a high-fat diet during rat pregnancy or suckling induces cardiovascular dysfunction in adult...

CALL FOR PAPERS Fetal Physiological Programming

A high-fat diet during rat pregnancy or suckling induces cardiovasculardysfunction in adult offspring

I. Y. Khan,1 V. Dekou,1 G. Douglas,1 R. Jensen,1 M. A. Hanson,2 L. Poston,1 and P. D. Taylor1

1Maternal and Fetal Research Unit, Division of Reproductive Health, Endocrinologyand Development, King’s College, London; and 2Centre for the Developmental Originsof Health and Disease, Princess Anne Hospital, Southampton, United Kingdom

Submitted 1 June 2004; accepted in final form 31 July 2004

Khan, I. Y., V. Dekou, G. Douglas, R. Jensen, M. A. Hanson, L.Poston, and P. D. Taylor. A high-fat diet during rat pregnancy orsuckling induces cardiovascular dysfunction in adult offspring. Am JPhysiol Regul Integr Comp Physiol 288: R127–R133, 2005. Firstpublished August 12, 2004; doi:10.1152/ajpregu.00354.2004.—Epi-demiological and animal studies suggest that diet-induced epigeneticmodifications in early life can contribute to development of themetabolic syndrome in adulthood. We previously reported features ofthe metabolic syndrome in adult offspring of rats fed a diet rich inanimal fat during pregnancy and suckling. We now report a study tocompare the relative effects of high-fat feeding during 1) pregnancyand 2) the suckling period in the development of these disorders. Asobserved previously, 6-mo-old female offspring of fat-fed dams suck-led by the same fat-fed dams (OHF) demonstrated raised bloodpressure, despite being fed a balanced diet from weaning. Femaleoffspring of fat-fed dams “cross fostered” to dams consuming acontrol diet during suckling (OHF/C) demonstrated raised bloodpressure compared with controls (OC) [systolic blood pressure (SBP;mmHg) means � SE: OHF/C, 132.5 � 3.0, n � 6 vs. OC, 119.0 �3.8, n � 7, P � 0.05]. Female offspring of controls cross fostered todams consuming the fat diet (OC/HF) were also hypertensive [SBP(mmHg) 131.0 � 2.5 mmHg, n � 6 vs. OC, P � 0.05]. Endothelium-dependent relaxation (EDR) of male and female OHF and OHF/Cmesenteric small arteries was similar and blunted compared with OC(P � 0.001). OC/HF arteries showed profoundly impaired EDR(OC/HF vs. OHF, P � 0.001). OHF/C and OC/HF demonstratedhyperinsulinemia and increased adiposity. Features of the metabolicsyndrome in adult offspring of fat-fed rats can be acquired bothantenatally and during suckling. However, exposure during pregnancyconfers adaptive protection against endothelial dysfunction inducedby maternal fat feeding during suckling.

blood pressure; endothelium; developmental programming

THE METABOLIC SYNDROME, AS defined by World Health Organi-zation criteria, affects as many as 34% of adults in the UnitedStates (3). Although adult diet and lifestyle risk factors un-doubtedly contribute to development of this increasingly com-mon disorder, epidemiological and animal studies now suggestthat the metabolic syndrome may also be acquired throughnutritional imbalance in early life (9, 23).

We previously reported that adult offspring of rats fed a dietrich in animal fat during pregnancy and suckling developvascular endothelial dysfunction and gender-specific hyperten-

sion (16). Population studies and investigations in experimen-tal animals have revealed critical periods when offspring aremost vulnerable to environmental influences, including mater-nal nutritional imbalance. In investigations of nutrition duringhuman pregnancy and suckling, in which cardiovascular andmetabolic functions have been assessed in the offspring in laterlife, some reports have identified critical periods during gesta-tion (25), whereas others emphasize the suckling period (7).The aim of the present study was to determine the relativeimportance of the gestational and suckling periods in inducingcardiovascular and metabolic disorders in offspring of rat damsfed a fat-rich diet. Studies in experimental animals using othernutritional interventions in pregnancy have generally indicatedan important role of the gestational period in the developmentof adulthood cardiovascular and metabolic disorders, as damsare frequently fed a standard chow diet immediately postpar-tum (12, 18, 22, 26, 31). Few have rigorously investigated therole of the suckling period.

