research paper : nutrient requirements and interactions

8/14/2019 research paper : Nutrient Requirements and Interactions

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Nutrient Requirements and InteractionsMaternal Dietary Carbohydrate Restriction and M ild-to-M oderate Exercise during Pregnancy M odifyAspects of Fetal Development in Rats1'2'3

M ONA COBRIN4 AND KRISTINE G KOSKI5School of D ietetics and H um an N utrition M cG ill U niversity MontrealQuebec H 9X 3V9 Canada

ABSTRACT To determine whether acute bouts of exercise during pregnancy would predispose the fetus toincreased risk if m aternal dietary carbohydrate w ere restricted, untrained pregnant rats were randomly assigned to a 0% (low), 12% (moderate) or 60% (high)glucose diet, and either rested or exercised daily for 20min from d 16 to term on a rodent treadmill at a mild(15.5 m /m in) or m oderate (24.3 m /m in) intensity. A 3 x3 nested factorial m odel w ith and w ithout food intake asa covariate was employed. Both greater exercise intensity and the lower levels of dietary carbohydrate indep en den tly d ecreased term m atern al liv er an d p lan tarisglycogen concentrations and increased plasm a lactatecon cen tration s. H ow ev er, sign ifica nt d ifferen ces d ue toexercise disappeared (except for plasm a lactate) withfood intake controlled for in the m odel, indicating thatene rg y d eficits m od ulated th ese e xercise effects. In co ntrast, fo r th e offsp rin g, w hen foo d in tak e w as co ntrolledfo r, a restricted lev el o f m atern al dietary carb ohy dratesignificantly low ered fetal w eight, plasm a glucose andi ns ulin c oncentr atio ns a nd liv er g ly cogen concentr atio nsmeasured at term . Exercise alone did not reduce meanfetal w eight if nested w eights w ithin a litter w ere used inthe statistical analysis. M ild to m oderate m aternal exe rc ise lowe re d o nly fe ta l p la sm a g lu co se c on ce ntra tio nsand only if m aternal food intake was not controlled for.These results indicate that acute exercise during pregn an cy can h av e d etrim en tal effects o n fe tal de velopmen to nly if d ie ta ry g lu co se is se ve re ly re stric te d. O th erw ise ,adequate glucose and energy in the maternal diet inuntrained pregnant rats during repeated bouts of acuteexercise seem to protect the fetus. J. Nutr. 125:1617-1627, 1995.IN EXING KEY W OR S

tissu e g ly co ge n p la sm a g lu co se r ats e ne rg y in ta ke

Exercise during pregnancy may induce a competition for glucose between exercising muscles and thegrowing fetus; previous studies have shown thatm aternal exercise dim inishes m aternal (Carlson et al.1986, Gorski 1983) and fetal (Gorski 1983) liver

glycogen, augments maternal skeletal muscleglycogen content (M ottola and Christopher 1991) andcom promises glucose intake by the fetus (Treadwayand Young 1989). Adequate glucose delivery to thefetus is critical because glucose is the principal m etabolic fuel metabolized by the grow ing embryo andfetus (Battaglia and Meschia 1978). G lucose is required for the in utero glycogen reserves that must bedeposited in the fetal liver and heart during the latterpart of gestation (Shelley 1961). Deficits in theseglycogen deposits have been associated w ith increasedperinatal m ortality (Shelley and Nelligan 1966). M orerecently, it was reported that the absence of a specificsource of dietary glucose lim ited in utero glycogendeposition, leading to high rates of neonatal m ortalitythat were reversed only if the level of glucose wasincreased in the maternal diet (Koski and H ill 1986and 1990, Lanoue et al. 1992).The purpose of this study was to determ inewhether different levels of dietary carbohydrate combined with various exercise intensities would perturbcarbohydrate hom eostasis in pregnancy and alter fetalgrowth and biochemical development. Thehypothesis was that acute bouts of exercise com bined

'Presented at the 36th Annual M eeting of the Canadian Federa tio n o f B io log ica l S cien ces, Ju ne 1 99 3, W in dso r, O nta rio (C ob rin ,M . & . K oski, K . G . (1993) T he interaction of dietary carbohydrateand exercise intensity during pregnancy on fetal growth and developm ent. Proc. C an. Fed. Biol. S oc. 36: 396 (abs.)j.

2T he financial support of the N atural S ciences and E ngineeringR esearch C ouncil of C anada (N SE RC OGP 0003623) is gratefullyacknowledged.'T heosts of publication of this article w ere defrayed in part bythe paym ent of page charges. T his article m ust therefore be herebym arked advertisem ent in accordance w ith 18 U SC section 1734solely to indicate this fact.4M . C ob rin w as th e rec ip ien t o f p os tg rad uate stu de nt fello wsh ipfrom the Fonds pour la Formation de Chercheurs et l'A ide duQube c( FCAR|, a Qube cp ro vi nc ia l f un di ng a ge nc y.5To whom correspondence and reprint requests should be addressed.

0 022 -3 16 6/9 5 $ 3.0 0 1 99 5 Am erica n In stitu te o f N utritio n.M anuscript received 12 July 1994. Initial review completed 24 August 1994. Revision accepted 22 Decem ber 1994.1617


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1618 COB R IN A ND K OSK Iw i th re stric te d mate rn al d ie tary c arboh ydrate in tak edurin g p re gnan cy cou ld c omp rom ise g lu co se d eliv e ryto the dev eloping fetus during late gestation in untrain ed p reg nan t rats. T h e sp ecif ic o bje ctiv es w e re 2 )to inv estigate w hether graded lev els of m aternal dietary glucose com bined w ith v arious degrees of exercise intensity in pregnant rat dam s w ould prov ok eb ioc hem i cal alte ratio ns in matern al p lasma glu co se,lactate and insulin and/or liv er and m u scle gly cogenconcentrations; 2} to determ ine w hether changes inth e matern al sy stem , w h ic h also in clu de d m e asu res o famn io tic f lu id g lu co se an d lactate , w o uld be ref lec tedin fetal indices such as litter siz e, pup w eight, liv erand heart gly cogen, and/or fetal plasm a glucose,lac tate and in su lin c on centratio ns; and 3 ) to e stab lishif any m easured m etabolic changes w ere m ediatedthrough dif ferences in energy intak e or w ere a resulto f a sp ecif ic d ietary g lu co se d ef icit. T h e p resen t stu dyis unique in that the lev el of m aternal dietary carbohy drate w as altered, f ood intak e (w hich is a prox y f orenergy intak e in isoenergetic diets) w as m easured,and signif icantly dif f erent ef f ects of ex ercise arere po rte d w h en f oo d in tak e w as an d w as n ot co ntro lle dfor as a cov ariate in the statistical m odel.

