ut hve sutly different mounts of glucose. The key difference etween these sugrs, however, is their chemicl structure. Sucrose ( discchride) must e hydrolysed prior to sorption, wheres the fructose nd glucose in HFCS-55 lredy exist s monoscchrides tht cn e sored without the need for enzymtic processing, which my hve significnt implictions for their intestinl sorption (Tppy, 2015). While excess consumption of oth sucrose nd HFCS-55 hs een ssocited with the development of oesity nd metolic disese in dults, much less is known out the effect of mternl consumption of these fructose-contining sugrs during pregnncy nd/or lcttion on the developing offspring. In ddition, there re currently no specific recommendtions regrding mternl consumption of dded sugrs during pregnncy or rest-feeding. Rodent studies hve egun to shed light on some of the dverse short- nd long-term effects of developmentl exposure to mternl consumption of pure fructose on the metolic helth of the offspring (Alzmendi et l. 2010; Vickers et l. 2011; Muki et l. 2014; Clyton et l. 2015; Rodríguez et l. 2015, 2016; Srı et l. 2015), nd this hs een the suject of severl reviews (Gorn et l. 2013; Regnult et l. 2013; Slood et l. 2014). However, reltively few studies hve investigted the effects of developmentl exposure to fructose-contining sugrs, sucrose or HFCS-55 (Bocrsly et l. 2012; D Alessndro et l. 2012, 2014; Smuelsson et l. 2013; Kendig et l. 2015). This is importnt given tht fructose is rrely consumed in isoltion, nd therefore, studies investigting sucrose nd HFCS-55 re more physiologiclly relevnt. In ddition to the specific nture of the developmentl insult, it is ecoming incresingly cler tht the timing of the insult is lso criticl for determining its progrmming effects. A numer of rodent studies hve shown tht the long-term progrmming effects of exposure to specific nutritionl insult during the prentl period differs from exposure to the sme insult during suckling (Armitge et l. 2005). Thus, it is importnt to determine the effects of exposure to mternl consumption of fructose-contining sugrs during the prentl nd suckling periods lone, in ddition to investigting the impct of exposure during the entire perintl period. To our knowledge, no previous studies hve compred the effects of exposure to sucrose or HFCS-55 during specific criticl windows of development on the shortnd longer-term metolic nd physiologicl outcomes in the offspring. Although studies hve shown tht developmentl exposure to fructose results in the ccumultion of intrheptic ft nd/or incresed expression of heptic lipogenic genes in offspring (Muki

3 J Physiol Perintl exposure to sucrose or HFCS et l. 2012; Rodríguez et l. 2013; Clyton et l. 2015), no studies hve investigted the impct of sucrose or HFCS-55 on heptic ft content or ftty cid composition. This is significnt, s the type of ftty cids stored in the liver, s well s the overll ft content, is known to influence heptic metolism nd the risk of developing diseses including non-lcoholic ftty liver disese (NAFLD) (Puri et l. 2007). We hve previously demonstrted tht mternl consumption of either sucrose or HFCS-55 prior to nd during pregnncy nd lcttion is ssocited with sugr-specific effects in the dm nd neworn offspring (Toop et l. 2015). In the current study we investigted the seprte effects of mternl consumption of sucrose or HFCS-55 during the prentl nd/or suckling periods on the short- (3 weeks) nd longer-term (12 weeks) outcomes in the offspring, specificlly ody weight nd ft mss, glucose tolernce, plsm hormone, lipid nd glucose concentrtions, nd heptic lipid content nd ftty cid composition. Methods Ethicl pprovl All procedures were pproved y the Animl Ethics Committee of SA Pthology (pprovl numer 26/12) for the University of South Austrli, nd this work complies with the niml ethics principles nd regultions of the Austrlin Code for the Cre nd Use of Animls for Scientific Purposes. The uthors ensured tht ll steps were tken to minimise the pin nd suffering of the nimls. A totl of 90 virgin femle nd 15 mle lino Wistr rts (8 weeks old) were otined from Lortory Animl Services (outred lino Wistr rts, colony source Hrln Sprgue Dwley from Indinpolis, IN, USA, imported 6 Novemer 2008; SA, Austrli) nd housed in individully ventilted cges under 12 h light drk cycletroomtempertureof22 C. Of the 90 femle rodents, 84 were mted over three cohorts, with n verge mting success rte of 90 ± 5%, resulting in 77 successful pregnncies. Of the 77 litters, 74 ± 12% were successfully cross-fostered to nother dm in the sme or different tretment group, resulting in 61 litters cross seven experimentl groups. Adult femle nd mle rts nd