To address this, we cross fostered pups from fat-fed dams tonormally-fed control dams and vice versa. One group of pupsfrom fat-fed dams was cross fostered to dams consumingstandard chow, and pups from another group of chow-fed damswere cross fostered at birth to dams fed the fat-rich diet.Comparisons were made with offspring exposed to the fat-richdiet both in pregnancy and during suckling and with controloffspring suckled by dams fed standard chow. All groups werefed standard chow postweaning. Radiotelemetry was employedto evaluate cardiovascular parameters in freely moving con-scious 180-day-old offspring, and isolated resistance arteryfunction was assessed by small vessel myography. Plasmaanalyses were carried out for determination of glucose ho-meostasis and lipid status.

MATERIALS AND METHODS

Animal Husbandry and Experimental Diets

Female Sprague-Dawley (100–120 days) rats were fed ad libitum,for 10 days before mating and throughout pregnancy, either a controldiet of a standard laboratory chow [5.3% fat (corn oil), 21.2% protein,49.2% carbohydrate, 4.6% fiber, 6.0 ash, 10.1% moisture, vitamins,and minerals; Rat and Mouse Diet no. 3, Special Diet Services,Witham, Essex, UK] or an experimental diet consisting of the stan-dard chow supplemented 20% wt/wt with animal lard with 20%

Address for reprint requests and other correspondence: P. D. Taylor,Division of Reproductive Health, Endocrinology and Development, St.Thomas’ Hospital, London SE1 7EH, UK (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 288: R127–R133, 2005.First published August 12, 2004; doi:10.1152/ajpregu.00354.2004.

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additional vitamins and minerals, protein, inositol, and choline tocorrect for the dilution [final composition 25.7% fat, 19.5% protein,34.7% carbohydrate, 3.5% fiber, 5.3% ash, 8.2% moisture (fats:palmitic acid 4.50%, stearic acid 1.99%, palmitoleic acid 0.12%, oleicacid 6.86%, linoleic acid 2.58%, �-linolenic acid 0.21%, arachidonicacid 0.19%); Special Diet Services] (29). The efficacy of supplemen-tation was confirmed by independent analysis of the diets (EclipseScientific Group, Cambridge, UK). At birth, half of the control litterswere cross fostered to a fat-fed dam and vice versa to form fourgroups [control offspring suckled by the same dam (OC), offspringfrom fat-fed dams suckled with the same dam (OHF), control off-spring suckled by a fat-fed dam (OC/HF), and offspring of fat-feddams suckled by a control dam (OHF/C)].

All offspring were fed standard chow from weaning. Animalweights were recorded from 48 h of age, when all litters were reducedto eight pups (4 male, 4 female). Food intake was recorded daily indams and weekly in offspring postweaning. Animals were maintainedin 12:12-h light-dark cycles at constant temperature and humidity.

Assessment of Offspring Blood Pressure, Heart Rate, and ActivityBy Radiotelemetry

Blood pressure, heart rate, and activity were assessed by radiote-lemetry (Dataquest IV, Data Sciences International). Briefly, themonitoring system consists of an implanted radiotelemetry probe witha fluid-filled catheter attached to a transducer/transmitter (radio fre-quency transducer model TA-11 PA-C40), a receiver panel, a consol-idation matrix, and a computer. Randomly selected littermates (1male, 1 female from each litter) at 180 days of age were administeredbuprenorphine (0.1 mg/kg sc) before surgery and anesthetized withisofluorane. The flexible catheter was secured in the abdominal aortaand the transmitter was transfixed to the abdominal wall. On recovery,rats were housed in individual cages and each cage was placed over areceiver panel with output to the computer. After 1-wk recoveryperiod, heart rate, systolic and diastolic blood pressures, and activitywere recorded for 10 s every 5 min for a week. Twelve hourly day andnight mean values were computed. Animals were then fasted over-night and blood samples were obtained by cardiac puncture andplasma stored at �80°C before analysis for lipids, glucose, andinsulin.

