Fetal origins of hyperphagia, obesity, and hypertension and
postnatal amplification by hypercaloric nutrition
Mark H.
Vickers,
Bernhard H.
Breier,
Wayne S.
Cutfield,
Paul L.
Hofman, and
Peter D.
Gluckman
Research Centre for Developmental Medicine and Biology, Faculty of
Medicine and Health Science, University of Auckland, 92019 Auckland, New Zealand
 |
ABSTRACT |
Environmental factors
and diet are generally believed to be accelerators of obesity and
hypertension, but they are not the underlying cause. Our animal model
of obesity and hypertension is based on the observation that impaired
fetal growth has long-term clinical consequences that are induced by
fetal programming. Using fetal undernutrition throughout pregnancy, we
investigated whether the effects of fetal programming on adult obesity
and hypertension are mediated by changes in insulin and leptin action
and whether increased appetite may be a behavioral trigger of adult
disease. Virgin Wistar rats were time mated and randomly assigned to
receive food either ad libitum (AD group) or at 30% of ad libitum
intake, or undernutrition (UN group). Offspring from UN mothers were
significantly smaller at birth than AD offspring. At weaning, offspring
were assigned to one of two diets [a control diet or a hypercaloric (30% fat) diet]. Food intake in offspring from UN mothers was significantly elevated at an early postnatal age. It increased further
with advancing age and was amplified by hypercaloric nutrition. UN
offspring also showed elevated systolic blood pressure and markedly
increased fasting plasma insulin and leptin concentrations. This study
is the first to demonstrate that profound adult hyperphagia is a
consequence of fetal programming and a key contributing factor in adult
pathophysiology. We hypothesize that hyperinsulinism and
hyperleptinemia play a key role in the etiology of hyperphagia, obesity, and hypertension as a consequence of altered fetal development.
appetite; insulin resistance; leptin resistance; cardiovascular
disease
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INTRODUCTION |
IMPAIRED FETAL
GROWTH is a major cause of perinatal morbidity and has long-term
clinical consequences. Increasing epidemiological evidence links low
birth weight to an increased risk of developing adult diseases,
including type 2 diabetes, hypertension, and cardiovascular disease
(4, 6, 14). Insulin resistance
has been documented in otherwise well, prepubertal children born with
intrauterine growth retardation (IUGR), suggesting that this may be one
of the earliest metabolic abnormalities present in these children (11). The epidemiological associations between an adverse
intrauterine environment and the later onset of adult metabolic and
cardiovascular disorders led to the concept of fetal programming and
the "fetal origins" hypothesis (5, 13,
16). This hypothesis proposes that an adverse intrauterine
environment alters the fetal metabolic and hormonal milieu, resulting
in developmental adaptations to ensure fetal survival. If these
adaptive responses, designed for survival in a substrate-limited fetal
environment, persist into postnatal life, it is proposed that they lead
to metabolic, cardiovascular, and endocrine disorders.
The present article is the first report of hyperphagia as a consequence
of fetal programming to use a model of fetal undernutrition that
resembles the clinical and metabolic abnormalities found in human IUGR.
The amplification of metabolic abnormalities in offspring of
undernourished dams by hypercaloric nutrition suggests that postnatal
environmental factors are important accelerators in the etiology of
adult-onset disease. Hyperinsulinism and hyperleptinemia seen in the
growth-retarded offspring may represent critical changes that lead to
the development of hyperphagia, obesity, and hypertension.
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MATERIALS AND METHODS |
Virgin Wistar rats (age 100 ± 5 days) were time mated
using a rat estrous cycle monitor to assess the stage of estrus of the animals before introducing the male. After confirmation of mating, rats
were housed individually in standard rat cages containing wood shavings
as bedding and with free access to water. All rats were kept in the
same room with a constant temperature maintained at 25°C and a
12:12-h light-dark cycle. Animals were assigned to one of two
nutritional groups (n = 15/group): 1)
undernutrition (30% of an ad libitum diet) of a standard diet
throughout gestation (UN group), and 2) standard diet ad
libitum throughout gestation (AD group). Food intake and maternal
weights were recorded daily until birth. After birth, pups were weighed
and litter size was recorded. Pups from undernourished mothers were
cross-fostered onto dams which received AD feeding throughout
pregnancy. Litter size was adjusted to 8 pups per litter to assure
adequate and standardized nutrition until weaning. After weaning, male
offspring from AD and UN mothers were divided into two balanced
postnatal groups to be fed either a standard diet (protein 19.9%, fat
5%, digestible energy 3,504 kcal/kg, protein energy/total energy
22.7%) or a hypercaloric diet (protein 28.5%, fat 30%, digestible
energy 4,922 kcal/kg, protein energy/total energy 22.7%). The mineral and vitamin contents in the two diets were identical and in accordance with the requirements for standard rat diets. Weights and food intake
of all offspring were measured daily for the first 2 wk and then every
2nd day. At day 100, systolic blood pressure measurements were recorded using tail-cuff plethysmography (19)
according to the manufacturer's instructions [a blood pressure
analyzer from IITC, Life Science (Woodland Hills, CA) was used]. At
least three clear systolic blood pressure recordings were taken per animal, and the coefficient of variation for repeated measurements was
<5%. At day 125, rats were fasted overnight, anesthetized with halothane, and killed by decapitation. Blood was collected into
heparinized vacutainers and stored on ice until centrifugation and
removal of supernatant for analysis. The Animal Ethics Committee of the
University of Auckland approved all animal work.