offspringt3nd12weeksofgewerekilledynoverdose of CO 2 followed y cervicl disloction. Neworn offspring tht were unle to e cross-fostered were killed y decpittion within 48 h of irth. Experimentl design As descried previously (Toop et l. 2015), following 1 week cclimtistion with d liitum ccess to stndrd lortory rt chow (Specilty Feeds, Glen Forrest, WA, Austrli; 14 kj g 1 ) nd wter, rts were rndomly ssigned to receive control diet with d liitum ccess to stndrd lortory rt chow nd wter (n = 25 dms), or d liitum ccess to chow nd n SSB contining either 10% (w/v) sucrose (CSR, Victori, Austrli; n = 19 dms) or HFCS-55 (Nture s Flvors, Ornge, CA, USA; n = 17 dms), mde fresh in the niml fcility using utoclved wter, nd replced every 48 h or when required (Fig. 1). Dms remined on this diet for t lest 4 weeks prior to mting, nd throughout pregnncy nd lcttion (Toop et l. 2015). After minimum of 4 weeks on their respective diets, ll dms were mted. Offspring were orn nturlly t dy 22.4 ± 0.07 of gesttion. Within 24 h of irth, litters were culled to eight pups per litter, four mles nd four femles where possile, nd ll offspring were cross-fostered to crete totl of seven experimentl groups: offspring exposed to mternl control diet during oth the prentl nd suckling periods (; n = 8 litters), offspring exposed Sucrose group HFCS-55 group Pregnncy Age (weeks) 0 Lcttion Birth Cross-fostering 3 12 IPGTT Postmortem Wening Post-wening Control Sucrose HFCS-55 IPGTT Postmortem Litters *n = 8 n = 11 n = 8 n = 8 *n = 8 n = 8 n = 9 n = 9 Figure 1. Schemtic of the tretment groups summrising exposure periods nd cross-foster design The offspring were orn to control dm nd cross-fostered to different control dm (week 0), nd these offspring were the controls for ll susequent nlyses. The group ws repeted in oth sucrose nd HFCS-55 nlyses, denoted y the sterisk ( ). In the sucrose exposure group, offspring were orn to sucrose-fed dm nd cross-fostered to different sucrose-fed dm; offspring were orn to sucrose-fed dm nd cross-fostered to control dm; nd offspring were orn to control dm nd cross-fostered to sucrose-fed dm. In the HFCS-55 exposure group, offspring were orn to n HFCS-55-fed dm nd cross-fostered to different HFCS-55-fed dm; offspring were orn to n HFCS-55-fed dm nd cross-fostered to control dm; nd offspring were orn to control dm nd cross-fostered to n HFCS-55-fed dm. At 3 weeks, n intrperitonel glucose tolernce test (IPGTT) ws performed on one mle nd one femle offspring per litter, with post-mortem conducted 1 dy lter. Offspring were wened nd fed control diet until 12 weeks, when nother IPGTT nd post-mortem ws conducted.

4 4382 C. R. Toop nd others J Physiol to mternl sucrose diet during oth the prentl nd suckling periods (; n = 11 litters), offspring exposed to mternl sucrose diet during the prentl period nd then mternl control diet during the suckling period (; n = 8 litters), offspring exposed to mternl control diet during the prentl period nd then mternl sucrose diet during the suckling period (; n = 8litters), offspring exposed to mternl HFCS-55 diet during oth the prentl nd suckling periods (; n = 8litters), offspring exposed to mternl HFCS-55 diet during the prentl period nd then mternl control diet during the suckling period (; n = 9 litters), or offspring exposed to mternl control diet during the prentl period nd then mternl HFCS-55 diet during the suckling period (; n = 9 litters) (Fig. 1). All pups were weighed every 2 dys from irth until 3 weeks of ge, t which point two mles nd two femles (where possile) were wened onto control diet with d liitum ccess to stndrd lortory rt chow nd wter. The wened offspring were weighed weekly until 12 weeks of ge. At 3 nd 12 weeks, one mle nd one femle per litter were killed with n overdose of CO 2 followed y cervicl disloction. Blood smples were collected vi crdic puncture, centrifuged t 1419 g for 10 min t 4 C, then plsm ws collected nd stored t 80 C. At oth 3 nd 12 weeks of ge, selected internl orgns including the hert, liver, drenls, kidneys, pncres nd viscerl (retroperitonel, gondl nd omentl depots) nd sucutneous dipose tissue depots were dissected out nd weighed. Liver smples were snp frozen in liquid nitrogen nd stored t 80 C for nlysis of lipid content nd composition. Intrperitonel glucose tolernce test Intrperitonel glucose tolernce tests (IPGTTs) were performed on one mle nd one femle offspring per litter 1 dy prior to the post-mortem t 3 nd 12 weeks of ge, s descried previously (Toop et l. 2015). Animls were fsted for t lest 15 h prior to the IPGTT, lthough offspring t 3 weeks of ge remined with the dm during the fsting period nd still hd ccess to rest milk. Blood glucose concentrtions