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Fetal Physiological Programming
1Maternal and Fetal Research Unit, Division of Reproductive Health, Endocrinology and Development, King's College, London; and 2Centre for the Developmental Origins of Health and Disease, Princess Anne Hospital, Southampton, United Kingdom
Submitted 1 June 2004 ; accepted in final form 31 July 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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blood pressure; endothelium; developmental programming
We previously reported that adult offspring of rats fed a diet rich in animal fat during pregnancy and suckling develop vascular endothelial dysfunction and gender-specific hypertension (16). Population studies and investigations in experimental animals have revealed critical periods when offspring are most vulnerable to environmental influences, including maternal nutritional imbalance. In investigations of nutrition during human pregnancy and suckling, in which cardiovascular and metabolic functions have been assessed in the offspring in later life, some reports have identified critical periods during gestation (25), whereas others emphasize the suckling period (7). The aim of the present study was to determine the relative importance of the gestational and suckling periods in inducing cardiovascular and metabolic disorders in offspring of rat dams fed a fat-rich diet. Studies in experimental animals using other nutritional interventions in pregnancy have generally indicated an important role of the gestational period in the development of adulthood cardiovascular and metabolic disorders, as dams are frequently fed a standard chow diet immediately postpartum (12, 18, 22, 26, 31). Few have rigorously investigated the role of the suckling period.
To address this, we cross fostered pups from fat-fed dams to normally-fed control dams and vice versa. One group of pups from fat-fed dams was cross fostered to dams consuming standard chow, and pups from another group of chow-fed dams were cross fostered at birth to dams fed the fat-rich diet. Comparisons were made with offspring exposed to the fat-rich diet both in pregnancy and during suckling and with control offspring suckled by dams fed standard chow. All groups were fed standard chow postweaning. Radiotelemetry was employed to evaluate cardiovascular parameters in freely moving conscious 180-day-old offspring, and isolated resistance artery function was assessed by small vessel myography. Plasma analyses were carried out for determination of glucose homeostasis and lipid status.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Female Sprague-Dawley (100–120 days) rats were fed ad libitum, for 10 days before mating and throughout pregnancy, either a control diet 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 standard chow supplemented 20% wt/wt with animal lard with 20% additional vitamins and minerals, protein, inositol, and choline to correct 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%, oleic acid 6.86%, linoleic acid 2.58%, 
-linolenic acid 0.21%, arachidonic acid 0.19%); Special Diet Services] (29). The efficacy of supplementation was confirmed by independent analysis of the diets (Eclipse Scientific Group, Cambridge, UK). At birth, half of the control litters were cross fostered to a fat-fed dam and vice versa to form four groups [control offspring suckled by the same dam (OC), offspring from fat-fed dams suckled with the same dam (OHF), control offspring suckled by a fat-fed dam (OC/HF), and offspring of fat-fed dams suckled by a control dam (OHF/C)].
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All offspring were fed standard chow from weaning. Animal weights were recorded from 48 h of age, when all litters were reduced to eight pups (4 male, 4 female). Food intake was recorded daily in dams and weekly in offspring postweaning. Animals were maintained in 12:12-h light-dark cycles at constant temperature and humidity.
Assessment of Offspring Blood Pressure, Heart Rate, and Activity By Radiotelemetry
Blood pressure, heart rate, and activity were assessed by radiotelemetry (Dataquest IV, Data Sciences International). Briefly, the monitoring system consists of an implanted radiotelemetry probe with a fluid-filled catheter attached to a transducer/transmitter (radio frequency transducer model TA-11 PA-C40), a receiver panel, a consolidation matrix, and a computer. Randomly selected littermates (1 male, 1 female from each litter) at 180 days of age were administered buprenorphine (0.1 mg/kg sc) before surgery and anesthetized with isofluorane. The flexible catheter was secured in the abdominal aorta and the transmitter was transfixed to the abdominal wall. On recovery, rats were housed in individual cages and each cage was placed over a receiver panel with output to the computer. After 1-wk recovery period, heart rate, systolic and diastolic blood pressures, and activity were recorded for 10 s every 5 min for a week. Twelve hourly day and night mean values were computed. Animals were then fasted overnight and blood samples were obtained by cardiac puncture and plasma stored at –80°C before analysis for lipids, glucose, and insulin.