M A TER IA LS A ND M ETHODSA ll procedures conform ed to guidelines for experim ental procedures set forth by the local anim alcare com mittee of M cGill Univ ersity and by theCanadian Council on A n im al Care (1984).E xper im en ta l design . A 3 x 3 fa ctor ia l design wa sused to study the interactiv e ef f ects of vary ing exercise intensity and lev el of dietary carbohy drateduring pregnancy on m aternal and fetal m easures.T ime -b re d Sp rague -Daw l ey p re gnan t rats (1 80 -2 00 g ,C harles R iv er, S t. C onstant, Q u b ec,Canada) werereceiv ed f rom d 0 (day f ollow ing im p regnation) to d 2of pregnancy (gestation length 22.5 d) and w ere imm ediately housed indiv idually in suspended w irecages in a tem p erature-controlled room (20 C)withf luorescent lighting (0700-1700 h). U pon arriv al,pregnant rats w ere random ized into three dietarytreatm e nts (0 , 1 2 or 6 0% d ietary g lu co se ). T h ese d ie ts

w e re f ed th ro ug ho ut p reg nan cy , an d d aily f oo d in tak eand body w eight gain w ere m easured. W ithin eachdietary group, dam s w ere again random iz ed to threedaily ex ercise protocols (20 m in/d f rom d 16 to d 21 ofpregnancy ): not ex ercised (rest), ex ercised at a m ildintensity (15.5 m /m in) on a rodent treadm ill, or ex ercised at a m oderate intensity (24.3 m /m in). A ll ratsw ere rested from the day they w ere receiv ed until d13 of pregnancy , w hen the acclim atiz ation protocolb egan . T h e g ro ups o f ex erc ised rats w e re h ab itu ated ,follow ing m inor m odif ications to a prev iouslyreport ed accl ima tiz ation pro toco l (Carls on e t al. 1986,Gorsk i 1983), by running on the rodent treadm ill

f rom d 13 to 15 of pregnancy inclusiv e at 8.10 m /m infor 10 m in/d.E xercise protocol. O u r exercise protocol wa smodif ie d f rom th ose emp lo yed in p rev io us p reg nan cy -e xercise stu die s (C arlso n et al. 1 98 6, G orsk i 1 98 3) inw hich the ex ercise w as lim ited to one 60-143-m inbo ut n ear term ; o ur ex erc isin g re gim e n in vo lv ed d ailyex ercise (from d 16 to term ) and spanned the entireperiod of fetal liv er gly cogen accum ulation, w hichtak es place f rom d 18 to term in pregnant rats (S helleyand N eligan 1966) and is k now n to be sensitiv e tod ie tary c arb oh ydrate restrictio n (K o sk i e t al. 1 98 6).Preg nan t rats w e re ex erc ised o n a ro den t treadm ill(from d 16 to term inclusiv e) at an intensity of either15.5 or 24.3 m /m in for 20 m in at 0% grade. T heseex ercise intensities w ere selected af ter a pilot studydeterm ined that the m ax im um running speed of untrained S prague-D aw ley pregnant rats at term w as24.3 m /m in for 20 m in at 0% grade; at this intensityour untrained term pregnant rats w ould not runbey ond the 20 m in due to lack of training and despiteav ersiv e stim u latio n. U sin g th e calc ulatio n f or w o rkin k g-m [(% grade) x (m /m in) x (durationm in) x bodym ass (g)]w ith 6 k g-m = 1 W , w e determ ined that ratsex ercised at 15.5 m /m in perform ed 16 W and thoseex ercised at 24.5 m /m in com pleted 28 W of w ork onav erag e at te rm .D iet se lec tio n a n d co mp osition . T h e ca r bo hyd ra tef re e, trig ly c erid e-b ase d d ie t and th e c on tro l c arb oh ydrate-rich diet are described in T able 1. T hese sam elev els of carbohy drate (0, 12 and 60% ) w ere used inprev iou s in ve stig atio ns (K o sk i et al. 1 98 6, K o sk i an d

TABL EC om po sitio n o f co ntr ol a n d ca r bo hydr a te r estr ictedtr ig lyc er id e ba sed g lu co se d ie ts 1

G lu co se lev elIngredient 0% 12% 60%Glucose2Soybeanoil3Cellulose4Casein5Vitaminmix6Mineralmix6DL-MethionineSodiumbicarbonateTotal,gMetabolizable energy, kj/g039.6441.3611.01.25.50.341.0100.0417.31234

'Dry w eight basis (g).2D ex trose (anhy drous) (IC N B iochem icals C anada, M ontr aQubec,Canada .3 Deg um m e d s oy be an o il (C an ad a Pac kers, M o ntr al, Qu be4 A lp hac el, IC N B io che m icals C anad a.

High nitrogen casein containing 86.5% protein (N x 6.25) or89.1% protein (N x 6.71) |IC N B iochem icals Canada).6S ee T able 1 in L anoue et al. (1992) f or com plete ingredient list.