were determined using hnd-held glucometer (FreeStyle Glucometer; Aott Dietes Cre, Victori, Austrli) from til vein lood smple ( 60 µl). A seline lood glucose concentrtion ws determined min prior to commencing the IPGTT. A 50% glucose solution (2 g kg 1 ; Pher, NSW, Austrli) ws then dministered vi intrperitonel injection. Til vein lood smples were collected immeditely prior to (0 min) nd 5, 10, 15, 30, 60 nd 120 min fter glucose dministrtion for determintion of lood glucose concentrtion. Are under the glucose curve (AUC) ws clculted using the trpezoidl rule. Determintion of plsm hormone nd metolite concentrtions Plsm glucose, triglyceride (TG), totl cholesterol, high-density lipoprotein (HDL) cholesterol (Thermo Electron, Pittsurgh, PA, USA) nd free ftty cid (FFA, lso known s non-esterified ftty cid; WAKO Pure Chemicl Industries Ltd, Osk, Jpn) concentrtions were determined using Konel 20X device (Thermo Fisher Scientific, Wlthm, MA, USA). Plsm insulin nd leptin concentrtions were mesured y immunossy with rt insulin (ALPCO Dignostics, Slem, NH, USA) nd leptin (Crystl Chem, Downers Grove, IL, USA) kits ccording to the mnufcturers specifictions. Internd intr-ssy coefficients of vrition were < 1% for ll ssys. Low-density lipoprotein (LDL) cholesterol ws clculted s the difference etween totl nd HDL cholesterol concentrtions. Determintion of heptic lipid concentrtion nd composition Heptic lipids were extrcted ccording to Folch et l. (1957) with minor modifictions (Bligh & Dyer, 1959; Tu et l. 2010). Briefly, 50 mg sections of liver from the right loe were homogenised in ice-cold 0.9% sline (Precelleys24, Bertin Technologies, Frnce). Totl lipids were extrcted with methnol/chloroform (2:1) nd dried under nitrogen. The remining residue ws weighed to determine the totl extrcted lipid content (%) from the weight of the lipid extrct (g) nd the weight of the liver smple (g). Following resuspension in 9:1 chloroform/methnol, the lipid extrct ws seprted y thin lyer chromtogrphy to isolte the FFA, TG nd phospholipid frctions, which were then trnsmethylted with 1% H 2 SO 4 in methnol t 70 C for 3 h. The resulting ftty cid methyl esters from ech lipid frction were then seprted nd quntified y gs chromtogrphy (6890; Hewlett-Pckrd, Plo Alto, CA, USA) equipped with cpillry column (50 m 0.32 mm internl dimeter) coted with 0.25 µm film thickness silic (BPX-70; SGE Anlyticl Science Pty Ltd, Victori, Austrli), which ws fitted with flme ionistion detector. The injector temperture ws set t 250 C nd the detector temperture t 300 C. Helium ws used s the crrier gs (35 cm s 1 ) nd the split-rtio ws 20:1. Ftty cid methyl esters were identified sed on the retention time nd pek re percentge of the unknown smples to commercil lipid stndrds (Nu-Check Prep Inc., Elysin, MN, USA) using ChemSttion softwre (Hewlett-Pckrd). From this, the concentrtion of totl FFA, phospholipid nd TG in the liver were determined, in ddition to the concentrtion of totl sturted ftty cids (SFAs), totl, omeg-7 nd omeg-9 monounsturted ftty cids

5 J Physiol Perintl exposure to sucrose or HFCS (MUFAs), nd omeg-3 nd omeg-6 polyunsturted ftty cids (PUFAs). Sttisticl nlysis All dt re presented s men ± SEM. Dt were ssessed for normlity using the Shpiro Wilk test. The nlysis ws split into two groups sed on offspring sugr exposure, in which the offspring were used s the controls for ll nlyses: the sucrose exposure group (,, nd offspring) nd the HFCS-55 exposure group (,, nd ) (Fig. 1). The effects of mternl consumption of sucrose or HFCS-55 during pregnncy nd/or lcttion on offspring ody weight t post-mortem, reltive orgn nd tissue weights, heptic lipid concentrtion nd composition, nd plsm metolite nd hormone concentrtions were initilly determined using two-wy ANOVA (nlyses split y sugr exposure, s defined ove) with mternl diet (during pregnncy nd/or lcttion) nd offspring sex s co-fctors. An effect of sex ws found for mny of the vriles reported, so ll dt were split for sex nd re-nlysed using one-wy ANOVA, followed y Bonferroni post hoc test where pproprite. The sttisticl significnce reported in the tles nd figures summrises the effect of tretment for mle or femle offspring for ech of the vriles reported, s determined y one-wy ANOVA. Differences etween mles nd femles (independent of tretment) identified y the two-wy ANOVA hve een identified in the tle nd figure legends. The effect of mternl sugr consumption in the sucrose exposure nd HFCS-55 exposure groups during pregnncy nd/or lcttion on offspring ody weight throughout the study period ws seprtely determined using nested repeted-mesures ANOVA. The effect of mternl diet on offspring glucose concentrtions during the