J. Anat. (2009) 215, pp36–51 doi: 10.1111/j.1469-7580.2008.00977.x
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Blackwell Publishing Ltd
REVIEW
Nutritional programming of disease: unravelling
the mechanism
Simon C. Langley-Evans
Division of Nutritional Sciences, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, UK
Abstract
Nutritional programming is the process through which variation in the quality or quantity of nutrients consumed
during pregnancy exerts permanent effects upon the developing fetus. Programming of fetal development is
considered to be an important risk factor for non-communicable diseases of adulthood, including coronary heart
disease and other disorders related to insulin resistance. The study of programming in relation to disease processes
has been advanced by development of animal models, which have utilized restriction or over-feeding of specific
nutrients in either rodents or sheep. These consistently demonstrate the biological plausibility of the nutritional
programming hypothesis and, importantly, provide tools with which to examine the mechanisms through which
programming may occur. Studies of animals subject to undernutrition in utero generally exhibit changes in the
structure of key organs such as the kidney, heart and brain. These appear consistent with remodelling of development,
associated with disruption of cellular proliferation and differentiation. Whilst the causal pathways which
extend from this tissue remodelling to disease can be easily understood, the processes which lead to this disordered
organ development are poorly defined. Even minor variation in maternal nutritional status is capable of producing
important shifts in the fetal environment. It is suggested that these environmental changes are associated with
altered expression of key genes, which are responsible for driving the tissue remodelling response and future
disease risk. Nutrition-related factors may drive these processes by disturbing placental function, including control
of materno-fetal endocrine exchanges, or the epigenetic regulation of gene expression.
Key words cardiovascular disease; metabolic syndrome; nutrition, pregnancy; programming.
Introduction
Modern patterns of disease are strongly related to patterns
of nutrition in the population. The major killers in all
developed countries are coronary heart disease, stroke
and cancer and these are growing in prevalence in all parts
of the world, as increasing wealth and global trade change
dietary patterns in countries where the population used to
subsist on simpler and largely plant-based diets. Risk of
cardiovascular disease and cancer is strongly influenced by
obesity and overweight, and hence the consumption of
energy dense, high-sugar, high-fat diets. Although for
cancer the contribution of specific dietary patterns is
proving difficult to elucidate (WCRF, 2007), it has been
overwhelmingly shown that cardiovascular disease is
promoted by diets rich in saturated fatty acids and low in
complex carbohydrates (Willett, 2006).
As scientists and lay people we accept that our adult lifestyle,
comprising diet and physical activity, smoking habits
and alcohol consumption, is one of the main determinants
of our long-term health and well-being. This is of course
only a part of the story and recent decades have brought
huge leaps forward in terms of understanding how these
lifestyle factors interact with the genome and allow inherited
factors to modulate that risk of disease. For example,
adults who carry the TT variant of the C677T polymorphism
of methyltetrahydrofolate reductase (MTHFR) are
more vulnerable to cardiovascular disease, but only if they
consume a diet that is low in folic acid (Klerk et al. 2002).
It is becoming increasingly clear that these interactions of
genes and environment begin to shape health and disease
at much earlier stages of life. For example, being breastfed
protects children from early-onset obesity (Arenz et al. 2004)
and hence may reduce risk of related disorders (Martin
et al. 2005). Disease in adulthood is in fact the product of
continual exposures to protective and disease-promoting
Correspondence
Simon C. Langley-Evans, Division of Nutritional Sciences, School of
Biosciences, University of Nottingham, Sutton Bonington,
Loughborough, LE12 5RD, UK.
E: Simon.Langley-Evans@Nottingham.ac.uk
Submitted as an Invited Review to accompany a presentation at the
Anatomical Society meeting in Nottingham, July 2008.
Accepted for publication 25 July 2008
Article published online 14 October 2008
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
37
factors throughout the lifespan. Exposures at early life
stages may be particularly important, as the plasticity of
growing and developing tissues means that they shape
the way in which the body responds to later challenges.
This review will set out the evidence that nutrition-related
factors in fetal life impact upon risk of diseases that do not
manifest for 40–60 years post-exposure.
The concept of nutritional programming
The term programming describes the process through
which exposure to environmental stimuli or insults during
critical phases of development brings about permanent
changes to the physiology or metabolism of the organism.
There are many examples of this process observable within
the natural world. One that is frequently cited describes the
mechanisms that determine sex in crocodilians. Alligators
and crocodiles lack sex chromosomes (Milnes et al. 2002).
Their eggs are laid into earth mounds within which exists
a temperature gradient. At most temperatures within the
nest the embryos will develop into females, whilst within
a very specific range of 4–5 °C the embryos are more likely
to become males. These effects are explained by the fact
that the temperature of the egg determines the expression
of genes encoding aromatases that are responsible
for synthesis of the sex steroids. These then govern the
physiological development of the embryos (Milnes et al.
2002). Under the definition provided above, this is clearly an
example of programming, as the stimulus is temperature,
the critical phase of development lies within the embryonic
period and the effect of the exposure is permanent.
Mammalian systems are also subject to programming
duringdevelopment.Programmingisafeatureoftheplasticity
of cell lines during embryonic and fetal life. For most types
of cell, plasticity is a short-lived characteristic, being only a
feature of the embryonic and fetal stages. These are the
critical developmental phases that are of greatest interest
in the context of nutrition and human health and disease.
The capacity to programme mammalian systems during early
life can be demonstrated as dramatically as the example of
reptilian temperature-dependent sex determination. Treatment
of newborn female rats with testosterone in the first
few days of life perturbs their lifelong reproductive function.