Assessment of Mesenteric Small Artery Function

One male and one female from each litter were studied. Rats werekilled by cervical dislocation, and body weights and fat depot weightswere recorded. Third-order branches of the mesenteric arcade weredissected and mounted in physiological salt solution on a small vesselmyograph as previously described (16). Arteries were submaximallyconstricted with norepinephrine (80% of maximal concentration) andresponses to acetylcholine (10�9-10�5 M) and nitric oxide (NO;10�8-10�5 M) were determined. NO concentrations were derived bydilution of a saturated solution.

Plasma Analysis

Fasting blood samples were obtained by cardiac puncture in the ratsstudied for telemetric recording. Rats were fasted overnight and killedby CO2 inhalation. Plasma glucose was measured by a routinelaboratory enzymatic UV test (HK/G6P-DH method; Cobas FaraCentrifugal analyzer) and insulin by ELISA (DRG InstrumentsGmbH). Plasma triglyceride and total cholesterol concentrations weremeasured by enzymatic colorimetric assays (UNIMATE 5 TRIG andUNIMATE 5 CHOL Roche/BCl, Sussex, UK).

Statistical Analysis

All values are given as means � SE. Statistical comparisons invascular studies were by repeated-measures ANOVA with Bonferronicorrection for multiple comparisons. EC50 values were determinedafter fitting data to a sigmoid curve (GraphPad Software, San Diego,CA) and analyzed by one-way ANOVA with Bonferroni correction.Statistical significance was assumed if P � 0.05. The study waspowered for differences in vascular function and blood pressure basedon previous studies. The initial ANOVA model incorporated sex as anindependent variable. Where this showed significance sexes wereanalyzed separately; otherwise male and female data were consideredtogether. Telemetry blood pressure data were compared over a weekby multiple regression with Generated Estimating Equations (Stataversion 6.0, StatCorp, College Station, TX) and by one-way ANOVAwith Bonferroni posttest for comparisons of mean day and nighttimevalues.

RESULTS

Data for the control offspring and offspring of dams fed afat-rich diet in pregnancy and suckling have been reportedpreviously (15).

Maternal Weight and Food Intake During Pregnancyand Weaning

Maternal weight was greater in fat-fed dams until day 16 ofgestation and also from birth until day 8 postpartum (repeated-measures ANOVA, P � 0.05 vs. controls; Fig. 1). Averagedaily food intake was significantly reduced in the fat-fed damsduring pregnancy [average daily food intake (g) days 0-20,25.6 � 0.99 for control, n � 10 vs. 21.16 � 0.94 for dams onthe lard diet, n � 11, P � 0.005]. Reduced food intake in thefat-fed dams resulted in a similar gross energy intake such thatdietary intake was effectively isocalorific compared with con-trol dams [average daily gross energy intake (kJ) 383.9 �14.85 for control, n � 10 vs. 414.7 � 18.42, n � 11 for damson the high-fat diet, P not significant].

In contrast, during suckling, despite a significant reductionin average daily food intake in the fat-fed dams [average dailyintake during suckling, days 22-42 (g), 55.15 � 1.26 g/day forcontrol, n � 10 vs. 49.9 � 0.69, for dams on the lard diet, n �11, P � 0.005], gross energy intake was hypercalorific com-pared with control dams [average daily gross energy intake (kJ)827.3 � 18.9 for control, n � 10 vs. 978.8 � 13.5, n � 11 fordams on the high-fat diet, P � 0.0001].