Assessment of Mesenteric Small Artery Function
One male and one female from each litter were studied. Rats were killed by cervical dislocation, and body weights and fat depot weights were recorded. Third-order branches of the mesenteric arcade were dissected and mounted in physiological salt solution on a small vessel myograph as previously described (16). Arteries were submaximally constricted with norepinephrine (80% of maximal concentration) and responses to acetylcholine (10–9-10–5 M) and nitric oxide (NO; 10–8-10–5 M) were determined. NO concentrations were derived by dilution of a saturated solution.
Plasma Analysis
Fasting blood samples were obtained by cardiac puncture in the rats studied for telemetric recording. Rats were fasted overnight and killed by CO2 inhalation. Plasma glucose was measured by a routine laboratory enzymatic UV test (HK/G6P-DH method; Cobas Fara Centrifugal analyzer) and insulin by ELISA (DRG Instruments GmbH). Plasma triglyceride and total cholesterol concentrations were measured by enzymatic colorimetric assays (UNIMATE 5 TRIG and UNIMATE 5 CHOL Roche/BCl, Sussex, UK).
Statistical Analysis
All values are given as means ± SE. Statistical comparisons in vascular studies were by repeated-measures ANOVA with Bonferroni correction for multiple comparisons. EC50 values were determined after 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 was powered for differences in vascular function and blood pressure based on previous studies. The initial ANOVA model incorporated sex as an independent variable. Where this showed significance sexes were analyzed separately; otherwise male and female data were considered together. Telemetry blood pressure data were compared over a week by multiple regression with Generated Estimating Equations (Stata version 6.0, StatCorp, College Station, TX) and by one-way ANOVA with Bonferroni posttest for comparisons of mean day and nighttime values.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Maternal Weight and Food Intake During Pregnancy and Weaning
Maternal weight was greater in fat-fed dams until day 16 of gestation and also from birth until day 8 postpartum (repeated-measures ANOVA, P < 0.05 vs. controls; Fig. 1). Average daily food intake was significantly reduced in the fat-fed dams during pregnancy [average daily food intake (g) days 0-20, 25.6 ± 0.99 for control, n = 10 vs. 21.16 ± 0.94 for dams on the lard diet, n = 11, P < 0.005]. Reduced food intake in the fat-fed dams resulted in a similar gross energy intake such that dietary intake was effectively isocalorific compared with control dams [average daily gross energy intake (kJ) 383.9 ± 14.85 for control, n = 10 vs. 414.7 ± 18.42, n = 11 for dams on the high-fat diet, P not significant].
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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 wet weight of intra-abdominal fat pads (retroperitoneal and perinephric) and gonadal fat lobes was increased when compared with controls (OC, P < 0.05). In females, significant differences in adiposity only occurred between offspring of the fat-fed dams suckled by their own dams (OHF) and controls (OC) (P < 0.05; Table 1).
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Males. There were no differences in blood pressure (Fig. 3A) or activity (Table 2) between the male offspring of control or fat-fed dams or cross-fostered litters. Heart rate was significantly lower in male OHF compared with OC (awake phase, P < 0.05; Fig. 3B). A similar reduction was apparent in both cross-fostered groups (OC/HF vs. OC, P < 0.05 and OHF/C vs. OC, P < 0.05; Fig. 3B).