Regions of the hypothalamus that control the reproductive
axis and archetypal female reproductive behaviours
are remodelled to resemble the male brain and the female
rats are rendered sterile (Arai & Gorski, 1968). The critical
period in which this androgenizing treatment is effective
is relatively short, but the effects are permanent.
In humans such precise experiments can obviously not
be recreated and demonstrations of programming effects
are often less convincing. Normal human physiology can,
however, be shown to respond to cues in the environment
that prevails during early development (Gluckman & Hanson,
2004). During the Second World War the Japanese military
considered the performance of their soldiers in hotter
climates, in relation to the areas of Japan that they were
recruited from. This led to the discovery that the number
of sweat glands is set soon after birth and cannot then be
further adjusted. When individuals were born in cooler
climates they activated a smaller number of sweat glands
than did individuals who were born in warmer climates.
The response to the environment during the early postnatal
period therefore brings about permanent changes to
physiology. These adaptations allow the individual born in
a warm climate to be optimally adapted for life in that
climate, whilst the individual from a cold climate will cope
less well with the heat.
The observation that the developing mammal has the
ability to respond to environmental cues, or physiological
insults, changes the way in which we should think about
the developmental process. Mammalian development
clearly is not entirely gene-led and is not purely a matter
of activating and switching off the expression of genes, in
well-ordered sequences. Adaptive responses triggered by
changes in the environment, as signalled through the
maternal system, can change the profile of genes that are
expressed at any given stage of development, with profound
and irreversible effects upon the physiology of the
fetus. Environmental influences upon apparently genetically
determined processes can be most simply observed
by looking at the effects of maternal physiology and
experience upon fetal growth. The growth trajectory of a
fetus is largely determined by the genes inherited from
both the mother and the father, but evolution has provided
mechanisms through which the genetically determined
growth rate can be constrained in response to maternal
cues. Classic experiments using embryo transfers in horses
and cattle show that the size of the mother is a primary
factor governing fetal growth. When Shetland mares carry
the foals resulting from Shire horse × Shetland pony
crosses, the genetically large offspring are born at a size
similar to the pure Shetland (Walton & Hammond, 1938).
This form of constraint ensures maternal survival, as it
prevents the development of a fetus that will become too
large to pass through the birth canal.
Similar observations can be made in human pregnancies,
where it is clear that very diverse signals can constrain
fetal growth. Factors which signal under-privilege or other
indicators of a less than optimal environment are generally
associated with lower weights at birth among human
babies. Socioeconomic class of the mother is more strongly
predictive of birth weight than any other single factor
(Bibby & Stewart, 2004). Social class is, however, only a
crude proxy indicator of maternal nutritional status,
family income, smoking habits, and access to healthcare
services. Several of these factors, for example smoking, are
known to influence fetal growth in their own right. Slowing
of fetal growth appears to be a common response to any
factor that may threaten the integrity of human pregnancy
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
38
or the health of the mother, including undernutrition,
maternal infection, multiple pregnancy and major psychological
trauma (Brown & Carlson, 2000; Smits et al. 2006).
Placing these concepts into the general context of the
aetiology of disease that was discussed at the start of this
review, it can be asserted that the early life exposures that
might manifest as constrained growth are part of a battery
of factors that are disease-promoting. These factors include
disease-susceptibility genes, adverse early life exposures, a
high-risk adult lifestyle (poor diet, sedentary lifestyle,
smoking) and ageing. Individual risk will be determined by
the interactions of these factors and the influences of
protective genes, optimal early life development and lowerrisk
adult lifestyle. Fetal growth constraint is possibly just
a readily assessed marker of the early life exposure to
adverse environmental cues. Whilst acknowledging that
more subtle changes to fetal physiology might occur,
markers of fetal growth retardation (low birthweight,
disproportion at birth) have been most widely used as the
basis of the evidence that human disease states are subject
to early life programming influences.
Epidemiological evidence for programming of
human disease
Some of the earliest evidence to support a role for the fetal
environment in determining risk of adult disease came from
studies of retrospective cohorts in England. Barker and
colleagues published a series of studies of a cohort of men and
women who had been born in the county of Hertfordshire
between 1911 and 1933 (Barker et al. 1989, 1990, 1993a; Hales
et al. 1991). Initial findings showed that, among men, risk
of death from coronary heart disease was inversely related
to weight at birth, with those men who were born at less
than 5.5 lbs being at double the risk of those who were
in excess of 9.5 lbs at birth. Risk factors for coronary heart
disease were similarly related to birth weight, with low
birth weight predicting higher blood pressure, higher
circulating clotting factors, and impaired glucose tolerance.
Indeed men of lower weight at birth were at massively
increased risk of type-2 diabetes and the metabolic
syndrome (Hales et al. 1991; Barker et al. 1993a).
Observations linking lower birth weight to disease later
in life were extended by studies of other cohorts that had
gathered more detailed information about infant characteristics
at birth. Greater risk of cardiovascular disease and
metabolic syndrome was shown to be associated with
reduced abdominal circumference (Martyn et al. 1995), a
large head circumference in relation to body length
(Barker et al. 1993b), and relative thinness (low weight in
proportion to body length) at birth (Barker et al. 1992). All
of these measurements provide evidence that intrauterine
growth was disproportionate and constrained in an
uneven manner. Several of the observations made using
retrospective cohorts were verified through consideration
of more contemporary populations. For example, glucose
intolerance in Indian children was found to be related to
birth characteristics (Yajnik et al. 1995), whilst thinness
at birth predicted greater blood pressure and other
physiological markers in English infants (Fall et al. 1995).