IPGTT ws determined y repeted-mesures ANOVA, split for sex, with mternl diet nd time s co-fctors. The repeted-mesures ANOVA ws performed using Stt12 (SttCorp LP, College Sttion, TX, USA). All other sttisticl nlyses were performed using SPSS (SPSS Inc., Chicgo, IL, USA). A proility of < 5% (P < 0.05) ws considered sttisticlly significnt in ll nlyses. Results Effects of perintl sucrose exposure Body weight nd growth. There ws no effect of prentl nd/or postntl sucrose exposure on offspring ody weight t ny time point etween irth nd 12 weeks of ge, nose til length, dominl circumference (dt not shown) or ody weight t post-mortem t either 3 weeks (Tle 1) or 12 weeks (Tle 2). Orgn weights. Femle, ut not mle, offspring prentlly exposed to sucrose () hd lower reltive hert weight compred to offspring, ut not offspring, t 3 weeks of ge (Tle 1). At 12 weeks, reltive hert weight ws lower in oth nd femles compred to femles (Tle 2). Reltive pncres weight tended (P = 0.051) to e lower in femles t 3 weeks compred to femles, nd nd mles hd significntly lower reltive pncres weights compred to mle offspringt12weeks.therewerenoeffectsofexposure to sucrose during either the prentl nd/or the suckling periods on reltive liver, drenl or kidney weights in either mle or femle offspring t 3 weeks (Tle 1) or 12 weeks (Tle 2) of ge. Body ft mss. Exposure to sucrose during the suckling period lone () ws ssocited with incresed reltive viscerl dipose tissue mss in femle, ut not mle, offspring t 3 weeks of ge compred to offspring, n effect driven primrily y significnt increse in the mss of the omentl depot (Tle 1). There were no other effects of sucrose exposure during the prentl or suckling periods on dipose tissue mss t 3 weeks of ge in either mle or femle offspring. There were no persistent effects of sucrose exposure during the suckling period in femle offspring t 12 weeks. However, 12-week-old femles exposed to sucrose during the prentl period only () hd significntly lower retroperitonel dipose tissue mss compred to offspring (Tle 2). In mles, however, those offspring exposed during the suckling period lone () hd lower reltive totl ft nd totl viscerl ft mss, nd reduced reltive gondl nd omentl ft mss t 12 weeks of ge compred to offspring (Tle 2). Glucose tolernce. No effects of prentl nd/or postntl sucrose exposure were oserved for glucose tolernce AUC in offspring t either 3 weeks (Tle 3) or 12 weeks (Tle 4). There were lso no significnt effects on individul lood glucose vlues t ny time points during the glucose tolernce test (dt not shown). Plsm glucose, hormone nd lipid concentrtions. At 3 weeks, plsm glucose concentrtions were significntly lower in oth mle nd femle offspring compred to offspring (Tle 3). Plsm insulin tended to e incresed in femles (P = 0.079) nd mles (P = 0.06). There ws trend towrd elevted plsm leptin concentrtions in offspring exposed to sucrose only during suckling () in oth mles (P = 0.091) nd femles (P = 0.056). The HDL cholesterol concentrtion nd the rtio of HDL/LDL cholesterol were incresed in offspring compred to offspring in oth mles nd femles. Plsm TG ws higher in 3-week-old mles

6 4384 C. R. Toop nd others J Physiol Tle 1. Body weight, reltive orgn weights nd reltive dipose tissue weights of 3-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 3 week mle n = 8 n = 11 n = 8 n = 8 n = 8 n = 8 n = 9 n = 9 Body weight (g) 45.4 ± ± ± ± ± ± ± ± Reltive liver (%) 3.74 ± ± ± ± ± ± ± ± Reltive hert (%) 0.63 ± ± ± ± ± ± ± ± Reltive drenl (%) ± ± ± ± ± ± ± ± Reltive kidney (%) 0.99 ± ± ± ± ± ± ± ± Reltive pncres (%) 0.18 ± ± ± ± ± ± ± ± Reltive totl dipose (%) 4.00 ± ± ± ± ± ± ± ± Reltive sucutneous 2.69 ± ± ± ± ± ± ± ± dipose (%) Reltive totl viscerl dipose (%) Reltive gondl dipose (%) Reltive retroperitonel dipose (%) Reltive omentl dipose (%) 0.91 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± week femle n = 8 n = 11 n = 8 n = 8 n = 8 n = 8 n = 8 n = 9 Body weight (g) 44.4 ± ± ± ± ± ± ± ± Reltive liver (%) 3.74 ± ± ± ± ± ± ± ± Reltive hert (%) 0.62 ± ± ± ± ± ± ± ± Reltive drenl (%) ± ± ± ± ± ± ± ± Reltive kidney (%) 1.05 ± ± ± ± ± ± ± ± Reltive pncres (%) 0.19 ± ± ± ± ± ± ± ± Reltive totl dipose (%) 3.94 ± ± ± ± ± ± ± ± Reltive sucutneous 2.74 ± ± ± ± ± ± ± ± dipose (%) Reltive totl viscerl 0.85 ± ± ± ± ± ± ± ± dipose (%) Reltive gondl 0.17 ± ± ± ± ± ± ± ± dipose (%) Reltive retroperitonel 0.20 ± ± ± ± ± ± ± ± dipose (%) Reltive omentl 0.48 ± ± ± ± ± ± ± ± dipose (%) Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) for ech sex within ech experimentl grouping. A significnt effect of offspring sex, independent of tretment, within ech experimentl grouping. Significnt P-vlues re in old.