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DIETA RY CA RBOHY DRA TE A ND EX ERCISE DUR IN G PREGN A NCY 1619Hill 1986 and 1990) that established the essentialityof m aternal dietary carbohy drate f or pregnancy . Eachisoenergetic diet w as calculated to contain 17.3 k j ofm etabolizable energy per gram of dry m atter, w hichm et the N R C requirem ent for pregnant rats (N R C1978). Protein w as present at a m inim al but adequatelev el of 11 % to supply the lev els of protein, nitrogenand essential am ino acids required by pregnant dam sand to avoid any ex cess protein that w ould prov idesupplem entary glucogenic am ino acids that w ouldreduce the sev erity of the carbohy drate def iciency ,- inearlier ex perim ents this lev el of protein producedpups w ith w eight at term not signif icantly dif ferentf rom that for controls fed higher lev els of protein(K osk i et al. 1986). A s w ith protein, the quantities ofv itam ins and m inerals w ere held constant and w eresupplied at four tim es the N RC requirem ents for thew ater-soluble v itam ins and 1.5 to tw o tim es the N RCrequirem ents for the fat-soluble v itam ins; theseam ounts perm itted adequate consum ption if foodintak e w ere v oluntarily restricted.T o m aintain glucose as the f irst lim iting nutrient,the low carbohydrate, high lipid diets w ere form ulated from the high carbohy drate reference control bysubstituting lipid (soy bean oil, 38 .6 k j/g ) isoen ergeti-cally for glucose (13.3 k j/g) and adding cellulose tom aintain a constant m etabolizable energy for eachk ilogram of diet. T rigly c ride w as chosen as the lipidbase because it serv es as a natural fat source, and theuse of a soy bean oil preserv es the gly cerol com ponent,w hich can act as a glucose precursor. T hus, the use ofintact trigly c ride as soy bean oil pro vided 4 % glucoseequiv alents as gly cerol in the glucose-f ree (0% ) diet,w hich is the m inim al am ount required to supportim plantation and m aintain pregnancy to term (K osk iet al. 1986). T he interm ediate 12% glucose diet w aschosen because this restricted lev el of m aternal dietary carbohy drate has produced fetal liv er gly cogenconcentrations that are approx im ately half those ofpregnant control anim als (K osk i et al. 1986). T he 60%glucose diet is the standard control diet used inreproductiv e studies. T he experim ental diets andw ater w ere freely av ailable, and daily food intak e andfood spillage w ere recorded. T otal f ood intak e w asthen calculated by sum ming the daily food intak es.T hus, cum ulativ e food intak e represented totalenergy intak e in our isoenergetic diets.Tissu e collection . Da ms were delivered of fetu sesby caesarian section on gestational d 22.5 (term ). A lldam s w ere k illed in a post-absorptiv e (fed) state af terthey w ere either ex ercised at a m ild or m oderateintensity or rested. Ex ercised rats w ere k illed im m ediately (t < 5 m in) af ter com pleting their designatedex ercise protocol. Dam s w ere anesthetiz ed w ithk etam ine-HCl (R ogarsetic, 30 m g/kg, R ogar/S TB ,L ondon, Ontario, Canada) injected into the jugularregion so as to avoid anesthetiz ing the fetuses.M aternal blood (5-7 m L) w as w ithdraw n by cardiacpuncture. A ll blood w as centrif uged and the plasm a

stored frozen (-20 C) until analy zed. Follow ingcardiac puncture, the abdom en w as opened and theliv er rem ov ed, f reez e-clam ped in liquid nitrogen andfroz en at -80 C. A l l uteri w ere ex am ined for im plantation and r sorption sites, then rem oved from theabdom en but k ept intact. A tuberculin sy ringe (1 cc,27 gauge) w as used to collect am niotic f luid, w hichw as stored f roz en at -20 C until analy zed. Indiv idualfetuses and placentas w ere then rem oved from them other and w eighed, and liv e fetuses w ere k illed byex sanguination. Fetal blood w as drained f rom the axillary artery v ia hepariniz ed capillary tubes. T he bloodw as centrifuged and the plasm a stored frozen at-20 C until analy zed. A ll fetal liv ers and hearts w ererem ov ed, f reez e-clam ped in liquid nitrogen and storedat -80 C. T he m aternal soleus and plantaris m usclesw ere rem oved from the hind leg, w eighed, f reeze-clam ped in liquid nitrogen and stored at -80 C untilanalyzed.An alytica l procedu res. All tissu e glycogen con centrations w ere determ ined by the m ethod of L o et al.(1970). M aternal and fetal plasm a glucose and amniotic f luid glucose w ere m easured by hexok inasedeterm ination (k it 16-U V , S igm a C hem ical, S t. L ouis,M O) using the A bbott V P S uper S y stem (Irv ing,T exas). T he procedure is sim ilar to that described byB ondar and M ead (1974). M aternal and fetal plasm alactate and am niotic f luid lactate w ere m easured bylactate dehy drogenase determ ination (k it no. 826-U V ,S igm a Chem ical) using the A bbott V P Super S y stem .T his procedure is based on that of Henry (1964).M aternal and f etal plasm a insulin w ere quantitativ elydeterm ined by R IA in w hich the antigen com petesw ith a constant am ount of radioactiv e tracer (I125-labeled insulin) for the binding sites of the antibodyusing S erono k its (R IA , B iodata, N CS Diagnostics,M ississauga, Ontario, Canada). T he insulin concentration w as obtained by interpolating from a standardcurve.Sta tistica l a na lysis. Th e exper im en ta l design wa s a3x 3 factorial random iz ed block design because therew ere three lev els of dietary glucose (0, 12 and 60%)and three lev els of ex ercise intensity (rest, m ild andm oderate). M aternal data w ere analy zed by A N O V Aand analy sis of cov ariance (S A S sof tw are, v ersion 6.0;SA S Institute, Cary , N C) in w hich cum ulativ em aternal food intak e, a prox y for energy in isoenergetic diets, w as treated as the covariate. A naly sis ofcov ariance w as necessary because food intak e v ariedaccording to the lev el of dietary carbohy drate. R esultsof m ain ef fects (diet, ex ercise, diet x ex ercise) w erepresented w ith and w ithout food intak e as a cov ariateso that the im pact of energy intak e on m odify ing thesignif icance of the m ain ef fects of diet and ex ercise onoutcom e m easures could be evaluated. A lim itation ofearlier ex ercise studies (Carlson et al. 1986, Gorsk i1983, M ottola and Christopher 1991, T readw ay andY oung 1989) w as that they did not m easure foodintak e or statistically control f or energy and/or carbohy drate intak e; th eref ore, th ey cou ld not dif ferentiatebetw een these dietary def icits.