Demonstrating these associations in young children was
important, as it showed that programmed changes to
physiology manifest almost immediately and do not
depend upon postnatal factors for their expression.
Epidemiological studies from the Scandinavian countries
have been especially useful in demonstrating programming
effects of early life upon disease risk. Two cohorts from
Helsinki, Finland, have enabled researchers to investigate
the interaction of prenatal and post-natal factors. Among
Helsinki men and women born between 1924 and 1944
the greatest risk of developing coronary heart disease and
diabetes was associated with being thin at birth and
relatively fat in later childhood (Ericksson et al. 2002a). This
has prompted the suggestion that fetal growth retardation
followed by rapid ‘catch-up growth’ in childhood is a
major risk factor for disease (Eriksson et al. 2002b).
The above examples show how health and disease in
adult life are related to factors that impact upon rates of
growth in fetal life. It is widely inferred that these
relationships are best explained by variation in maternal
nutrition. Figure 1 shows the fetal origins of adult disease
hypothesis and shows the central role of less than optimal
maternal nutrition as one of the drivers of programming
processes. However, epidemiological support for this
hypothesis is rather tenuous as there are few data to directly
link exposure to undernutrition to later ill-health. This
weakness is to some extent offset by studies of the Dutch
Hunger Winter, which occurred towards the end of the
Second World War. The Nazi blockade of food rations to
Western Holland in retaliation for strikes by railway workers,
resulted in widespread hunger over a period of 6 months.
At the height of the famine the adult ration delivered only
500–600 kcal per day. Birth weights among babies affected
by famine in late gestation were approximately 250 g
lower than those of babies born before or conceived after
the famine (Roseboom et al. 2001). Surprisingly, and rather
in contrast to the fetal origins of adult disease hypothesis,
the babies whose mothers were caught by famine in the
first trimester of gestation, were heavier at birth than the
population average before and after the period of famine.
Follow-up studies of the babies born during the Dutch
famine do, however, strongly support a role for nutrition
in programming disease risk. Adults exposed to the famine
in utero suffered more ill-health than their contemporaries
whose mothers had not been affected by the famine.
Exposure to famine in early gestation was associated with
greater prevalence of coronary heart disease, with raised
concentrations of circulating lipids, blood-clotting factors
and more obesity than in individuals who were not
exposed to the famine (Roseboom et al. 2001).
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
39
Animal models provide proof of principle
The epidemiological studies described above are highly
suggestive of an association between early life nutrition
and later disease risk, but remain a long way off establishing
the causality of any such link. Work in this area has
been subjected to robust criticism and concerns have been
raised about the fact that retrospective analyses of these
historical cohorts are inevitably confounded by many
different factors, about failures to correct adequately
analyses for confounders and about the possibility of a
publication bias in the nutritional programming literature
(Joseph & Kramer, 1996; Paneth et al. 1996; Huxley et al.
2002). Most importantly, the link between maternal
nutrition and fetal growth is far from clear cut and the
majority of studies of women living in developed countries
show little or no association between maternal intakes of
energy, macro- and micronutrients and infant birth weight
(Mathews et al. 1999; Langley-Evans & Langley-Evans, 2003).
A few studies have shown direct associations between
measured intakes of nutrients in pregnancy and risk
factors for disease in the resulting offspring. For example,
follow-up of a study of nutrition in pregnancy conducted
in Aberdeen in the 1940s, showed that elevated blood
pressure in middle-aged men was predicted by a low
intake of animal protein if the mothers were consuming a
high-carbohydrate diet (Campbell et al. 1996). Similarly, a
US study has shown that blood pressure in infants was
related to maternal calcium intake in pregnancy (Gillman
et al. 2004).
Such studies are far from convincing, however, and even
the remarkable findings of the definitive ‘natural experiment’
of the Dutch Hunger Winter can be questioned.
During the Dutch famine, women still had access to
black-market foodstuffs so the severity of undernutrition
may have been patchy. Moreover, the stress of wartime
and the Occupation may explain some of the findings
attributable to undernutrition (Stein, 2004). It is also
overly simplistic to assume that maternal intake is a good
indicator of the nutritional environment experienced by
the fetus. Intake is just one element of the fetal supply
line, which is also related to maternal nutrient stores and
body composition, maternal age, maternal physical activity
and energy expenditure, blood flow to the placenta,
efficiency of placental nutrient transfer and the action of
hormones such as the insulin-like growth factors, which
regulate nutrient partitioning between maternal and
fetal compartments (Harding, 2004).
Epidemiology may be a particularly poor tool for the study
of nutritional programming. It is clear that intervention
studies to explore the issue would be unethical and
impractical. Properly designed prospective cohort studies
will, by definition, require five or six decades of followup
to consider any possible association between maternal
nutrition, body composition or other factors, and the
development of disease. Animal studies therefore become
the only practical way forward for this area of research.
Work with suitable models not only allows the plausibility
of the programming hypothesis to be demonstrated, it also
permits measurement of invasive endpoints, consideration
of programming effects across the full lifespan, and evaluation
of the possibility of intergenerational effects of
undernutrition in pregnancy (Langley-Evans et al. 2006).