Offspring Body Weight and Food Intake

Body weight and food intake were similar between groups(Fig. 2). In male offspring of all experimental groups (OHF,OC/HF, OHF/C), adiposity as assessed by the combined wetweight of intra-abdominal fat pads (retroperitoneal and peri-nephric) and gonadal fat lobes was increased when compared

R128 IN UTERO AND SUCKLING EXPOSURE TO DIETARY FAT

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with controls (OC, P � 0.05). In females, significant differ-ences in adiposity only occurred between offspring of thefat-fed dams suckled by their own dams (OHF) and controls(OC) (P � 0.05; Table 1).

Radiotelemetry Monitoring of Offspring Blood Pressure,Heart Rate, and Activity

Males. There were no differences in blood pressure (Fig. 3A)or activity (Table 2) between the male offspring of control orfat-fed dams or cross-fostered litters. Heart rate was signifi-cantly lower in male OHF compared with OC (awake phase,P � 0.05; Fig. 3B). A similar reduction was apparent in bothcross-fostered groups (OC/HF vs. OC, P � 0.05 and OHF/Cvs. OC, P � 0.05; Fig. 3B).

Females. Systolic blood pressure (SBP) was significantlyraised in female offspring of fat-fed dams when compared withcontrols, as described previously (16) [mean nighttime (awakephase) SBP (mmHg) over 7 days OHF, 132.5 � 3.0, n � 7 vs.

OC, 119.0 � 3.8, n � 7, P � 0.05; Fig. 4A]. OHF/C alsodemonstrated increased SBP (132.5 � 3.0, n � 6 vs. OC, P �0.05; Fig. 4A) as did OC/HF (131.0 � 2.5 mmHg, n � 6 vs.OC, P � 0.05; Fig. 4A and Table 2). Diastolic blood pressurewas also raised in female OHF (P � 0.05 vs. control; Fig. 4Aand Table 2) and OHF/C (P � 0.05 vs. OC; Fig. 4A) but wasnot significantly increased in OC/HF (vs. OC, P not signifi-cant). Activity and heart rate were not different among thefemale groups (Table 2 and Fig. 4B).

Vascular Function

Male and female. No differences in endothelium-dependentresponses were observed between male and female offspringwithin the different groups; hence male and female data wereconsidered together. All offspring exposed to the fat diet ingestation or during suckling demonstrated blunted acetylcho-line-induced relaxation in mesenteric arteries compared withcontrols (by repeated-measures ANOVA, OHF vs. OC, P �

Fig. 1. Maternal body weight and food intake incontrol (‚, n � 10) and fat-fed dams duringpregnancy and lactation (Œ, n � 11, *P � 0.05,repeated-measures ANOVA). Values are ex-pressed as means � SE.

Fig. 2. Growth rate and food intake in male and female offspring of control dams (OC, E, n � 6), lard-fed dams (OHF, F, n � 6), and cross-fostered groups(OC/HF, �, n � 6 and OHF/C, �, n � 6). Values are expressed as means � SE.

R129IN UTERO AND SUCKLING EXPOSURE TO DIETARY FAT


0.0005; OHF/C vs. OC, P � 0.0001). Relaxation was furtherimpaired in OC/HF (vs. OC, P � 0.0001; OC/HF vs. OHF,P � 0.0005; Fig. 5A). Sensitivity to acetylcholine was unaf-fected because pEC50 values were similar between groups(Table 3), but maximal responses were significantly impairedin OHF and OHF/C relative to control (OHF vs. OC, P �0.001; OHF/C vs. OC, P � 0.001) and further reduced inOC/HF (vs. OC, P � 0.001; male and female OC/HF vs. OHF,P � 0.001; Table 3).

Endothelium-independent relaxation as assessed by re-sponses to NO (aqueous solution) was not different betweenexperimental groups (P not significant by repeated-measuresANOVA; Fig. 5B).

Plasma Analyses

There were no significant differences in lipid profiles orplasma glucose between offspring from the different experi-mental groups compared with controls at 180 days of age(Table 4). No differences in plasma insulin concentrationswere observed between male and female offspring within thedifferent experimental groups; hence male and female datawere considered together. A significant increase in fasting

plasma insulin concentration was observed in all “fat-exposed”groups relative to controls (plasma insulin OHF, 1.77 � 0.26,n � 12 vs. OC, 0.75 � 0.09, n � 12, P � 0.003; OC/HF,1.47 � 0.31, n � 11 vs. OC, P � 0.05; OHF/C, 1.58 � 0.24,n � 10 vs. OC, P � 0.02).