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1620 COBR IN A ND K OSK IW hen there w as no statistically signif icant interaction betw een dietary glucose lev el and ex ercise intensity , the m ain ef f ects of diet and of ex ercise w ereanaly zed w ithout an interaction term in the m odelstatem e nt. A signif icant interaction w as f ound onlyf or m aternal liv er w eight and f etal plasm a lactate.W h en fetal data w ere recorded by indiv idual f etus

(i.e., f etal and placenta w eight, f etal liv er and heartgly cogen), the data w ere nested. T he nesting of datam eant that although the data w ere ex am ined by indiv idual f etus, the v ariability of these data w as attributed to the litter. T hus each pup w as not treatedas an independent observ ation because the dam w asthe experim ental unit in this study . How ever, alth ou gh u nn ested d ata are n ot statistic ally re lev an t too ur mo del, th ey are rep orted h ere so th at compariso nscan be m ade to prev ious inv estigations that hav ere po rte d re su lts u sin g u nn este d f etal d ata. A l l v alue sw ere considered statistically signif icant at the P


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DIETA RY CA RB OHY DR A TE A ND EX ERCISE DUR IN G PR EGN AN CY 1621

TABLE

Compar ison of the si gni fi cance of mai n ef fects di et, exer ci se, di et x exer ci se) on mater nal and f etal metabol ic measur es i npr egnant r ats and their of fspr ing wi th and without f ood i ntake as a covar iate in the stati stical model 1W ithout f oodntakeDependent

variableMaternalLiverweightg)Glycogen(mg/g|Heartweightg|Soleusweightg)Glycogen(mg/g)Plantarisweightg)Glycogen(mg/g)PlasmaGlucose

(mmol/L)Lactate|mmol/L)Insulin(pmol)AmnioticGlucose(mmol/L|Lactate(mmol/L)FetalPlasmaGlucose

(mmol/L)Lactate(mmol/L)Insulin(pmol)Liverglycogenmg/g)Heartglycogen |mg/g)Diet0 .00010.00010.02030.00360.00010.01970.0001NS0.00270.00010.00010.03400.0001NS0.0201NA 2NA ExerciseNS20.0583NSN SN S

x0 .0747NSNSNSNSNS0 .0822NSNS0 .0835NSNSNS0 .009NSNANADie t0 .0foodntakeExerciseNSNSNS0.0776NSN

x0 .0479NSNS

1R esults (P v alues) f rom A N O V A : diet, m ain ef fect of lev el of dietary carbohy drate (0, 12, 60% glucose); ex ercise, m ain ef fect of ex erciseintensity (rest, m ild (15 m /m in), m oderate (23 m /m in|]; D x E, interaction of tw o m ain ef fects of diet and ex ercise. W ith food intak erepresents the level of signif icance w hen energy w as treated as a covariate in the m odel: ex ercise, diet, D x E, food intak e.2N S = nonsignif icant (assum es P > 0.100). N A = not applicable.

2 When energy intake was included in thestatistical m odel, liv er and plantaris gly cogen concentrations w ere not signif icantly low ered by our exercise protocols; only m aternal plasm a lactate concentration w as m odif ied by ex ercise. S im ilarly ,m aternal liv er, soleus, and plantaris gly cogen concentrations and m aternal plasm a lactate w ere not signif icantly af fected by the lev el of carbohydrate in them aternal diet if food intak e w as in the statisticalm odel. In contrast, signif icant reductions in liv er andsoleus w eight, m aternal plasm a insulin, and am nioticf luid glucose and lactate concentrations rem ainedw hen food intak e w as treated as a covariate, indicating that dif ferences for these latter variablesw ere produced by the specif ic m aternal dietary carbohy drate def icit w hereas dif ferences in the form er, particularly the ef fect of ex ercise regim ens on liv er andplantaris gly cogen concentrations, w ere produced bythe energy def icit.3 As fo r fe ta l measur ements inc re as ed mate rna lex ercise and dietary glucose restriction m odif ied f etalplasm a glucose in spite of the fact that the dam w aseugly cem ic. W ith food intak e as a covariate, restrictions in m aternal dietary glucose resulted in signif icantly low er f etal plasm a glucose and insulin w hereasex ercise had no ef fect, indicating that insuf f icient

m aternal energy intak e had produced the earlier signif icant change in fetal plasm a glucose follow ing m ildto m oderate ex ercise in the pregnant rat dam .

Materna l b od y w eig htsB oth the lev el of dietary carbohydrate (P < 0.0001)and ex ercise intensity (P < 0.0139) signif icantly inf luenced m aternal w eight gain,- cum ulativ e w eightgain decreased as the lev el of dietary carbohydratedecreased, and dam s ex ercised at a m oderate intensityw eighed signif icantly less than those rested or ex ercised at a m ild intensity (T able 2). Initial bodyw eights (m ean = 200 g) had not been signif icantlydif f erent am ong the treatm ent groups.

Reproduct ive performanceR eproductiv e perform ance of the dam s w asm easured by the follow ing indicators: the num ber ofim plantations (12 1), the r sorption rate (1 I/litter), the stillbirth rate (0/litter) and the num ber ofliv e fetuses (11 I/litter). N either dietary carbohydrate lev el nor ex ercise intensity inf luenced any of


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1622 COB R IN A ND K OSK Ithese variables; the values w ere not dif ferent am ongthe groups (data not show n), indicating that none ofthe signif icant dif ferences w ere caused by v ariation inlitter siz e.R esults of the ef fects of dietary carbohydrate lev eland ex ercise intensity on fetal w eights are sum mariz ed in T able 4. T he signif icance of both nested andunnested pup w eight at term (d 22.5) is reported sothat com parisons can be m ade to prev ious inv estigations, all of w hich have used unnested fetal data only ,thus assum ing that each fetus represented an independent observ ation. W h en our data w ere unnested,both diet (P < 0.0001) and ex ercise (P < 0.0002) w erefound to hav e signif icant ef fects on term fetalw eights. How ev er, af ter nesting, only carbohy dratelev el produced signif icant dif ferences in f etal w eightsam ong all dietary treatm ent groups. T he lack of signif icant dif ferences from our ex ercise protocols oncethe data w ere nested indicated that our ex erciseregim ens produced higher variability am ong pupsw ithin a litter than am ong litters.Mater na l me ta bo lic va r ia bles

Pl asma. M ater nal pl asma gl ucose was not si gni f icantly altered by diet or by ex ercise (T able 5).How ev er, m aternal plasm a lactate w as signif icantlyhigher in ex ercised v s. rested dam s, w hereas m aternalplasm a insulin w as af fected by diet and not ex ercise,but not in a dose-dependent m anner as m ight beex pected. Dam s fed the 12% glucose diet had signif icantly low er non-fasting plasm a insulin concentrations than dam s fed the 0 and 60% diets,- concentrations for the latter groups did not signif icantly dif fer.