7 J Physiol Perintl exposure to sucrose or HFCS Tle 2. Body weight, reltive orgn weights nd reltive dipose tissue weights of 12-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 12 week mle n = 8 n = 11 n = 8 n = 8 n = 8 n = 8 n = 9 n = 9 Body weight (g) ± ± ± ± ± ± ± ± Reltive liver (%) 3.87 ± ± ± ± ± ± ± ± Reltive hert (%) 0.34 ± ± ± ± ± ± ± ± Reltive drenl (%) ± ± ± ± ± ± ± ± Reltive kidney (%) 0.64 ± ± ± ± ± ± ± ± Reltive pncres (%) 0.15 ± ± ± ± ± ± ± ± Reltive totl ft (%) 7.56 ± ± ± ± ± ± ± ± Reltive sucutneous ft (%) 2.73 ± ± ± ± ± ± ± ± Reltive totl viscerl ft (%) 4.80 ± ± ± ± ± ± ± ± Reltive gondl ft (%) 1.76 ± ± ± ± ± ± ± ± Reltive retroperitonel ft (%) 1.86 ± ± ± ± ± ± ± ± Reltive omentl ft (%) 1.19 ± ± ± ± ± ± ± ± week femle n = 8 n = 11 n = 8 n = 8 n = 8 n = 8 n = 9 n = 9 Body weight (g) ± ± ± ± ± ± ± ± Reltive liver (%) 3.53 ± ± ± ± ± ± ± ± Reltive hert (%) 0.40 ± ± ± ± ± ± ± ± Reltive drenl (%) ± ± ± ± ± ± ± ± Reltive kidney (%) 0.68 ± ± ± ± ± ± ± ± Reltive pncres (%) 0.17 ± ± ± ± ± ± ± ± Reltive totl ft (%) 9.54 ± ± ± ± ± ± ± ± Reltive sucutneous ft (%) 2.84 ± ± ± ± ± ± ± ± Reltive totl viscerl ft (%) 6.53 ± ± ± ± ± ± ± ± Reltive gondl ft (%) 3.09 ± ± ± ± ± ± ± ± Reltive retroperitonel ft (%) 1.95 ± ± ± ± ± ± ± ± Reltive omentl ft (%) 1.39 ± ± ± ± ± ± ± ± Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) for ech sex within ech experimentl grouping. A significnt effect of offspring sex, independent of tretment, within ech experimentl grouping. Significnt P-vlues re in old.

8 4386 C. R. Toop nd others J Physiol Tle 3. Glucose tolernce AUC nd plsm glucose, hormone nd lipid concentrtions of 3-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 3 week mle n = 7 n = 11 n = 8 n = 7 n = 8 n = 8 n = 8 n = 9 Blood glucose AUC ± ± ± ± ± ± ± ± (mmol l 1 min 1 ) Glucose (mmol l 1 ) 12.9 ± ± ± ± ± ± ± ± Insulin (ng ml 1 ) 0.20 ± ± ± ± ± ± ± ± Leptin (ng ml 1 ) 4.59 ± ± ± ± ± ± ± ± TG (mmol l 1 ) 2.31 ± ± ± ± ± ± ± ± Totl cholesterol (mmol l 1 ) 3.66 ± ± ± ± ± ± ± ± HDL cholesterol (mmol l 1 ) 0.60 ± ± ± ± ± ± ± ± LDL cholesterol (mmol l 1 ) 3.06 ± ± ± ± ± ± ± ± HDL/LDL rtio 16:84 16:84 23:77 8: :84 19:81 16:84 10: week femle n = 8 n = 11 n = 8 n = 8 n = 8 n = 8 n = 8 n = 9 Blood glucose AUC (mmol ± ± ± ± ± ± ± ± l 1 min 1 ) Glucose (mmol l 1 ) 13.5 ± ± ± ± ± ± ± ± Insulin (ng ml 1 ) 0.41 ± ± ± ± ± ± ± ± Leptin (ng ml 1 ) 4.29 ± ± ± ± ± ± ± ± TG (mmol l 1 ) 2.05 ± ± ± ± ± ± ± ± Totl cholesterol (mmol l 1 ) 3.51 ± ± ± ± ± ± ± ± HDL cholesterol (mmol l 1 ) 0.57 ± ± ± ± ± ± ± ± LDL cholesterol (mmol l 1 ) 2.96 ± ± ± ± ± ± ± ± HDL/LDL rtio 15:85 18:82 21:79 10: :85 21:79 21:79 12: Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) for ech sex within ech experimentl grouping. AUC, re under the curve; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Significnt P-vlues re in old.

9 J Physiol Perintl exposure to sucrose or HFCS Tle 4. Glucose tolernce AUC nd plsm glucose, hormone nd lipid concentrtions of 12-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 12 week mle n = 8 n = 5 n = 8 n = 8 n = 8 n = 8 n = 9 n = 9 Blood glucose AUC ± ± ± ± ± ± ± ± (mmol l 1 min 1 ) Glucose (mmol l 1 ) 13.4 ± ± ± ± ± ± ± ± Insulin (ng ml 1 ) 2.78 ± ± ± ± ± ± ± ± Leptin (ng ml 1 ) 7.49 ± ± ± ± ± ± ± ± TG (mmol l 1 ) 1.85 ± ± ± ± ± ± ± ± Totl cholesterol (mmol l 1 ) 1.93 ± ± ± ± ± ± ± ± HDL cholesterol (mmol l 1 ) 1.00 ± ± ± ± ± ± ± ± LDL cholesterol (mmol l 1 ) 0.96 ± ± ± ± ± ± ± ± HDL/LDL rtio 50:50 60:40 57:43 54: :50 67:33 63:37 61: week femle n = 8 n = 5 n = 8 n = 8 n = 8 n = 8 n = 8 n = 9 Blood glucose AUC ± ± ± ± ± ± ± ± (mmol l 1 min 1 ) Glucose (mmol l 1 ) 13.6 ± ± ± ± ± ± ± ± Insulin (ng ml 1 ) 2.33 ± ± ± ± ± ± ± ± Leptin (ng ml 1 ) 5.84 ± ± ± ± ± ± ± ± ± TG (mmol l 1 ) 1.36 ± ± ± ± ± ± ± ± Totl cholesterol (mmol l 1 ) 1.96 ± ± ± ± ± ± ± ± HDL cholesterol (mmol l 1 ) 1.06 ± ± ± ± ± ± ± ± LDL cholesterol (mmol l 1 ) 0.90 ± ± ± ± ± ± ± ± HDL/LDL rtio 54:46 62:38 64:36 55: :46 70:30 74:26 66: Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) for ech sex within ech experimentl grouping. A significnt effect of offspring sex, independent of tretment, within ech experimentl grouping. AUC, re under the curve; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