DISCUSSION

This study evaluated the relative roles of the in utero andsuckling periods in the induction of cardiovascular and meta-bolic dysfunction in an animal model employing fat feeding inpregnancy and lactation (16). We demonstrated independenteffects of maternal fat feeding during pregnancy and sucklingbecause blood pressure, endothelial function, and plasma in-sulin were abnormal not only in offspring whose dams wereexposed to the maternal fat-rich diet during pregnancy but alsowhen the fat-rich diet was confined to the suckling period.Importantly, because endothelial function was most severelycompromised in offspring whose dams consumed the fat-richdiet only in suckling, it appears that prior exposure in uteroconfers a degree of protection against the insult of a fat-richdiet postpartum.

Fig. 3. A: systolic and diastolic blood pres-sure (SBP and DBP) in male offspring ofcontrol dams (OC, E, n � 6), fat-fed dams(OHF, F, n � 6), and in cross-fostered groups(OC/HF, �, n � 6 and OHF/C, �, n � 6). B:heart rate in male offspring of control dams(OC, E, n � 6), fat-fed dams (OHF, F, n �6), and in cross-fostered groups (OC/HF, �,n � 6 and OHF/C, �, n � 6). *P � 0.05, OCvs. OHF. †P � 0.05, OC vs. OHF/C. ‡P �0.05, OC vs. OC/HF for a period of 1 wk bygenerated estimated equation (GEE). bpm,Beats/min.

Table 1. Body weights and fat distribution in offspring of OC, OHF, and OC/HF, OHF/C

OC (n � 10) OHF (n � 10) OC/HF (n � 8) OHF/C (n � 8)

MalesBody wt, g 641.31�18.60 681.95�16.80 631.78�20.30 601.28�23.37§Fat deposition

Abdominal lobes, g 13.26�1.89 20.22�3.30 21.19�3.45 18.91�1.80As %body wt 2.07�0.30 2.97�0.48 3.35�0.55 3.15�0.30Gonadal lobes, g 10.55�1.80 13.22�2.19 12.26�1.95 13.00�2.10As %body wt 1.65�0.28 1.94�0.32 1.94�0.31 2.16�0.35Total fat depots, g 23.99�2.33 33.44�3.01 33.45�3.11 31.91�2.48As %body wt 3.74�0.25 4.90�0.30* 5.30�0.40† 5.31�0.31‡

FemalesBody wt, g 341.41�8.43 348.68�9.27 340.27�7.02 332.44�8.85Fat deposition

Abdominal lobes, g 4.89�2.01 8.99�2.19 7.41�2.11 6.59�1.15As %body wt 1.43�0.40 2.58�0.63 2.18�0.52 1.98�0.30Gonadal lobes, g 2.48�1.99 6.48�3.31 4.39�0.99 4.94�1.90As %body wt 0.73�0.43 1.86�0.45 1.29�0.25 1.50�0.47Total fat depots, g 7.37�2.29 15.47�3.21 11.80�1.89 11.53�2.13As %body wt 2.16�0.50 4.44�0.51* 3.47�0.29 3.47�0.44

Values are means � SE. *P � 0.05, control dams (OC) vs. lard-fed dams (OHF). †P � 0.01, OC vs. cross-fostered group (OC/HF). ‡P � 0.01, OC vs.cross-fostered group (OHF/C). §P � 0.05, OHF vs. OHF/C by ANOVA with Bonferroni posttest.



The importance of the in utero period in determining latercardiovascular risk has been demonstrated in several animalmodels including maternal protein undernutrition (18) andglobal dietary undernutrition (22, 31) in pregnancy in whichhypertension and/or insulin resistance develops in adult off-spring despite the dams being fed a normal diet postdelivery. Ithas also been suggested that the preimplantation period iscrucial (17). The role of the suckling period has been lessfrequently investigated but has major implications for postnatalnutrition, growth, and development.