Programming in animals
Studies of nutritional programming using animal models
have been ongoing since the early 1990s and one of the
Fig. 1 The fetal origins of adult disease
hypothesis. Adverse environmental cues from
the mother are signalled to the developing
fetus. The prevailing conditions may be
sufficiently harsh to result in the loss of the
pregnancy, or alternatively the fetus may mount
adaptive responses to ensure immediate
survival. One of these responses may be a
slowing of growth that will ultimately result in
lower weight at delivery. Other aspects of the
adaptive response, which may be localized to
specific organs and tissue types, will serve to
modify physiology and metabolism and hence
tissue functions. The trade-off for overcoming
the challenge in fetal life may be increased risk
of disease later in life.
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
40
most striking features of the body of literature that has
accumulated since then is the consistency of effects (LangleyEvans,
2006). The models that have been developed are
immensely varied and have largely been based on rats and
mice, or sheep. Within these species it has been possible
to examine the impact of variation in the maternal diet
at different stages of gestation, and then consider the
impact upon the developing fetus and the mature offspring
that are generated. The range of nutritional insults
that have been utilized is diverse and can be loosely
grouped into models that seek to restrict global nutrition
(i.e. all nutrients consumed by the mother), macronutrient
intake or micronutrient intake, or models that seek to
interfere with delivery of nutrients to the fetus without
impacting on maternal diet, or models that attempt to
deliver an excess of nutrients (Table 1).
Initially many of these experiments were developed to
model the low birth weight hypothesis in its simplest form.
It is well established that restricting maternal food intake,
or ligating a uterine artery (Nüsken et al. 2008), during
rodent pregnancy, will lead to fetal growth retardation, so
this global undernutrition approach seemed a natural
way of considering cross-species correlates of the kind of
observations noted with cohorts such as the Hertfordshire
cohort. Woodall and colleagues (Woodall et al. 1996)
initially published studies of the offspring of rats subject to
a severe food restriction during pregnancy. Rat dams fed
just 30% of normal rations produced offspring with
markedly lower birth weight, and these animals went on
to develop elevated blood pressure by 7 months of age.
Moreover, if the offspring subject to fetal growth retardation
were fed a hypercaloric, obesity-promoting diet in their
adult life, they showed a greater propensity for fat gain,
which appeared to be partly explained by programming
effects upon levels of energy expenditure physical activity
(Vickers et al. 2003).
One of the major criticisms of the global nutrient
restriction models is that they have little relationship to
contemporary problems in human nutrition. Other experimental
approaches have sought to examine nutritional
programming effects independently of any impact of the
maternal diet upon fetal growth rates, instead focusing on
common nutrient deficiencies and problems in human
populations. Iron deficiency anaemia is the most prevalent
micronutrient deficiency on a global scale (Stoltzfus, 2003).
It is estimated that approximately two thirds of the world
population suffers some degree of iron deficiency, and
pregnant women in both developed and developing
countries are particularly at risk. The feeding of an irondeficient
diet to female rats prior to and during pregnancy
results in elevated blood pressure in their offspring, an
endpoint that is associated with changes in cardiac
development during the fetal period (Gambling et al. 2003;
Andersen et al. 2006). Studies of the offspring of irondeficient
dams also show that fatty acid metabolism is subject
to long-term programming effects (Zhang et al. 2005).
The prevailing concerns about the nutrition of women
in developed countries are focused more on nutritional
excess rather than deficiency of specific nutrients. Surprisingly,
whilst experiments which restrict the nutrition of
pregnant animals almost universally result in high blood
pressure, glucose intolerance, insulin resistance and a
greater propensity to become obese (Langley-Evans, 2006),
experiments where the nutritional manipulation is in the
opposite direction have almost exactly the same outcomes.
The feeding of diets which are high in fat during rat
pregnancy is associated with elevated blood pressure and
vascular endothelial dysfunction in the resulting offspring
(Khan et al. 2003, 2005). Fetal exposure to a high protein diet
has been shown to induce defects of energy expenditure
that lead to obesity (Daenzer et al. 2002).
Maternal obesity may also be an important programming
influence with major relevance to modern societies.
Obesity changes many aspects of the environment
experienced by the fetus, including the quality and
quantity of nutrients delivered across the placenta and the
endocrine milieu. Obese women are, of course, at greater risk
of most complications of pregnancy, including gestational
diabetes, pre-eclampsia and pre-term delivery (Jensen
et al. 2003). Bayol and colleagues sought to examine the
impact of feeding a ‘junk-food’ diet, comprising highly
palatable human foods, to rats during pregnancy and
Table 1 Animal models of nutritional programming. A diverse series of experiments have been performed in rodents and large animals to demonstrate
the biological plausibility of disease programming through modification of maternal or fetal nutrition
General approach Specific intervention Species used
Induction of low birth weight Global nutrient restriction Rat, sheep
Uterine ligation Rat, guinea pig
Over-nutrition of mother Induction of maternal obesity Rat, mouse
High-fat, high-cholesterol die Rat, rabbit
High protein diet Rat
Undernutrition of mother Micronutrient deficiency Rat, mouse
(Ca, Fe, Na, Zn)
Macronutrient restriction Rat, mouse
(protein)
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
41
lactation. Offspring exposed to such a dietary pattern
were themselves more obese than controls (Bayol et al.
2007). Samuelsson et al. (2008), induced obesity in mice
several weeks prior to mating and then studied the impact
of maternal obesity upon several endpoints in their offspring.
Mice exposed to the obese maternal environment
had higher blood pressure, greater adiposity and insulin
resistance in comparison with controls.