Insulin-like growth factor I (IGF-I) in rat blood plasma was
measured using the IGF-binding protein-blocked RIA described previously
(7, 8), and plasma insulin was measured by
RIA (19). A double-antibody RIA was developed and
validated for measurement of leptin in rat plasma. An antibody was
raised in rabbits against a synthetic fragment (aa 30-45) of
bovine leptin. The standard preparation for the RIA was rm-leptin (no.
CR-6781, Crystal Chem, Houston, TX) used in concentrations
ranging from 0.5 to 20 ng/ml. Samples were assayed undiluted or
diluted 1:2-1:4 in assay buffer (0.05 M PBS, pH 7.4, containing
0.1 M NaCl, 0.5% BSA, 10 mM EDTA, and 0.05% NaN3).
Briefly, 100 µl of primary antibody (1:25,000) were added to tubes
containing 100 µl of sample or standard. After incubation for 24 h at 4°C, 100 µl of tracer (125I-labeled rm-leptin,
20,000 counts/min per tube) were added to all tubes, followed by a
further incubation for 24 h at 4°C. A second antibody technique
was used to separate bound from free ligand (8). Rat
plasma samples showed parallel displacement to the standard curve, and
recovery of unlabeled rm-leptin was 101.4 ± 2.7% (SE,
n = 26). The half-maximally effective dose, or
ED50, was 0.37 ng/ml, and the intra-assay coefficient of
variation was 5% (all samples were measured within a single assay).
Plasma glucose concentrations were measured using a YSI Glucose
Analyzer (model 2300, Yellow Springs Instrument, Yellow Springs, OH).
Blood plasma glycerol and free fatty acids were measured by diagnostic kits (Sigma no. 337 and Boehringer-Mannheim no. 1383175, respectively). Statistical analyses were carried out using SigmaStat (Jandel Scientific, San Rafael, CA) and StatView (version 5, SAS Institute, Cary, NC) statistical packages. Differences between groups were determined by two-way ANOVA. Plasma leptin data were also analyzed by
analysis of covariance (ANCOVA), with unadjusted fat pad weight as a
covariate. Data are shown as means ± SE. Statistical significance was accepted at the P < 0.05 level.
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RESULTS |
There was a small reduction in maternal body weights compared with
day 1 of gestation in pregnant UN group females until
day 15 of gestation. From day 15 of gestation, UN
dams gained weight and had achieved premating weights by the time of
parturition. Litter size was not significantly different between the
two groups (AD 13.6 ± 0.6, UN 12.6 ± 1.1). Maternal
undernutrition resulted in fetal growth retardation, reflected by
significantly decreased body weight at parturition in the offspring
from UN dams (AD 6.04 ± 0.46 g, UN 3.9 ± 0.38 g,
P < 0.0001). From parturition until weaning at
day 22, body weights remained significantly lower in the UN
offspring (AD 52.5 ± 1 g, UN 33.8 ± 2 g,
P < 0.001). Total body weights remained significantly
lower in UN offspring for the remainder of the study. Caloric intake
was calculated over three distinct periods: from weaning until puberty
(22-40 days), postpuberty (60-80 days), and mature adulthood
(100-125 days). UN offspring showed hyperphagia at each age period
(Fig. 1). Importantly, ANOVA revealed
statistical interactions between programming and diet that became
significant during the postpubertal period and increased even further
during the adult age period. Systolic blood pressures in UN offspring
at ~100 days of age were significantly (P < 0.0001)
higher than in AD offspring on both the control and hypercaloric diets.