Exposure to the fat-rich diet during gestation alone wasassociated with raised systolic and diastolic blood pressures inadult female offspring. The suckling of control animals withfat-fed dams also led to adulthood elevation of blood pressure,but this was confined to a rise in the systolic pressure. Studiesin genetically hypertensive rats have also implied that raisedblood pressure may, in part, be acquired during the sucklingperiod (1, 5). Further relevant studies include those that impli-cate in utero stress in development of adulthood behavioral andfunctional disturbances. Offspring of rats subjected to a stress-ful stimulus during pregnancy but cross fostered to normaldams develop raised adulthood systolic arterial pressure andenhanced reactivity to restraint stress (14). In common with thepresent study, female offspring were the most affected, whichmay indicate a similarity of mechanism between these models.

Ozanne and Hales (24) recently investigated the life span ofmice exposed to protein restriction in utero or during suckling.Offspring of protein-restricted dams cross fostered to controldams and reared on a “cafeteria” diet had markedly reducedlongevity compared with controls, corroborating the sugges-tion that in utero dietary deprivation may lead to a phenotypeconferring survival advantage only in a time of nutritionalrestriction (11). However, control mice fed a low-protein dietduring suckling showed increased longevity whatever the sub-sequent diet, suggesting a sustained and predominant benefit ofrestricted nutrition in the suckling period.

Endothelial dysfunction was present in small mesentericarteries of all offspring whose dams consumed the fat-rich diet,showing that this disorder, in common with raised SBP, canoriginate either in utero or during suckling. Endothelial dys-function may give rise to, or be a consequence of, insulinresistance (13) and could reflect activation of inflammatorypathways as a result of increased adiposity (4).

The arteries from control offspring suckled by fat-fed damsdemonstrated almost complete failure of relaxation to acetyl-choline. This suggests that the developing vasculature is par-ticularly sensitive to dietary insult postnatally but also impliesthat exposure to the fat diet in utero conferred protection fromthe insult of fat feeding during the suckling period. Thisaccords with the suggestion that the fetus can mount a bene-

Fig. 4. A: SBP and DBP in female offspringof control dams (OC, E, n � 6), fat-fed dams(OHF, F, n � 6), or cross-fostered groups(OC/HF, �, n � 6 and OHF/C, �, n � 6).*P � 0.05, OC vs. OHF. †OC vs. OHF/C.‡P � 0.05, OC vs. OC/HF for a period of 1wk by GEE. B: heart rate in female offspringof control dams (OC, E, n � 6), fat-fed dams(OHF, F, n � 6), or cross-fostered groups(OC/HF, �, n � 6 and OHF/C, �, n � 6).

Table 2. Cardiovascular parameters and activity in offspring of OC, OHF, and OC/HF, OHF/C at 180 days of age

Males Females

OC(n � 7)

OHF(n � 7)

OC/HF(n � 6)

OHF/C(n � 6)

OC(n � 7)

OHF(n � 7)

OC/HF(n � 6)

OHF/C(n � 6)

MAP, mmHgNight 114.3�2.9 112.2�3.0 109.4�4.0 112.0�2.4 102.1�1.9 110.3�2.2* 106.8�2.2 111.6�2.4‡Day 111.0�2.0 110.1�2.5 106.1�3.7 110.2�2.2 99.3�1.6 107.6�2.0* 103.5�1.9 108.7�2.3‡

Heart rate, beats/minNight 398.3�5.2 355.4�9.5* 362.5�9.4† 359.9�8.6‡ 410.9�4.1 399.1�5.9 393.9�10.5 417.0�5.3Day 362.1�7.3 323.0�8.9* 359.9�8.6† 319.1�8.2‡ 369.6�4.7 362.9�7.6 358.3�9.17 367.2�6.5