Low protein diets and rodent pregnancy
Work in our laboratory has focused upon maternal protein
restriction during rat and mouse pregnancy. This experimental
model is by far the best characterized and most
widely studied of all of the animal models of nutritional
programming. As shown in Fig. 2, availability of protein
varies considerably between regions across the world. In
all, 65% of world population are at risk of protein intakes
below the UK Reference Nutrient Intake for pregnancy
and rely heavily on lower quality, plant protein sources.
Within developed countries there is considerable variation
in maternal protein intake, and younger mothers in lower
socioeconomic groups are more likely to consume lower
protein intakes (Langley-Evans & Langley-Evans, 2003).
The feeding of a low protein (LP) diet in rat pregnancy
produces only subtle effects on fetal growth (LangleyEvans
et al. 1996a) and consistently induces persistent high
blood pressure (Langley & Jackson, 1994) and impairments
of renal development (Langley-Evans et al. 1999) in the
offspring. LP-exposed offspring are hypertensive relative
to control animals from as early as weaning (LangleyEvans
et al. 1994) and this effect persists throughout their
adult lives (Langley-Evans & Jackson, 1995, Fig. 3). Changes
in blood pressure appear to be related to dysfunction of
the renin-angiotensin system. Studies have shown elevated
activity of angiotensin converting enzyme and
altered renal expression of the angiotensin II receptors
(Sherman & Langley-Evans, 1998, 2000; McMullen et al.
2004). Treatment of LP-exposed offspring with angiotensin
converting enzyme inhibitors and angiotensin II
receptor antagonists during the suckling period appears
to reverse the programming effect of the maternal diet
(Sherman & Langley-Evans, 2000).
Rats exposed to LP diets in utero have a shorter lifespan
(Aihie-Sayer et al. 2001) and show evidence of disturbed
glucose homeostasis (Fernandez-Twinn et al. 2005), vascular
dysfunction (Torrens et al. 2006), increased susceptibility
to oxidative injury (Langley-Evans & Sculley, 2005), impaired
immunity (Calder & Yaqoob, 2000), altered feeding
behaviour (Bellinger et al. 2004) and increased central fat
deposition (Bellinger et al. 2006). Studies of the impact of
fetal protein restriction upon longevity and mechanisms
of ageing in the rat have been particularly enlightening.
Rats exposed to a maternal low protein diet during fetal
development show little evidence of metabolic abnormalities
at 9 months of age, although it is established that blood
pressure is elevated and there are renal abnormalities well
before this stage. By 18 months of age, however, the rats
develop hepatic steatosis, have normoglycaemia but raised
plasma insulin (indicating insulin resistance), are profoundly
hypertriglyceridaemic and are hypercholesterolaemic
Fig. 2 Protein consumption is highly variable on a worldwide scale.
Data shows per capita availability of protein from plant and animal
sources calculated from the 2004 Food and Agriculture Organization,
global food balance sheets. Intakes of animal protein and lower quality
plant protein sources are shown for countries grouped into regions
defined by the World Health Organization. The most densely populated
regions (SE Asia and China, S Asia) can be seen to have less protein
available than richer regions (N America and Europe). The UK RNI is the
Reference Nutrient Intake, which defines the level of protein intake
for pregnancy at which deficiency in a population is highly unlikely.
Although all regions have protein availability above this figure, actual
intakes are likely to be lower as food balance sheets overestimate
the amount actually consumed and cannot differentiate between
sub-groups in the population (e.g. rich vs. poor, urban vs. rural).
Fig. 3 Systolic blood pressure is elevated in the offspring of rats fed a
low protein diet in pregnancy. Pregnant rats were fed an 18% casein
control diet or a 9% casein low protein diet throughout gestation. At
birth all dams were fed the same standard chow diet, which was also
used to wean the offspring. Blood pressure was measured using an
indirect tail-cuff method. Data are shown as mean ± SE for n = 5–12
observations per group at each time point. * indicates a significant
difference between control and low protein-exposed animals (P < 0.05).
Data compiled from Langley & Jackson (1994) and Langley-Evans &
Jackson (1995).
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
42
(Erhuma et al. 2007). Work by Ozanne and colleagues has
yielded similar findings, despite differences in the precise
nature of the dietary restriction protocol applied in early
life. Rats exposed to a maternal low protein diet throughout
the fetal and suckling periods develop insulin resistance
resulting from insulin signalling defects, but only in
old age (males at 18 months and females at 21 months)
(Ozanne et al. 2003; Fernandez-Twinn et al. 2005). Together,
these observations suggest that protein undernutrition
in early life is able to programme an insulin-resistant
phenotype which develops with ageing. As with the
human metabolic syndrome, the combined influence of
fetal undernutrition and ageing produces rats that are
insulin resistant, hypertensive, at risk of obesity and
exhibit microalbuminuria.
The programming effects of a low protein diet in rat
pregnancy are also noted in mice. This creates exciting
opportunities for the investigation of programming using
the full range of molecular tools that are available, including
transgenic strains. Our laboratory has used this approach
to study nutritional programming of atherosclerosis, a
problem that cannot be investigated in rats, which are
atherosclerosis-resistant. The apoE*3 Leiden mouse is a
strain that carries a naturally occurring mutation in the
human ApoE gene, on a C57Bl/6J background (Groot et al.