T hese indiv idual dif ferences ex isted w hether foodintak e w as or w as not included in the statisticalm odel (T able 5). Plasm a lactate concentrations, asexpected, w ere signif icantly higher in ex ercised v s.rested dam s. A ll plasm a m easurem ents w ere perform ed in post-absorptiv e (fed) pregnant rat darns.T issue. T he gl ycogen concentr ati ons of two exercising m uscles show ed contrasting ef fects of diet andex ercise and, depending on w hether food intak e w as acov ariate, ex ercise and/or the specif ic dietary carbohy drate def icit m odif ied the statistical signif icance ofthe m ain ef fects of diet and ex ercise (T able 5). T hesoleus m uscle w ith its predom inantly slow tw itchox idativ e f ibers w as not af fected by the acute ex erciseprotocols, and only w hen food intak e w as not included as a covariate did a sev ere (0 or 12%) dietarycarbohy drate restriction signif icantly low er the concentration of gly cogen in this m uscle. For thepredom inantly fast tw itch plantaris m uscle, w hichrelies on the short-term gly coly tic sy stem for energy ,the gly cogen concentration w as af fected by both exercise and diet, w ith the highest intensity ex erciseand the low er lev els of glucose in the m aternal dietproducing signif icantly low er plantaris m usclegly cogen concentrations, but only if food intak e w asnot controlled for in the statistical analy sis. T hus, theelim ination of signif icant ex ercise and diet ef fects,once food intak e w as included in the statisticalm odel, dem onstrated that for plantaris gly cogen concentrations, the absence of suf f icient food intak e andnot the lev el of carbohydrate in the m aternal diet orthe ex ercise intensity per se had produced the earliers igni fi can t d if f erenc es .M aternal liv er gly cogen concentrations (T able 5)w ere not signif icantly low ered by the m ild-m oderate

T BLET he ef fect of gr aded l ev isof di etar y gl ucose an d ex er ci se i nten si ty on i ndi vi du al f etal wei gh ts i n ter m r at pu ps1 2

S tat is ti cal anal y si sExerciseintensityRest

MildModeratePooled3Maternal

dietarylucose0(103)

3.8 (173) 3.3 (135) 3.3 (41 1) 3 .5 0 .0 70.060.070.02a(130)

1185)' (123)1438)125.1

0.045 .0 0 .044 .8 0 .075 .0 0 .02bleve l

(%)g

(127)(USI(1261(368)605.4 5.2 5.2 5 .3 0.0 4

0.040.050.02CPooled3(360)4 .8 0 .02( 47 3) 4 .5 0 .0 2(384) 4 .5 0 .02ef fec ts4Die tExerciseD x EPNe ste d0 .0 00 1s

0.090650.3730sUnnested0.000160.000260.75946'V alues are least square m eans S EM ,w ith num ber of fetuses in parentheses.2W i thin a row , v alues are tested f or m ain ef fect of diet: dif ferent low er-case letter superscripts indicate signif icant dif ferences. W i thin a

colum n, v alues are tested f or m ain ef fect of ex ercise; dif ferent capital letter superscripts indicate signif icant dif ferences.3 W hen th ere w as n o sign if ic an t in teractio n e ff ect o f d iet an d e xerc ise (D x E ), th e statistical an aly sis treate d all d ietary c arb oh yd rate g ro up sat each lev el of ex ercise as a single group and treated all ex ercise groups at each lev el of carbohy drate as a single group; the signif icance isreported for these pooled m eans w hen food intak e w as included as a cov ariate.4R esults f rom A N O V A : diet, m ain ef fect of lev els of dietary carbohy drate; ex ercise, m ain ef fect of ex ercise intensity ,- D x E , interaction ofthe tw o m ain ef fects (diet and ex ercise).sS ignif icance (P v alue) w hen indiv idual fetal w eights are nested w ithin a litter and the dam is treated as the ex perim ental unit.

S ignif icance (P value) w hen indiv idual fetal w eights are not nested and each fetus is considered as an independent observation.


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DIETA RY CA RB OHY DR A TE A N D EX ER CISE DUR IN G PR EGN A N CY 1623

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C C n C CJ3Ti. >. 3 3>K o Z Z c S PC C'* ^&.2


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1624 COB R IN A ND K OSK Iex ercise protocols. How ever, w hen food intak e w asnot in the statistical analy sis, the restriction ofglucose in the m aternal diet resulted in signif icantlylow er liv er gly cogen concentrations, indicating, asprev iously show n for plantaris gly cogen concentrations, that adequate energy intak e play ed an important role in m aintaining these m aternal tissueg ly cogen concent rat ions .

F eta l meta bo lic va r ia ble sP la sma . When individua l va lues were compa r ed(T able 5), f etal plasm a glucose w as signif icantly low erin dam s fed 0% dietary glucose than in dam s fed 12 or60% dietary glucose, w hereas fetal glucose w ashigher, but still eugly cem ic, in of f spring from exercised v s. rested dam s. How ever, w hen food intak e w astreated as a cov ariate, only dietary carbohydratem odif ied fetal plasm a glucose, w ith signif icantly

low er concentrations occurring in fetuses from dam sfed the 0% glucose diet. A signif icant interactiv eef fect of diet and ex ercise w as found for fetal plasm alactate (T able 6) such that pups from dam s fed 12%glucose and m ildly ex ercised had low er plasm a lactateconcentrations than pups from dam s rested or m oderately ex ercised, w hereas pups from dam s fed the 60%glucose diet and m oderately ex ercised had concentrations signif icantly low er than those of all othergroups. Fetal insulin responded to dietary carbohydrate lev el but not to ex ercise intensity (T able 5).Fetuses from dam s fed 0% dietary carbohydrate hadsignif icantly low er insulin concentrations than didfetuses from dam s fed 60% dietary carbohydrate, but