Hypercalorically fed AD and UN offspring had significantly
(P < 0.001, Fig. 2)
elevated systolic blood pressure compared with offspring fed the
control diet.

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Fig. 1.
Caloric intake per day in offspring from ad libitum-fed
(AD) and undernutritionally fed (30% AD diet, UN) mothers, during 3 distinct postnatal age periods (of ~3 wk each), fed either a control
or a hypercaloric diet. Data are means ± SE and were analyzed by
two-way ANOVA. Prepubertal period: programming effect P < 0.005, hypercaloric diet effect P < 0.005, and
programming × diet interaction not significant. Postpubertal
period: programming effect P < 0.005, hypercaloric
diet effect P < 0.0001, and programming × diet
interaction P < 0.05. Mature adult period: programming
P < 0.0001, hypercaloric diet P < 0.0001, and programming × diet interaction P < 0.0005.
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Fig. 2.
Systolic blood pressure and fasting plasma insulin
concentrations at day 100 of age in AD and UN offspring fed
either a control or a hypercaloric diet. Data are means ± SE and
were analyzed by two-way ANOVA. Effect of fetal programming
P < 0.0001, effect of hypercaloric diet
P < 0.001. There were no significant statistical
interactions.
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All organ and tissue weight data presented at the time
animals were killed (125 days of age) are expressed as a percentage of
body weight unless otherwise stated. Body weights of UN offspring on
either diet were significantly (P < 0.001) lower than
those of AD offspring, and body weights were significantly
(P < 0.05) increased on the hypercaloric diet (Table
1). Nose-to-anus lengths were
significantly (P < 0.005) shorter in UN offspring and
were not affected by hypercaloric feeding (Table 1). UN offspring had
larger retroperitoneal fat pads relative to body weight than AD
offspring (P < 0.05). Hypercaloric nutrition
significantly (P < 0.001) increased retroperitoneal
fat pad weights in both groups of animals (Table 1). UN offspring had
significantly (P < 0.005) smaller kidneys than AD
offspring (Table 1). Heart and spleen weights were not significantly
different between UN and AD offspring and were not affected by diet. UN
offspring had significantly (P < 0.005) smaller livers
than AD offspring, and liver weight relative to body weight was
decreased (P < 0.05) by hypercaloric nutrition (Table
1).
Plasma IGF-I concentrations were not different between UN and AD
offspring and were not affected by diet (Table
2). UN offspring had significantly
(P < 0.001) higher fasting insulin concentrations, which were markedly increased by hypercaloric nutrition
(P < 0.005, Fig. 2). UN offspring had significantly
(P < 0.005) higher fasting leptin concentrations. UN
offspring fed a hypercaloric diet had markedly (P < 0.005) elevated plasma leptin concentrations, and a significant
(P < 0.05) programming × diet interaction was
observed (Fig. 3). When unadjusted fat
pad weight was used as a covariate in ANCOVA analysis, there was no
significant difference between AD and UN animals, and plasma leptin was
therefore proportional to retroperitoneal fat content. Fasting glucose
concentrations were not different between AD and UN offspring, and no
significant effect of diet was observed (Table 2). There was no
difference in plasma glycerol concentrations between UN and AD
offspring, although hypercaloric nutrition significantly
(P < 0.05) elevated plasma glycerol in both UN and AD
offspring. Programming or diet did not affect the plasma free fatty
acid concentrations (data not shown).

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Fig. 3.
Plasma leptin concentrations at 125 days of age in AD and
UN offspring fed either a control or a hypercaloric diet. Data are
means ± SE and were analyzed by two-way ANOVA. Effect of fetal
programming P < 0.001, effect of hypercaloric diet
P < 0.0005, and programming × diet interaction
P < 0.05.