1996). This results in impaired clearance of lipoproteins
from the plasma, raised plasma lipid levels and greater
susceptibility to developing atherosclerosis when the mice
are fed diets rich in cholesterol. Whilst other transgenic
mouse strains, for example the LDL receptor knockout
mouse, develop atherosclerosis even when fed a standard
chow diet, the ApoE*3 Leiden mouse develops lesions only
when fed a cholesterol-rich diet. This makes this strain
ideal for studies evaluating the influence of diet upon
atherosclerosis and coronary heart disease. Feeding a low
protein diet to wild-type mice crossed with ApoE*3 Leiden
males, resulted in ApoE*3 Leiden offspring that, when fed an
atherogenic diet from weaning, developed atherosclerotic
lesions that were more than twice as large as those exhibited
by mice exposed to a protein-replete diet in fetal life
(Yates et al. 2008).
Unravelling the mechanism of programming
Tissue remodelling
Normal tissue development in mammalian systems
involves the key processes of proliferation and differentiation.
All organs and tissues develop from a relatively
small pool of progenitor cells in the embryo. These progenitor
lineages are expanded in organogenesis during a
proliferative phase. The precise timing of this proliferative
phase will vary between tissues. Some organs such as the
heart are formed very early in development, whereas others,
for example the kidney, begin to grow at later stages.
Proliferation is followed by a differentiation phase, during
which the pool of cells formed earlier takes on the characteristic
morphology and functions associated with the
organ. The simplest explanation for how nutrition might
programme physiology and function of organs is that the
maternal response to under- or overnutrition interferes
with either the proliferation or differentiation steps in
tissue and organ development (Brameld et al. 1998).
The impact of disruption to either process would be
expected to yield characteristic changes within a tissue.
Adverse conditions during proliferation would not impact
upon the differentiation of the cells within a tissue, and
the affected organ would be expected to be smaller
(fewer cells in total), but with a normal profile of cell types.
In contrast, a tissue subject to adverse conditions during
differentiation would be of normal size but would have an
altered profile of cell types and potentially fewer functional
units. Examples of both scenarios can be clearly seen
in animal models of nutritional programming.
The kidneys of rats exposed to low protein diets in utero
provide a good example of remodelling. On gross examination
there is little difference between the kidneys of
control and low protein-exposed animals and the organs
are of similar size. However, determination of nephron
number reveals a marked reduction of the order of 30–
40% in the nutrient-restricted animals (Langley-Evans
et al. 1999; Vehaskari et al. 2001). Similar effects on
nephrogenesis are noted in sheep subjected to global
undernutrition in utero (Gopalakrishnan et al. 2005). A
reduction in the number of functional units, whilst
maintaining normal tissue mass and cell number, strongly
suggests a major influence of undernutrition during the
differentiation of the specialized structures. In humans,
nephron number has been correlated with weight at birth
(Hughson et al. 2003). Populations in which there is a high
degree of poverty are noted to manifest differences in renal
morphometry. Among Australian aboriginal populations,
for example, rates of chronic renal failure are more than
20-fold more common than in the Caucasian population
and renal volume (a proxy for nephron number) tends to
be lower (Singh & Hoy, 2004).
The brain is also a target for remodelling in response to
variation in maternal nutrition. Fetal exposure to a maternal
low protein diet results in a reduced density of capillaries
within the cerebral cortex of mature offspring (BennisTaleb
et al. 1999). When low protein feeding was extended
to cover both the fetal and suckling periods in rats, there
were profound changes to the gross structure of the brain.
Hypothalamic centres involved in appetite regulation
were enlarged, suggesting changes during proliferation,
but differentiation was also clearly affected as neurons
expressing peptides (galanin and neuropeptide Y) responsible
for regulating appetite were present at much lower
densities (Plagemann et al. 2001). Given the role of the
hypothalamus in homeostasis, even relatively subtle
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
43
changes, of the kind identified by these studies, could have
huge repercussions for a wide range of basic physiological
and metabolic functions. The pancreas from rats exposed
to low protein diets during pregnancy and lactation also
shows evidence of both disordered proliferation and
differentiation. Undernutrition reduces the number of
functional units (β-cells and islets of Langerhans) present.
The islets that are present are smaller and have a reduced
vascular supply due to a reduction in the density of the
pancreatic capillary network (Snoeck et al. 1990; Dahri
et al. 1991).
Remodelling of the heart has not been as clearly
demonstrated as in the tissues described above. However,
variation in the size of the heart has been noted in offspring
exposed to maternal undernutrition. Increased heart size
was noted in cultured day 10.5 rat embryos from female
rats fed iron-deficient diets for 4 weeks before mating and
throughout pregnancy (Andersen et al. 2006). In contrast,
exposure to a low protein diet in fetal development
impairs heart growth in late gestation. This is in keeping
with a general impairment of truncal growth that is noted in
low protein-exposed fetuses. Neonates exposed to protein
restriction in utero exhibit thinning of the left ventricular
wall, and their hearts contain fewer cardiomyocytes
(Cheema et al. 2005). This suggests that remodelling
similar to that noted in other organs is taking place. Crucially,
this remodelling appears to promote left ventricular
hypertrophy in adult life (Tappia et al. 2005). Male rats
subject to protein undernutrition in fetal life are more
vulnerable to the effects of ischaemia–reperfusion (Elmes
et al. 2007).
The remodelling processes described above are almost
certainly the proximal causes of the disease phenotypes
that are programmed by nutrition. Changes to the
structure and function of tissues and organs will drive
disordered physiology through a variety of means. For
example, in the kidney it is believed that chronic renal
disease and hypertension are products of the process
shown in Fig. 4. A programmed deficit of nephrons leads
to a vicious cycle in which rising local blood pressures are
required to maintain renal perfusion and fluid homeostasis,
and yet in turn these higher pressures promote further
nephron loss. However, all such processes are secondary to
the true drivers of programming. The most important
questions to be addressed remain unanswered. We do not
yet fully understand which process or processes are
responsible for remodelling of tissue development.