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Male and female. No differences in endothelium-dependent responses were observed between male and female offspring within the different groups; hence male and female data were considered together. All offspring exposed to the fat diet in gestation or during suckling demonstrated blunted acetylcholine-induced relaxation in mesenteric arteries compared with controls (by repeated-measures ANOVA, OHF vs. OC, P < 0.0005; OHF/C vs. OC, P < 0.0001). Relaxation was further impaired in OC/HF (vs. OC, P < 0.0001; OC/HF vs. OHF, P < 0.0005; Fig. 5A). Sensitivity to acetylcholine was unaffected because pEC50 values were similar between groups (Table 3), but maximal responses were significantly impaired in OHF and OHF/C relative to control (OHF vs. OC, P < 0.001; OHF/C vs. OC, P < 0.001) and further reduced in OC/HF (vs. OC, P < 0.001; male and female OC/HF vs. OHF, P < 0.001; Table 3).
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Plasma Analyses
There were no significant differences in lipid profiles or plasma glucose between offspring from the different experimental groups compared with controls at 180 days of age (Table 4). No differences in plasma insulin concentrations were observed between male and female offspring within the different experimental groups; hence male and female data were 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).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The importance of the in utero period in determining later cardiovascular risk has been demonstrated in several animal models including maternal protein undernutrition (18) and global dietary undernutrition (22, 31) in pregnancy in which hypertension and/or insulin resistance develops in adult offspring despite the dams being fed a normal diet postdelivery. It has also been suggested that the preimplantation period is crucial (17). The role of the suckling period has been less frequently investigated but has major implications for postnatal nutrition, growth, and development.
Exposure to the fat-rich diet during gestation alone was associated with raised systolic and diastolic blood pressures in adult female offspring. The suckling of control animals with fat-fed dams also led to adulthood elevation of blood pressure, but this was confined to a rise in the systolic pressure. Studies in genetically hypertensive rats have also implied that raised blood pressure may, in part, be acquired during the suckling period (1, 5). Further relevant studies include those that implicate in utero stress in development of adulthood behavioral and functional disturbances. Offspring of rats subjected to a stressful stimulus during pregnancy but cross fostered to normal dams develop raised adulthood systolic arterial pressure and enhanced reactivity to restraint stress (14). In common with the present study, female offspring were the most affected, which may indicate a similarity of mechanism between these models.
Ozanne and Hales (24) recently investigated the life span of mice exposed to protein restriction in utero or during suckling. Offspring of protein-restricted dams cross fostered to control dams and reared on a "cafeteria" diet had markedly reduced longevity compared with controls, corroborating the suggestion that in utero dietary deprivation may lead to a phenotype conferring survival advantage only in a time of nutritional restriction (11). However, control mice fed a low-protein diet during suckling showed increased longevity whatever the subsequent diet, suggesting a sustained and predominant benefit of restricted nutrition in the suckling period.
Endothelial dysfunction was present in small mesenteric arteries of all offspring whose dams consumed the fat-rich diet, showing that this disorder, in common with raised SBP, can originate either in utero or during suckling. Endothelial dysfunction may give rise to, or be a consequence of, insulin resistance (13) and could reflect activation of inflammatory pathways as a result of increased adiposity (4).
The arteries from control offspring suckled by fat-fed dams demonstrated almost complete failure of relaxation to acetylcholine. This suggests that the developing vasculature is particularly sensitive to dietary insult postnatally but also implies that exposure to the fat diet in utero conferred protection from the insult of fat feeding during the suckling period. This accords with the suggestion that the fetus can mount a beneficial "predictive adaptive" response (8), in this case in anticipation of consumption of a fat-rich diet during suckling. It is suggested that the fetus makes responses, based on prenatal signals, about the environment from the mother, which will confer adaptive advantage postnatally if the prediction is accurate. If the prediction is incorrect because the postnatal nutritional environment changes, then the responses are maladaptive and disease risk increases. Work from our group provides recent support for this hypothesis by demonstrating that endothelial function in adulthood is normal when offspring of dams that consumed a fat-rich diet during pregnancy and suckling are themselves fed the fat diet from weaning to adulthood (15). The blood pressure, however, remains elevated in the female offspring. A similar study showed that piglets whose sows consumed an "atherogenic" diet in pregnancy are themselves protected from the atherogenic effects if the same diet is fed to them postweaning (21).