DBP, mmHgNight 92.2�3.9 90.9�3.1 85.5�4.2 90.6�3.0 81.1�1.4 91.2�1.7* 85.1�1.5 89.8�2.4‡Day 89.9�3.6 88.1�3.0 84.7�4.0 87.1�3.1 80.0�1.0 88.6�1.5* 83.4�1.3 87.4�2.7‡

Locomotor activityNight 2.9�0.3 3.1�0.3 2.7�0.3 2.7�0.4 4.7�0.5 3.7�0.4 3.6�0.5 3.8�0.4Day 2.0�0.2 1.7�0.2 1.8�0.2 1.8�0.2 2.3�0.3 1.9�0.2 2.4�0.5 1.8�0.2

Values are means � SE. *P � 0.05, OC vs. OHF. †P � 0.05, OC vs. OC/HF. ‡P � 0.05, OC vs. OHF/C. MAP, mean arterial pressure; DBP, diastolic bloodpressure. Locomotor activity is expressed in arbitrary units.



ficial “predictive adaptive” response (8), in this case in antic-ipation of consumption of a fat-rich diet during suckling. It issuggested that the fetus makes responses, based on prenatalsignals, about the environment from the mother, which willconfer adaptive advantage postnatally if the prediction is ac-curate. If the prediction is incorrect because the postnatalnutritional environment changes, then the responses are mal-adaptive and disease risk increases. Work from our groupprovides recent support for this hypothesis by demonstratingthat endothelial function in adulthood is normal when offspringof dams that consumed a fat-rich diet during pregnancy andsuckling are themselves fed the fat diet from weaning toadulthood (15). The blood pressure, however, remains elevatedin the female offspring. A similar study showed that pigletswhose sows consumed an “atherogenic” diet in pregnancy arethemselves protected from the atherogenic effects if the samediet is fed to them postweaning (21).

The neonatal rat is delivered at a much earlier stage ofdevelopment than the human infant, and care must be exercisedin drawing parallels with studies of nutrition in neonatalchildren. However, Lucas and colleagues (7, 19), in studies ofpremature infants whose developmental stage at delivery ismuch closer to that of neonatal rats, reported that feedingformula milk (rich in fats) compared with breast milk isassociated with increased blood pressure and reduced endothe-lium-dependent dilation in early adulthood. This is analogousto the present study in which the poorest endothelial functionwas evident in those rats exposed to high fat during sucklingalone.

Rearing rats in small litters (restricted to 4 pups) leads tooverfeeding during suckling and produces phenotypic similar-ities to the present model, such as increased adiposity, hyper-

insulinemia, leptin resistance, obesity (27), and altered lipidprofiles (10) in adulthood. These studies emphasize the impor-tant phenotypic induction influences of the suckling period.

In the normotensive male offspring, significant bradycardiawas observed in all experimental groups, whereas heart ratewas normal in the hypertensive females. This implies that thefemales may mount an inadequate baroreceptor response toelevated blood pressure. Reduction in baroreceptor reflex sen-sitivity is associated with both the metabolic syndrome (2) andessential hypertension (20).

The abnormalities in the 180-day-old offspring occurredwithout any associated changes in the plasma lipid profile, aspreviously reported in this model at this age (16). Olderanimals develop a clearly abnormal glucose and lipid profile(16). Plasma glucose concentrations tended to be raised in maleand female offspring of fat-fed dams but not in the cross-fostered groups. However, fasting plasma insulin was signifi-cantly raised in all fat-exposed groups. We recently showedinsulin resistance in older animals by euglycemic hyperinsu-linemic clamp (30). Hyperinsulinemia appears therefore to beinduced independently during both the in utero period and thesuckling period.