The role of the placenta
The placenta might conceivably play a major role in
mediating the programming effects of maternal under- or
overnutrition. Placental factors are inevitably involved to
some extent, as perturbations in the maternal environment
must be transmitted across the placenta to affect the
fetus. The relationship between placental function and
fetal nutrition is complex. The nutrients that are transferred
from the maternal to the fetal compartment may
not reflect actual maternal intakes as some, for example
amino acids, can be synthesized de novo within the
placenta (Cleal & Lewis, 2008). In some cases, fetal demand
for energy and nutrients might be met by mobilization of
maternal stores and transfer by placenta. In other cases,
low maternal intakes of some nutrients, for example folic
acid, may disproportionately impact upon the fetus as
the maternal requirements will take priority. Moreover,
the ability of the placenta to provide the fetus with the
substrates it requires will depend upon the quality of the
placentation, the size of the placenta, the demands of
the placenta itself for nutrients, and the extent of the
Fig. 4 Programming of low nephron number is
a potential driver of hypertension and renal
disease. Lower nephron number resulting from
undernutrition during fetal development
cannot be recovered in postnatal life as
nephrogenesis is complete by birth in humans
and most other species. This results in reduced
functional capacity for the kidneys, forcing
increases in local blood pressure to maintain
glomerular filtration and fluid homeostatic
functions. Rising pressures cause further
damage and loss of nephrons and the kidney
enters a vicious cycle of rising pressures and
progressive tissue injury. Eventually, function
will be degraded and systemic blood pressure
will rise and the individual may enter a state of
renal failure. Adapted from Mackenzie &
Brenner (1995).
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
44
maternal adaptations to pregnancy that should increase
cardiac output to support placental perfusion (Harding,
2004).
The contribution of the placenta to the maternal–fetal
nutrient supply line is therefore difficult to assess in the
context of programming. It is conceivable, however, that
deficits or imbalances in the quality or quantity of maternal
nutrition could contribute to programming simply by
restricting fetal substrate supply. The impact of unsuccessful
placentation upon fetal growth, for example, can be readily
observed in conditions such as pre-eclampsia. Here, problems
with the invasion of the placenta during the early stages
of its development not only contribute to the hypertensive
disorder experienced by the mother. The reduced placental
perfusion that accompanies pre-eclampsia is also associated
with fetal growth retardation (Poston, 2006).
In addition to the contribution to the maternal–fetal
supply line, the placenta is a major source of endocrine
signals. Whilst some of these play a role in maintaining the
pregnancy, others are likely to be involved in modulating
the fetal growth rate and the maturation of the brain and
other organs within the fetus. For example, the placenta
secretes corticotrophin releasing hormone, which is known
to have an important role in the maturation of the fetal
hypothalamic–pituitary adrenal axis and, via production
of glucocorticoids from the fetal adrenal, the maturation
of the lungs. Many of the hormones produced by the
placenta serve to control the flow of blood through the
placenta and hence optimize the delivery of substrates to
the fetal tissues. Maternal undernutrition and other
stressors may modulate patterns of placental hormone
secretion and hence alter the uterine environment
experienced by the fetus, compromise the integrity of the
placenta itself, alter gestation length and modify fetal
growth rates.
Despite these putative effects, the role of the placenta
in programming has not been investigated in great depth,
either in humans or in any of the animal models. It is
apparent, however, that the placenta is highly sensitive to
variation in the maternal diet. We have consistently shown
that feeding a low protein diet in rat pregnancy initially
promotes an increased rate of placental growth (LangleyEvans
et al. 1996a). Placentas from rats culled at 14 days’
gestation, for example, were larger when mothers were
fed a low protein diet, particularly if the undernutrition
was targeted at the first 7 days of pregnancy (LangleyEvans
& Nwagwu, 1998). By day 20 gestation, differences
in placental weight associated with protein restriction are
not apparent, but the fetal : placental ratio differs from
that of controls. Observations of this sort are suggestive of
effects of undernutrition upon placentation, but are far
from convincing.
Mice fed low protein diets in pregnancy also manifest
gross differences in placentation, but these effects appear
different to those noted in rats. At day 14.5 gestation,
pregnant mice fed low protein diets had smaller fetuses
but placentas of similar size to controls (Rutland et al.
2007). By day 18.5, both fetal and placental weight were
reduced. Stereological examination of placental structure
revealed changes in the lengths of maternal and fetal
blood vessels within the labyrinthine layers of the placenta
(Fig. 5). Low protein feeding was associated with perturbation
of the expression of cadherin and β-catenin in
the vascular endothelium. These adhesion molecules are
regulators of junctional integrity and permeability (Dejana,
1996). Their relative absence from junctional sites in the
low protein-exposed placenta may indicate that undernutrition
may render the placenta more permeable. Put
together, the reduction in length of the labyrinthine
vessels and the down-regulation of the adhesion molecules
suggests that maternal undernutrition causes vascular
dysfunction in the placenta. This could certainly contribute
Fig. 5 Maternal low protein diets in mouse pregnancy impair the
development of blood vessels in the labyrinthine layers of the placenta.
Pregnant mice were fed an 18% casein (Control) or 9% casein (low
protein) diet from conception until sacrifice at either day 14.5 or 18.5 of
gestation. Stereological analysis revealed differences in the length of
maternal and fetal vessels in later pregnancy. (A) Maternal vessel length;
(B) fetal vessel length. Data are shown as mean ± SE for n = 5
observations at each time point. * indicates a significant difference
between control and low protein fed groups (P < 0.05). Data redrawn
from Rutland et al. (2007).