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DISCUSSION |
Two novel observations are of particular importance for our
understanding of the mechanisms and the physiological basis of human
biology and disease. First, fetal undernutrition induced inappropriate
hyperphagia in adult life. Second, postnatal hypercaloric nutrition
amplified the metabolic abnormalities induced by fetal undernutrition,
which included hyperinsulinism, hyperleptinemia, hypertension, and
obesity. Although several rodent models of hyperphagia have been
reported, our study is the first to show the development of profound
adult hyperphagia as a consequence of fetal programming. The mechanism
by which hyperphagia is induced by fetal programming is not clear, but
the hyperleptinemia and increased fat pad mass seen in offspring from
UN mothers suggest a state of leptin resistance. Elevated plasma
concentrations of leptin, as seen in offspring from UN mothers, would
normally decrease appetite (9, 10). However,
amplification of the hyperphagia occurred in this group with a
hypercaloric diet, and this was paralleled by a highly significant
programming × diet interaction in plasma leptin concentrations. These data suggest that the increased appetite simply did not reflect
appetite-driven catch-up growth and likely reflected inappropriate hyperphagia as a consequence of fetal programming. Although increased plasma insulin levels are normally associated with reduction in appetite (17), the hyperinsulinism seen in the UN
offspring is likely to reflect insulin resistance and reduced insulin
action, as seen in children born with IUGR (11). Thus
reduced insulin action may further contribute to inappropriate
stimulation of appetite. Offspring from UN mothers that were fed on a
hypercaloric diet showed the highest plasma insulin concentrations and
developed marked hyperphagia. Thus we propose that fetal undernutrition induces impaired neuroendocrine regulation in which hyperleptinemia, hyperinsulinism, and insulin and leptin resistance may lead to hyperphagia, obesity, and hypertension during adult life.
Our animal model closely resembles the clinical and metabolic
abnormalities seen in humans born with low birth weight.
Epidemiological data suggest that children born with IUGR also have an
increased risk of developing obesity. This was most clearly shown in
the Dutch Famine Study, in which poor nutrition in the first trimester of pregnancy resulted in increased rates of obesity in 19-yr-old males
(15). Although the programming effects due to fetal
undernutrition were anticipated in our study, it was surprising to see
marked amplification of hyperphagia, hyperinsulinism, hyperleptinemia, and hypertension in offspring from UN mothers with the postnatal hypercaloric diet. The mechanism underlying the amplification of
hyperphagia by the hypercaloric diet remains to be determined. However,
diet-induced obesity will exacerbate insulin resistance with
compensatory hyperinsulinism; thus the present study suggests that
environmental factors, such as a hypercaloric diet, can amplify the
metabolic and cardiovascular abnormalities in programmed offspring. The
amplification of the effects of fetal programming may be a result of
hyperinsulinism. Whereas insulin sensitivity by definition reflects
insulin action on glucose and carbohydrate metabolism, it is now
established that not all insulin-receptive tissues become insulin
resistant (1, 2). Thus secondary
hyperinsulinism can have stimulating and undesirable effects, such as
chronic renal sodium retention and increased sympathetic nervous system activity, whereas resistance in other tissues, such as the endothelium, can result in impaired vasodilation (3, 18).
Although it is likely that insulin resistance or hyperinsulinism is
involved in the blood pressure abnormalities seen in offspring from
undernourished mothers, other factors may also contribute. In
particular, the kidneys in the growth-retarded animals were smaller,
suggesting that there may be a renal component to the hypertension
(12).
In summary, this study in a model of fetal undernutrition confirms the
clinical and metabolic abnormalities found in both low birth-weight
humans and other animal models of IUGR. However, this is the first
report of hyperphagia in an animal model of IUGR, and the first study
to investigate the effects of a hypercaloric diet on postnatal sequelae
of fetal programming. The amplification of the metabolic and
cardiovascular abnormalities by hypercaloric nutrition seen in these
growth-retarded animals suggests that postnatal environmental factors
are important in the etiology of adult-onset disease. We propose that
leptin and insulin resistance, and the resulting hyperleptinemia and
hyperinsulinism seen in the growth-retarded animals, are critical in
the development of hyperphagia, obesity, and hypertension. The findings
of the present study extend and reinforce the theory of fetal
programming, the phenomenon that the intrauterine environment can have
significant long-term health sequelae in offspring. They also suggest
that health care funding may be better spent on preventing health
problems during pregnancy than in waiting until metabolic and
cardiovascular disorders manifest, years or even decades later.
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ACKNOWLEDGEMENTS |
We thank Christine Keven, Andrzej Surus, and Janine Street for
expert technical assistance.
 |
FOOTNOTES |
We acknowledge support from the Health Research Council of New Zealand
and the National Child Health Research Foundation.
Address for reprint requests and other correspondence: B. H. Breier, Research Centre for Developmental Medicine and Biology, Faculty of Medicine and Health Science, The Univ. of Auckland, Private
Bag 92019, Auckland, New Zealand (E-mail
bh.breier{at}auckland.ac.nz).
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. §1734 solely to indicate this fact.
Received 1 October 1999; accepted in final form 8 February 2000.
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