As reported previously (29), the dams ate significantly lessof the high-fat diet during pregnancy such that their intake wasin fact isolcalorific to controls. Nontheless, the overall fatintake was approximately fourfold greater in fat-fed dams thancontrols. As the fat was substituted for other constituents of thediet, and although vitamins, micronutrients, and protein wereadded to compensate, there was a 14% reduction in carbohy-

Fig. 5. A: endothelium-dependent relaxationto acetylcholine in third-order mesenteric ar-teries of male and female offspring of controldams (OC, E, n � 16), fat-fed dams (OHF, F,n � 16), and cross-fostered groups (OC/HF,�, n � 16 and OHF/C, �, n � 16). B:endothelium-independent relaxation to aque-ous nitric oxide in male and female offspringof control dams (OC, E, n � 14), fat-fed dams(OHF, F, n � 14), and cross-fostered groups(OC/HF, �, n � 9 and OHF/C, �, n � 9).*P � 0.0005, OC vs. OHF. †P � 0.0001,OHF/C vs. OC. ‡P � 0.0001, OC vs. OC/HF.§P � 0.0005, OC/HF vs. OHF in male off-spring by repeated-measures ANOVA.

Table 3. Endothelium-dependent relaxation in smallmesenteric arteries in male and female offspring of OC,OHF, and OC/HF, OHF/C at 180 days of age

OC(n � 15)

OHF(n � 15)

OC/HF(n � 15)

OHF/C(n � 15)

Maximum, %Norepinephrinepreconstriction

2.75�2.48 27.45�3.43* 61.38�4.25†§ 27.97�5.45‡

pEC50

acetylcholine7.65�0.07 7.51�0.12 7.40�0.27 7.34�0.17

Values are means � SE. *P � 0.001, OC vs. OHF. †P � 0.001, OC vs.OC/HF. ‡P � 0.05, OC vs. OHF/C. §P � 0.001, OHF vs. OC/HF.

Table 4. Plasma glucose and lipid profiles in male andfemale offspring of OC, OHF, and OC/HF, OHF/Cat 180 days of age

OC(n � 6)

OHF(n � 6)

OC/HF(n � 6)

OHF/C(n � 6)

MalesGlucose, mmol/l 21.73�1.57 29.91�1.88 27.17�3.11 22.15�2.57Cholesterol, mmol/l 2.70�0.19 3.04�0.20 2.95�0.11 3.11�0.28Triglyceride, mmol/l 1.04�0.17 1.30�0.09 1.18�0.12 1.21�0.25HDL, mmol/l 1.58�0.17 1.81�0.10 1.66�0.13 1.79�0.17

FemalesGlucose, mmol/l 12.38�2.44 18.08�3.59 10.78�0.89 11.68�2.23Cholesterol, mmol/l 2.70�0.19 2.75�0.24 3.12�0.16 2.87�0.22Triglyceride, mmol/l 1.04�0.17 1.11�0.17 1.15�0.08 1.21�0.17HDL, mmol/l 1.58�0.17 1.53�0.12 2.02�0.07 1.75�0.15

Values are means � SE. HDL, high-density lipoprotein.



drate content compared with controls. We cannot entirelyexclude therefore the possibility that the diet-induced pheno-type is attributable to an imbalance in the carbohydrate-to-fatratio.

This animal model has certain similarities with other devel-opmental models, all of which lead to a phenotype withfeatures of the metabolic syndrome (11), which, it has beensuggested, may imply a common underlying mechanism. Ma-ternal activation of the hypothalamic pituitary adrenal axis (28)and maternal hyperinsulinemia (29) have each been implicated.

Because certain features of the metabolic syndrome wereacquired by rat offspring whose mothers consumed a fat-richdiet either in utero and/or during suckling, this study demon-strates a broad window of developmental susceptibility to adietary imblalance of a kind prevalent both among populationsof developed countries and those of some developing countriesundergoing rapid economic transition. The data suggest that“predictive adaptive” responses occur in utero to protectagainst a subsequent “dietary challenge” in the postnatal pe-riod. The observations presented provide strong support for thedevelopmental origins of adult disease.

ACKNOWLEDGMENTS

We thank P. Lumb, J. Judah, and G. Fulcher for technical assistance.

GRANTS

This study was supported by the British Heart Foundation. L. Poston issupported by Tommy’s the Baby Charity.

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a high-fat diet during rat pregnancy or suckling induces cardiovascular dysfunction in adult...

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