10 4388 C. R. Toop nd others J Physiol compred to nd, ut not, mles (Tle 3). There were no significnt differences in plsm FFA concentrtions etween tretment groups t 3 weeks of ge (mles, P = 0.164; femles, P = 0.368; Fig. 2A). At 12 weeks, however, plsm FFAs were significntly incresed in mle offspring compred to offspring (P = 0.036), with trend towrd the sme effect in femles (P = 0.088; Fig. 2B). There were no other effects of perintl sucrose exposure on plsm hormones, glucose or lipids t either 3 weeks (Tle 3) or 12 weeks (Tle 4) of ge. Heptic lipid content nd ftty cid composition. There were no overll effects of exposure to sucrose during either the prentl nd/or the suckling periods on totl heptic ft content (mles, P = 0.371; femles, P = 0.141; Fig. 3A) or heptic TG, FFA or phospholipid concentrtions t 3 weeks of ge (Tle 5). At 12 weeks, significnt effect of tretment ws detected for totl liver ft content in femle offspring (P = 0.032), nd for phospholipid concentrtion in mle offspring (P = 0.036; Tle 6), ut the Bonferroni post hoc djustment filed to identify ny significnt differences etween tretment groups. Heptic ftty cid composition ws significntly ltered y perintl sucrose exposure in oth mle nd femle offspring t 3 weeks. Heptic totl MUFA content ws incresed in femle offspring compred to offspring (Tle 5). In terms of individul MUFAs, heptic n-7 MUFA content ws higher in oth mle nd femle offspring compred to offspring, nd n-7 MUFA ws lso higher in femles compred to femles (mles, P = 0.012; femles, P = 0.001; Fig. 3B). In femles, offspring lso hd incresed totl heptic n-9 MUFA concentrtions compred to offspring (P = 0.033; Fig. 3C), ut this effect ws not present in mles. Heptic totl n-3 PUFA content ws higher in femles compred to the nd groups, nd ws lso higher in compred to offspring in mles (Tle 5). There were no effects of mternl sucrose consumption on heptic SFA or n-6 PUFA content in either mle or femle offspring t 3 weeks (Tle 5). There were no significnt differences etween groups for the content of ny individul ftty cids t 12 weeks of ge in either mle or femle offspring (Tle 6). A Plsm FFA (meq l 1 ) C Plsm FFA (meq l 1 ) B Mle Femle Mle Femle D Mle Femle Mle Femle Plsm FFA (meq l 1 ) Plsm FFA (meq l 1 ) Figure 2. Plsm free ftty cid concentrtions Plsmfree fttycid (FFA) concentrtions ofa,3-week-old ndb, 12-week-old offspring exposed to sucrose, nd C, 3-week-old nd D, 12-week-old offspring exposed to HFCS-55 during the prentl nd/or suckling periods. Dt re presented s men ± SEM., Different lower-cse letters denote men vlues tht were significntly different from ech other (P < 0.05, one-wy ANOVA) within ech experimentl grouping (sucrose or HFCS-55 exposure groups) nd within ech sex. There were no significnt effects of offspring sex, independent of tretment.

11 J Physiol Perintl exposure to sucrose or HFCS Effects of perintl HFCS-55 exposure Body weight nd growth. There were no effects of HFCS-55 exposure during the prentl nd/or suckling periods on offspring ody weight t ny time point etween irth nd 12 weeks of ge, nose til length or dominl circumference (dt not shown), or on ody weight t post-mortem t 3 weeks (Tle 1) or 12 weeks (Tle 2) of ge. Orgn weights. While there were no effects of HFCS-55 exposure on reltive orgn weights t 3 weeks (Tle 1), t 12 weeks of ge femle offspring hd higher reltive liver weight when compred to oth nd offspring, n effect not oserved in mles (Tle 2). There were no effects of HFCS-55 exposure during either the prentl or the suckling periods on reltive hert, drenl, kidney or pncres weights in 12-week-old offspring, independent of sex (Tle 2). Body ft mss. Exposure to HFCS-55 during the suckling period lone ws ssocited with incresed reltive omentl ft mss in oth mle nd femle offspring t 3 weeks of ge compred to offspring (Tle 1). In mles, totl reltive ft mss nd reltive sucutneous ft mss were lso oth higher in compred to offspring, ut not compred to offspring, with trend towrd incresed reltive viscerl ft mss in mle offspring exposed to HFCS-55 during suckling, regrdless of prentl exposure (P = 0.056). At 12 weeks of ge, however, mles exposed to HFCS-55 during oth the prentl nd the suckling periods () hd lower reltive retroperitonel ft mss nd lso tended (P = 0.063) to hve lower reltive totl viscerl ft mss compred to offspring (Tle 2). No other effects of perintl HFCS-55 exposure on reltive ft mss were oserved in offspring t 3 or 12 weeks. Glucose tolernce. There ws no effect of HFCS-55 exposure on glucose tolernce AUC t either 3 weeks (Tle 3) or 12 weeks (Tle 4). There were lso no effects on ny individul lood glucose vlues during the glucose tolernce test for offspring t 3 or 12 weeks of ge (dt not shown). Plsm glucose, hormone nd lipid concentrtions. At 3 weeks of ge, plsm FFA concentrtions were