neither group dif fered from the values for fetuses ofdam s fed the 12% glucose diet.Tissue. F eta l liver a nd hea r t glycogen werem easured as indicators of fetal carbohydrate stores(T able 5). Fetal liv er gly cogen concentration w as inf luenced by the lev el of carbohydrate in the m aternaldiet but not by m ild to m oderate ex ercise and onlyw hen food intak e w as a cov ariate in the m odel. Fetuses of dam s fed 60% carbohydrate had liv ergly cogen lev els signif icantly higher than those odam s fed 12% carbohydrate, w hich in turn had fetalliv er gly cogen v alues signif icantly higher than thoseof fetuses of dam s fed 0% carbohydrate. S im ilarly ,fetal heart gly cogen w as also signif icantly af fected bythe lev el of dietary glucose, but not by our ex erciseprotocols w hen food intak e w as a covariate; fetusesfrom dam s fed 0 or 12% dietary glucose had signif icantly low er lev els of heart gly cogen than fetusesfrom dam s fed the control (60%) glucose diet.

DISCUSSIONIn our study w e attem pted to dif ferentiate betw eenthose ef fects that w ere m ediated by energy restrictionand those that could be attributed either to ex erciseand/or to the lev el of dietary carbohydrate. W e alsochose to use untrained pregnant rats to elim inate anypossible m etabolic adaptation resulting f rom training(e.g., enhanced gly cogen repletion follow ing high intensity ex ercise). A s a m ain treatm ent ef fect, ourex ercise protocol produced signif icantly low ered

gly cogen concentration only in the fast tw itch plantaris m uscle, w hich is as expected for the non-

TAB LET he effect o f gra de d levels o f d ieta ry g lu co se a nd ex ercise in ten sity o n feta l p la sm a l clate 2

S tat is ti cal anal y si s3ExerciseintensityMaternal

dietary g luc ose le ve l%)01260MaineffectsF*P4mmol/LRestMildModerate(9)

4.9 .6|8)5.5 .7(11)4 .8 0 .6a(5)

A6.9(10)C4.4(6|

A6 . 6

0.80.6

0.8ab 7

V i .7(7)A6 .00.7(6)B3.2 0.8bDietExerciseD x E1.32(0.61)1.67(0.30)3.69(2.60)0.2739(0.54

'V alues are m eans S EM , with num ber of dam s in parentheses.^ W ithin a row , v alues are tested f or m ain ef fect of diet; dif ferent low er-case letter superscripts indicate signif icant dif ferences. W i thincolum n, v alues are tested f or m ain ef fect of ex ercise; dif ferent capital letter superscripts indicate signif icant dif ferences.^ R esults f rom A N O V A : diet, m ain ef fect of lev els of dietary carbohy drate; ex ercise, m ain ef fect of ex ercise intensity ; D x E , interaction othe tw o m ain ef fects (diet x ex ercise).4N um bers in parentheses represent F and P v alues w hen energy w as treated as a cov ariate in the m odel: ex ercise, diet, diet x ex ercise, f oodintake.


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DIETA RY CA RBOHY DRA TE A ND EX ERCISE DUR IN G PR EGN AN CY 1625pregnant anim al. How ever, w hen food intak e w ascontrolled for in the statistical m odel, this dif ferencew as nonsignif icant. T he only other variable to beconsistently altered by ex ercise w as plasm a lactate,w hich is expected in ex ercising anim als and has beenreported by others (Carlson et al. 1986). For m ostm aternal v ariables, including m aternal liv er andm uscle gly cogen concentrations, our results show edthat a signif icant m ain ef fect of diet w as nonsignif icant w hen food intak e w as treated as a covariate,indicating that the reduction in food intak e caused anenergy def icit that resulted in low m uscle and liv ergly cogen concentrations in rats f ed the carbohy drate-restricted diets. T hese results suggest that theprim ary concern in the m aternal sy stem is for adequate energy and that w ith adequate energy intak e,acute daily ex ercise late in pregnancy produces nodeleterious m aternal m etabolic def ects.In the fetal sy stem , how ever, outcom es that w eresignif icantly altered by diet (plasm a glucose and insulin and liv er and heart gly cogen concentrations)w ere still signif icantly low er w hen food intak e w astreated as a covariate. T his dem onstrates that for thefetus, m aternal dietary carbohydrate intak e is as important as energy , and an adequate lev el of m aternaldietary glucose is critical for f etal dev elopm ent. W esuggest that an adequate energy intak e of a carbohydrate-restricted diet during pregnancy w ill not supplysuf f icient glucose for f etal dev elopm ent, w hereasdaily bouts of m ild to m oderate ex ercise in untrainedpregnant rats w ill not be deleterious to fetal dev elopm ent if m aternal energy intak e is adequate.Prev ious experim ents using single bouts of acuteex ercise near term in f ed pregnant rats hav e presentedconf licting results. B oth Gorsk i (1993) and Carlson etal. (1986) observ ed that ex ercise lasting 60 m in at 12m /m in, 0% grade (-35 W ) caused no signif icant decline in m aternal blood glucose (rest v s. 60 m in) butdid result in signif icantly low er m aternal liv ergly cogens on d 21 of gestation. How ever, low er fetalplasm a glucose and f etal liv er gly cogen w ere reportedby Gorsk i (1983) but not by Carlson et al. (1986). Ourresults, using sequential daily bouts of m ild tom oderate ex ercise (16-28 W ), supported the observation of Carlson et al. (1986) that m aternal plasm aglucose concentrations and m aternal and fetal liv ergly cogen concentrations w ere unchanged by ex ercise,-how ev er, w e did observ e a signif icant decrease in fetalplasm a glucose that vanished once m aternal f oodintak e w as considered as a cov ariate. T o ex plain theirearlier contrasting results (Carlson et al. 1986 v s.Gorsk i 1983) the authors had suggested interstraindif ferences in response to ex ercise and/or dif ferencesin experim ental protocol... W e could add the follow ing com ments in light of our in utero f indings. Itis possible that the com parison of near-term [i.e.,gestational day s 20.5 and 21.5 (Carlson et al. 1986)]w ith term data [gestational length 22.5 day s, Gorsk i