The neonatal rat is delivered at a much earlier stage of development than the human infant, and care must be exercised in drawing parallels with studies of nutrition in neonatal children. However, Lucas and colleagues (7, 19), in studies of premature infants whose developmental stage at delivery is much closer to that of neonatal rats, reported that feeding formula milk (rich in fats) compared with breast milk is associated with increased blood pressure and reduced endothelium-dependent dilation in early adulthood. This is analogous to the present study in which the poorest endothelial function was evident in those rats exposed to high fat during suckling alone.
Rearing rats in small litters (restricted to 4 pups) leads to overfeeding during suckling and produces phenotypic similarities to the present model, such as increased adiposity, hyperinsulinemia, leptin resistance, obesity (27), and altered lipid profiles (10) in adulthood. These studies emphasize the important phenotypic induction influences of the suckling period.
In the normotensive male offspring, significant bradycardia was observed in all experimental groups, whereas heart rate was normal in the hypertensive females. This implies that the females may mount an inadequate baroreceptor response to elevated blood pressure. Reduction in baroreceptor reflex sensitivity is associated with both the metabolic syndrome (2) and essential hypertension (20).
The abnormalities in the 180-day-old offspring occurred without any associated changes in the plasma lipid profile, as previously reported in this model at this age (16). Older animals develop a clearly abnormal glucose and lipid profile (16). Plasma glucose concentrations tended to be raised in male and female offspring of fat-fed dams but not in the cross-fostered groups. However, fasting plasma insulin was significantly raised in all fat-exposed groups. We recently showed insulin resistance in older animals by euglycemic hyperinsulinemic clamp (30). Hyperinsulinemia appears therefore to be induced independently during both the in utero period and the suckling period.
As reported previously (29), the dams ate significantly less of the high-fat diet during pregnancy such that their intake was in fact isolcalorific to controls. Nontheless, the overall fat intake was approximately fourfold greater in fat-fed dams than controls. As the fat was substituted for other constituents of the diet, and although vitamins, micronutrients, and protein were added to compensate, there was a 14% reduction in carbohydrate content compared with controls. We cannot entirely exclude therefore the possibility that the diet-induced phenotype is attributable to an imbalance in the carbohydrate-to-fat ratio.
This animal model has certain similarities with other developmental models, all of which lead to a phenotype with features of the metabolic syndrome (11), which, it has been suggested, may imply a common underlying mechanism. Maternal activation of the hypothalamic pituitary adrenal axis (28) and maternal hyperinsulinemia (29) have each been implicated.
Because certain features of the metabolic syndrome were acquired by rat offspring whose mothers consumed a fat-rich diet either in utero and/or during suckling, this study demonstrates a broad window of developmental susceptibility to a dietary imblalance of a kind prevalent both among populations of developed countries and those of some developing countries undergoing rapid economic transition. The data suggest that "predictive adaptive" responses occur in utero to protect against a subsequent "dietary challenge" in the postnatal period. The observations presented provide strong support for the developmental origins of adult disease.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-hydroxysteroid dehydrogenase, and fetal programming. Kidney Int 57: 1412–1417, 2000.[CrossRef][ISI][Medline]
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, n = 10) and fat-fed dams during pregnancy and lactation (
, n = 11, *P < 0.05, repeated-measures ANOVA). Values are expressed as means ± SE.
, n = 6), lard-fed dams (OHF, 
, n = 6), and cross-fostered groups (OC/HF, 
, n = 6 and OHF/C, 
, n = 6). Values are expressed as means ± SE.
P < 0.05, OC vs. OHF/C. 
P < 0.05, OC vs. OC/HF for a period of 1 wk by generated estimated equation (GEE). bpm, Beats/min.
P < 0.0005, OC/HF vs. OHF in male offspring by repeated-measures ANOVA.