higher in offspring exposed to HFCS-55 only during the prentl period () compred to offspring in oth mles nd femles (mles, P = 0.008; femles, P = 0.013; Fig. 2C). This sme elevtion of plsm FFAs in the compred to the group ws lso oserved t 12 weeks of ge in oth mles nd femles (mles, P = 0.006; femles, A Heptic ft content (%) D Heptic ft content (%) B Mle Femle Mle Femle Mle Femle Heptic omeg-7 (mg 100 g 1 ) Heptic omeg-7 (mg 100 g 1 ) E Mle c Femle c c C Heptic omeg-9 (mg 100 g 1 ) F Heptic omeg-9 (mg 100 g 1 ) Mle Mle Femle Femle Figure 3. Heptic lipid concentrtions of 3-week-old offspring Totl heptic ft A, omeg-7 B, nd omeg-9 C, content of 3-week-old offspring exposed to sucrose, nd totl heptic ft D, omeg-7 E, nd omeg-9 F, content of 3-week-old offspring exposed to HFCS-55 during the prentl nd/or suckling periods. Dt re presented s men ± SEM.,,c Different lower cse letters denote men vlues tht were significntly different from ech other (P < 0.05, one-wy ANOVA) within ech experimentl grouping (sucrose or HFCS-55 exposure groups) nd within ech sex. There were no significnt effects of offspring sex, independent of tretment.

12 4390 C. R. Toop nd others J Physiol Tle 5. Heptic lipid concentrtions in 3-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 3 week mle n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 TG (mg 100 g 1 ) 1774 ± ± ± ± ± ± ± ± FFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± Phospholipid 2232 ± ± ± ± ± ± ± ± (mg 100 g 1 ) SFA (mg 100 g 1 ) 1380 ± ± ± ± ± ± ± ± MUFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-3 (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-6 (mg 100 g 1 ) 1240 ± ± ± ± ± ± ± ± week femle n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 TG (mg 100 g 1 ) 1079 ± ± ± ± ± ± ± ± FFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± Phospholipid 2395 ± ± ± ± ± ± ± ± (mg 100 g 1 ) SFA (mg 100 g 1 ) 1172 ± ± ± ± ± ± ± ± MUFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-3 (mg 100 g 1 ) ± ± ± ± ± ± ± ± 36.3 ˆ0.050 n-6 (mg 100 g 1 ) 1039 ± ± ± ± ± ± ± ± Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) within ech experimentl grouping. A significnt effect of offspring sex, independent of tretment, within ech experimentl grouping. While the one-wy ANOVA identified significnt effect of tretment, no significnt differences were detected etween tretments y the Bonferroni post hoc test. TG, triglyceride; MUFA, monounsturted ftty cid; SFA, sturted ftty cid; FFA, free ftty cid; n-3, omeg-3; n-6, omeg-6.

13 J Physiol Perintl exposure to sucrose or HFCS Tle 6. Heptic lipid concentrtions in 12-week-old mle nd femle offspring exposed to sucrose or HFCS-55 during the prentl nd/or suckling periods P-vlue P-vlue 12 week mle n = 6 n = 5 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 Totl liver ft content (%) 8.1 ± ± ± ± ± ± ± ± TG (mg 100 g 1 ) 1463 ± ± ± ± ± ± ± ± FFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± Phospholipid (mg 100 g 1 ) 2367 ± ± ± ± 21 # ± ± ± ± SFA (mg 100 g 1 ) 1275 ± ± ± ± ± ± ± ± MUFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-3 (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-6 (mg 100 g 1 ) 1362 ± ± ± ± ± ± ± ± n-7 (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-9 (mg 100 g 1 ) ± ± ± ± ± ± ± ± week femle n = 6 n = 5 n = 6 n = 6 n = 6 n = 6 n = 6 n = 6 Totl liver ft content (%) 4.4 ± ± ± ± ± ± ± ± TG (mg 100 g 1 ) 1883 ± ± ± ± ± ± ± ± FFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± Phospholipid (mg 100 g 1 ) 2555 ± ± ± ± 42 # ± ± ± ± SFA (mg 100 g 1 ) 1618 ± ± ± ± ± ± ± ± MUFA (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-3 (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-6 (mg 100 g 1 ) 1471 ± ± ± ± ± ± ± ± n-7 (mg 100 g 1 ) ± ± ± ± ± ± ± ± n-9 (mg 100 g 1 ) ± ± ± ± ± ± ± ± Dt re presented s men ± SEM. The reported P-vlue indictes the effect of tretment within ech sex, s determined y the one-wy ANOVA., Different superscript letters denote men vlues tht were significntly different from ech other (P < 0.05) within ech experimentl grouping. A significnt effect of offspring sex, independent of tretment, within ech experimentl grouping. # A significnt interction etween tretment nd sex, s determined y the two-wy ANOVA. While the one-wy ANOVA identified significnt effect of tretment, no significnt differences were detected etween tretments y the Bonferroni post hoc test. TG, triglyceride; MUFA, monounsturted ftty cid; SFA, sturted ftty cid; FFA, free ftty cid; n-3, omeg-3; n-6, omeg-6; n-7, omeg-7; n-9, omeg-9.