(1989) and present study ] m ay contribute to som e ofthe confusion in data interpretation because it is accepted that fetal liv er gly cogen m ay be m obiliz ed inutero, but only at term . Hence, the absence of dif ferences in gly cogen concentrations in the fetal liv ers inthe Carlson study m ay be due to perform ing m easurem ents follow ing only a single bout of ex ercise priorto, and not at, term . T he absence of an ef fect ofex ercise on gly cogen concentrations m easured at termin our study (w hich did m odify fetal, but not m aternalplasm a glucose) m ay result f rom the m ildness of theex ercise intensity ev en though it w as perform ed atterm . T herefore, it is still possible that high intensityex ercise during the entire period of fetal gly cogenaccum ulation could signif icantly m odif y tissue stores,as observ ed in the Gorsk i (1983) study , in w hich thefed dam s exercised to exhaustion at term had profound hypogly cem ia, v ery low m aternal liv ergly cogen lev els, and low fetal liv er gly cogen concentrations. A n other consideration in the interpretationof the ex isting studies concerns the sam pling procedures and the subsequent statistical analy ses. C arlsonet al. (1986) and Gorsk i (1983) used m ultiple t tests inw hich each fetus w as treated as an indiv idual observation ev en though the treatm ent has been applied tothe dam . T here is high indiv idual variation w ithin alitter, and consequently the lack of a randomselection of a representativ e sam ple from each litterand the lack of application of correct statistics (one-or tw o-w ay A N OV A follow ed by a m ore conservativ em ultiple com parison test, giv en the nature of theex perim ental designs) m ay hav e resulted in acceptingsignif icant dif ferences that did not ex ist.In the present study , m oderate ex ercise duringpregnancy resulted in signif icantly low er gly cogenconcentrations in the m aternal plantaris m uscle andgreater m aternal lactate concentrations, indicatingthat the ex ercise protocol w as suf f iciently intensiv eto produce the com monly expected observations.How ev er, the elim ination of the signif icant ef fects ongly cogen concentrations w hen food intak e w as controlled for in the statistical m odel dem onstrates anim portant role for energy in m odulating m usclegly cogen lev els. In contrast to our observ ation, others(B aldw in et al. 1973, B lak e and Hazelw ood 1971,M ottola and Christopher 1991) have reported increased gly cogen concen trations in the gastrocnem iusm uscle follow ing ex ercise; how ever, this occurredsev eral hours af ter ex ercise, w hereas our m easurem ents w ere m ade im m ediately af ter the ex ercisebout, before any repletion, thus explaining the low erthan average reported values (M ottola andChristopher 1991) in our study .T here w as no ef fect of ex ercise on reproductiv eperform ance in our study or the studies by Carlson etal. (1986) or Gorsk i (1983). T his contrasts w ith theresults of studies by Garris et al. (1985) and W ilson


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1626 COB R IN A ND K OSK Iand G isolf i (1980), in w hich rat dam s ex ercised regularly during pregnancy had a signif icant decrease inthe num ber of liv e births per litter, decreasedp lacen tal we ights , s uppre ss ion o f u te ropl acen tal b loodf low , an d an in cre ase d f etal m o rality rate in ex erc isedg ro up s. O ur f ailu re to sh ow a re sp onse to reg ular d ailyex ercise m ay be due to the f act that the intensity and/or duration of ex ercise w as not as high or as long asthat reported in the studies of G arris et al. (1995) orW ilson and G isolf i (1980), in w hich the pregnant ratsex ercised for 1 h/d for a longer tim e span duringpregnancy.Pre vio usly , a stu dy f rom th is lab orato ry (K o sk i an dFe rgusso n 1992) f ound th at amn io tic f lu id g lu co se andlac tate in cre ase d sig nif ic an tly as th e le v el o f c arb oh ydrate increased in the m aternal diet. A l though therew as a signif icant ef f ect of diet on am niotic f luidglucose and lactate in the present study , acute m ild tom oderate ex ercise did not produce any signif icantchanges in these tw o v ariables. On the basis of thisex ercise study , w e w ould suggest that am n iotic f luidglucose or lactate w ould not be a sensitiv e indicatoro f m ild to mo de rate ex erc ise stre ss d uring p reg nan cy .A l though m aternal w eight gain w as signif icantlylow er w ith in cre ased e xercise in te nsity in this stu dy ,f etal w eight, w hen indiv idual pup data w ere nested,w as not signif icantly altered by ex ercise. T his w assu rp risin g b ecause it h as b een assum ed th at mate rn alw e ig ht lo ss, e v en if p re cip itate d b y in cre ase d e x erc ise ,w ould be ref lected in the fetus. How ever, in tw odif ferent studies (M ottola and Christopher 1991,Mo tto la et al. 1 98 9), f etal b od y w e ig ht f rom ex erc iseddam s w as either not dif ferent or increased in theex ercised dam s w hen com pared w ith the nonex er-c ise d con tro l g ro up . In c on trast, se v eral o th er stu die sreported a reduction in f etal body w eight as a result ofm aternal ex ercise (G ilbert et al. 1981; L ongo et al.1978, N elson et al. 1983; T erada 1974). T hese observ ations are sim ilar to our present f indings w hen pupw eights, w hich w ere unnested, w ere signif icantly low ered by both dietary carbohy drate restriction andin cre ase d e x erc ise in te nsity . T h ese d isp arate re su ltssuggest that f etal w eight m ay not be a reliably sensitiv e indicator of ex ercise stress, and w ithoutu nif orm me th od olo gical an d statistical co ntro ls, th eissue cannot be fully resolv ed at this tim e.T his study show ed that f etal and m aternal compartm ents do not respond sim ilarly to the diet andex ercise ef f ects, as dem onstrated by dif feringresponses in m aternal and fetal w eight gain andplasm a glucose, lactate and insulin. W e suggest thatm aternal and fetal responses should be ex am inedseparately , and suggest that caution be used in theex trapolation of results from one com partm ent toanother, because it cannot be assum ed that fetalresponses are ref lectiv e of those occurring in them aternal sy stem . B ecause a signif icant m odif yingef f ect of food intak e w as found in this study , w e also

suggest that ex ercise ef fects reported in prev iousstudies could hav e resulted f rom inadequate energyintak e. W e conclude that m ild to m oderate ex ercisealone, w hen accom panied by adequate energy andcarbohy drate intak e, does not pose a threat to themo the r o r the deve loping f e tu s. Further inves tigati on sare needed to determ ine if this is also true w henex ercise is of a higher intensity .