14 4392 C. R. Toop nd others J Physiol P = 0.039), nd t this ge, FFA concentrtions were lso higher in compred to offspring in mles (Fig. 2D). At 3 weeks of ge, oth the HDL cholesterol concentrtion nd the rtio of HDL/LDL cholesterol were lower in mles compred to ll other groups, nd in femles when compred to nd only (Tle 3). At 12 weeks of ge, femle offspring hd higher HDL cholesterol compred to femles. Plsm LDL cholesterol ws decresed in mle nd femle nd offspring compred to offspring, ccompnied y n increse in HDL/LDL rtio (Tle 4). Plsm insulin tended to e incresed in 12-week-old mles (P = 0.07), ut there were no other effects of perintl HFCS-55 exposure on plsm glucose, leptin or TG concentrtions. Heptic lipid content nd ftty cid composition. Exposure to HFCS-55 during the suckling period only () ws ssocited with incresed totl liver ft content in 3-week-old mles compred to, nd offspring (P < 0.001), with trend (P = 0.074) towrd this sme effect in femles (Fig. 3D). Mle offspring hd lower phospholipid content compred to mles only (Tle 5). At 3 weeks of ge, femle offspring hd elevted totl MUFAs compred to femles (Tle 5). In ddition, the n-7 MUFA content of the liver ws incresed in femles exposed to HFCS-55 during the suckling period ( nd ) compred to offspring (P < 0.001), nd this effect ws more pronouncedinthninoffspring(fig.3e). Femle offspring lso hd n incresed heptic n-9 MUFA content compred to offspring (P = 0.004), n effect tht ws not oserved in mles (P = 0.348; Fig. 3F). While the Bonferroni post hoc djustment did not identify ny significnt differences etween tretment groups, heptic n-3 PUFAs ppered to e elevted in 3-week-old femles (P = 0.05), with similr trend oserved for heptic n-6 PUFAs (P = 0.059) in the sme offspring. At 12 weeks, there were no significnt chnges in totl heptic lipid content nor the content of ny of the individul ftty cids, despite orderline effect on heptic n-7 MUFA concentrtion in mles exposed to HFCS-55 during suckling (P = 0.052). Discussion Despite fructose-contining sweeteners eing ssocited with detrimentl helth impcts in dults, the effect of mternl consumption of SSBs on the developing offspring is reltively unknown. We previously reported tht mternl sucrose nd HFCS-55 consumption were oth ssocited with incresed energy intke in dms, ut tht the metolic impct of these sugrs differed, with sucrose-fed dms exhiiting incresed diposity nd dyslipidemi, while those tht consumed HFCS-55 tended to develop hyperinsulinemi (Toop et l. 2015). In this previous study, mternl HFCS-55 lso resulted in lower plsm FFA concentrtions in oth the dm nd their neworn offspring (Toop et l. 2015). The current study uilds on these previous findings y following up the offspring exposed perintlly to either sucrose or HFCS-55 t 3 nd 12 weeks of ge. We hve identified severl effects, prticulrly in reltion to ody ft mss, plsm FFAs nd heptic lipid composition, nd hve shown tht these re dependent on oth the specific sugr involved nd the timing of the exposure (i.e. prentlly or during the suckling period). Neither developmentl exposure to sucrose nor HFCS-55 hd ny significnt impct on offspring ody weight t 3 or 12 weeks of ge. Therefore, mternl sugr consumption did not pper to either impir or ccelerte the postntl growth trjectory of the offspring efore or fter wening. This is consistent with the findings reported y Kendig et l. (2015), who lso reported no impct of mternl consumption of 10% (w/v) sucrose everge during pregnncy on the ody weight of wenling offspring. Other studies tht hve investigted the effects of mternl sucrose consumption during pregnncy nd lcttion (D Alessndro et l. 2012, 2014; Smuelsson et l. 2013), or sucrose or HFCS consumption during either the pregnncy or lcttion periods (Bocrsly et l. 2012), hve generlly reported incresed ody weight of offspring, dependent on oth sex nd ge. The discrepncies etween studies my e explined y differences in mternl sugr consumption, exposure window nd the ge t which offspring were evluted. The mjor findings of the current study relte to the significnt impct of perintl sucrose nd HFCS-55 exposure on offspring ft mss nd heptic lipid profile. The postntl window is the mjor period in which rodents deposit white dipose tissue (Tylor & Poston, 2007), nd consistent with other cross-fostering models investigting theeffectofexposuretomternloesity(gorskiet l. 2006) or consumption of high-ft (Sun et l. 2012) or cfeteri (Gugusheff et l. 2013) diet during different developmentl windows, exposure to either sucrose or HFCS-55 during the suckling period ws found to hve greter impct on dipose tissue deposition thn exposure efore irth. Thus, exposure to either sucrose or HFCS-55 during the suckling period lone, ut not during the prentl period, ws ssocited with incresed viscerl ft deposition in the 3-week-old offspring. This suggests tht offspring consumed incresed energy during this period, potentilly from incresed ft nd/or energy content of the mternl milk. While these prmeters were not mesured in the current study, Chicco et l. (2016) reported tht mternl consumption of high-sucrose diet ws ssocited with incresed sturted ft content of the milk. The incresed ft mss oserved in 3-week-old