L IT ER A T UR E CIT EDBaldw in, K . M ., R eitm an, J. S ., T erjung, R . L ., W inder, W . W . &H ollosz y, J. O . (1973| S ubstrate depletion in dif ferent ty pes ofm uscle and liv er during prolonged running. A m . J. Phy siol. 225:1045-1050.B attaglia, F. C . & M eschia, G . (1978) Principal substrates of f etalm etabolism . Phy siol. R ev . 58: 499-527.B lak e, C. A . & Hazelw ood, R . L . (1971) Effect of pregnancy andex ercise on actom yosin, nucleic acid and gly cogen content ofthe rat heart. Proc. S oc. Ex p. B iol. M ed. 136: 632-636.B on dar, P.J.L . & M e ad , D . C . (1 97 4) E valu atio n o f g lu co se-6 -p ho s-phate dehy drogenase f rom leuconostoc m esenteroides in thehex ok inase m ethod for determ ining glucose in serum . Clin.C hem . 20: 586-590.Canadian Council on A nim al Care (1984) Guide to the Care andUse of Experim ental A nim als, V olum es I and II. N ational L ibrary of C anada, O ttaw a, O ntario, C anada.Carlson, K . I., Y ang, H. T ., B radshaw , W . S ., Conice, R . K . &W inder, W . W . (1986) Ef fect of m aternal ex ercise on f etal liv erglycogen late in gestation in the rat. J. A ppi. Phy siol. 60:1254-1258.Garris, D . R ., K asperek , G. J., Ov erton, S . V . & A lligood, G. R(1985) Ef fects of exercise on fetal-placental grow th anduteroplacental blood f low in the rat. B iol. N eonate 47: 223-229.G ilbert, R . D ., N elson, P. & L ongo, L . D . (1981) L ong term m aternalex ercise in guinea pigs: ef fects of f etal grow th and dev elopm entand placental dif fusing capacity . A m . J. O bstet. G ynecol. 140123-127.Gorsk i, J. (1983) Ef fect of ex ercise on m etabolism of energy substrates in pregnant m other and her f etus in the rat. In: B iochemistry of Ex ercise (K nuttgen, H . G ., V ogel, J. A . & . Portm ans, J.eds.), pp. 229-233. H um an K inetics Publishers, C ham paign, ILH en ry , R . J. (1 964 ) C lin ical C hem is try Prin cip le s an d T ec hn ic s,pp. 664-666. Harper and R ow , N ew Y ork , N Y .K osk i, K . G . & Fergusson, M . A . (1992) A m n iotic f luid com positionresponds to changes in m aternal dietary carbohydrate and isrelated to m etabolic status in term fetal rats. J. N utr. 122:385^92.K osk i, K . G., Hill, F. W . &. Hurley , L . S . (1986) Ef fect of lowc arb oh yd rate die ts d urin g p re gn an cy o n e m bry oge nesis and f etalgrow th and dev elopm ent in rats. J. N utr. 116: 1922-1937.K osk i, K . G. & Hill, F. W . (1986) Ef fect of low carbohy drate dietsduring pregnancy on parturition and postnatal surv iv al of thenew born rat pup. f . N utr. 116: 1938-1948.K osk i, K . G. & Hill, F. W . (1990) Ev idence for a critical periodduring late gestation w hen m aternal dietary carbohy drate isessential f or surv iv al of new born rats. J. N utr. 120: 1016-1027.L an ou e, L ., M i niaci, S . & K o sk i, K . G . (1 99 2] Plac en tal c om p ositiondoes not respond to changes in m aternal dietary carbohy drateintak e in rats. } . N utr. 122: 2374-2382.Lo, S ., R ussell, J. C. & Tay lor, A . W . (1970) Determ ination ofgly cogen in sm all tissue sam ples. J. A p pi. Phy siol. 28: 234-236.Longo, L . D., Hew itt, C . W . & Loring, R .H.W . (1978) T o w hatex tent does m aternal ex ercise af fect fetal ox yg nation anduterine blood f low ? Fed. Proc. 37: 905 (abs.).M ottola, M . F., B agnali, K . M . & B eicastro, A . N . (1989) Ef fects of


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DIETA RY CA RBOHY DRA TE A ND EX ERCISE DUR IN G PREGN A NCY 1627stren uou s m ate rnal ex ercise o n f etal o rgan w eig hts an d sk eletaldev elopm ent in rats. J. D ev . Phy siol. 11: 111-115.M ottola, M . F. & Christopher, P. D. (1991) Ef fects of m aternalex ercise on liv er and sk eletal m uscle gly cogen storage inpregnant rats. J. A p pi. Phy siol. 71: 1015-1019.N ational R esearch Council |1978) N utrient requirem ents of thelaboratory rat. In: N utrient R equirem ents of L aboratoryA n im als, pp. 7-37. N ational A c adem y of S ciences, W ashington,DC.

N elson, P. S ., G ilbert, R . D . & L ongo, L . D . (1983) Fetal grow th andp lace ntal d if fu sing c ap acity in g uine a p ig s f ollo w in g lo ng -te rmm aternal ex ercise. J. D ev . Phy siol. 5: 1-10.

S helley , H . J. (1961) G ly cogen reserv es and their changes at birthand in anox ia. B r. M ed. B ull. 17: 137-143.S helley , H . J. & N elligan, G . A . (1966) N eonatal hy pogly cem ia. B r.M ed. B ull. 22: 34-39.T erada, M . (1974) E ffect of phy sical activ ity bef ore pregnancy onf etu ses o f m ic e e xe rcise d f orcib ly d urin g p reg nan cy . T erato lo gy10: 141 -144 .T readw ay , J. L . & Y o ung, J. C . (1989) D ecreased glucose uptak e inthe f etus af ter m aternal ex ercise. M ed. S ci. S ports E xercise 21:140-145.W ilson, N . C. & Gisolf i, C. V . (1980) Ef fects of ex ercising ratsduring pregnancy , f . A p pi. Phy siol. 48: 34-40.

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