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
45
to disordered maternal–fetal substrate supply and hence
drive fetal tissue remodelling.
A series of studies by Powell and Jansson have shown
that expression of placental glucose and amino acid transporters
and hence the supply of substrates to the fetus
may be modulated by maternal nutritional status. Jansson
et al. (2006) reported that low protein feeding in rats
down-regulated the placental system A amino acid transporter
by as much as 80% in mid-gestation and suggested
that this effect may have been driven by lower concentrations
of insulin, leptin and IGF-1 in the maternal circulation.
In contrast, treatment of pregnant rats with six injections
of glucose (2 g kg–1
body weight to induce hyperglycaemia)
on days 10 and 11 of gestation, did not alter expression of
the system A or system L amino acid transporter, but despite
this did reduce the capacity of placentas to transport
[14
C]methyl-aminoisobutyric acid, a function mediated by
system A (Ericsson et al. 2007). This suggests that maternal
metabolic control in early pregnancy could be an important
determinant of feto-placental growth and placental
function later in pregnancy.
Glucocorticoids and programming
In addition to acting as the conduit for nutrient exchange
between mother and fetus and as a source of hormones,
the placenta acts as a critical barrier, preventing the access
of potentially harmful substances to the fetal compartment.
For substances in the aqueous phase, this is relatively
simple as only those for which transporters exist will be
able to cross the placenta. For fat-soluble materials,
movement to the fetal compartment should be free and
unimpeded, but it is clear that the placenta has systems in
place to regulate the entry of at least some compounds.
The glucocorticoids are steroid hormones which are
important mediators of stress responses and metabolic
functions. Like all steroid hormones, binding of hormones
to glucocorticoid receptors can initiate the transcription of
gene targets through binding of receptor complexes to
glucocorticoid response elements. These are widespread
within the genome. There is a very large concentration
gradient for glucocorticoids across the placenta, which can
be as great as 1000 : 1, indicating that the placenta actively
prevents them from passing from mother to fetus. This
function is carried out by 11β-hydroxysteroid dehydrogenase
2 (11βHSD2), which in placenta converts active glucocorticoids
such as cortisol to inactive forms (Edwards et al. 1996). The
presence of 11βHSD2 ensures that the fetal tissues are
exposed to only low levels of glucocorticoid. This enables
the fetal hypothalamic–pituitary–adrenal axis to develop
independently of maternal influences and, importantly,
ensures that fetal gene expression is not overly influenced by
factors that impact upon maternal glucocorticoid secretion.
Glucocorticoids are routinely used in obstetric practice
to promote maturation of the fetal lungs when preterm
delivery is likely. Antenatal glucocorticoids promote
secretion of pulmonary surfactant. These therapeutic uses
circumvent the barrier function of 11βHSD2 as the administered
hormones are synthetic glucocorticoids such as
dexamethasone and betamethasone, which are poor
substrates for the enzyme. Studies of rodents and sheep
suggest that fetal exposure to these hormones can have
potent programming effects upon renal development and
later blood pressure (Benediktsson et al. 1993; Celsi et al.
1998; Dodic et al. 2002). The strong similarities between
the phenotypes associated with antenatal glucocorticoid
exposure and nutritional insults have prompted consideration
that undernutrition may act as a stressor that alters
the endocrine cross-talk between mother and fetus.
Indeed, the feeding of a low protein diet in rat pregnancy
resulted in reduced expression and activity of placental
11βHSD2 (Langley-Evans et al. 1996b). As shown in Fig. 6,
this has led to the hypothesis that undernutrition promotes
overexposure of the fetal tissues to glucocorticoids
of maternal origin, and this drives tissue remodelling and
the development of the programmed phenotype (LangleyEvans,
1997a).
Support for this hypothesis can be derived from
experiments that have sought to blockade or mimic the
relationship between maternal undernutrition and
placental 11βHSD2. When rat dams fed low protein diets
are treated with metyrapone in early to mid-gestation,
effectively performing a pharmacological adrenalectomy,
their offspring fail to develop the programmed phenotype
of hypertension and renal insufficiency (McMullen&LangleyEvans,
2005). Replacement doses of corticosterone restore
the programming effect of the diet. The programming
effects of protein restriction are therefore, to some extent,
dependent upon an intact hypothalamic–pituitary–
adrenal axis in the mother. Inhibition of 11βHSD2 using
carbenoxolone during late gestation programmes raised
blood pressure in the offspring of rats fed a control diet
(Langley-Evans, 1997b).
The processes through which glucocorticoid overexposure
promotes widespread programming of physiology,
metabolism and disease risk are not well understood.
There are many reports of glucocorticoid target genes
being up-regulated in a range of tissues in animals exposed
to undernutrition in utero (Langley-Evans et al. 1996c;
Desai et al. 1997; McMullen et al. 2004), but in almost all
cases these observations have been made in adult animals
and therefore well downstream of primary programming
events. However, glucocorticoids are known to bring
about the maturation of tissues, favouring differentiation
over proliferation. It may therefore be possible that in the
context of tissue remodelling they lead to production of
smaller organs with an altered profile of functional units.
Certainly in the kidney, even very brief exposure to
synthetic glucocorticoids reduces nephron number (Dodic
et al. 2002).
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
46
Epigenetic regulation of gene expression
The expression of genes is known to be regulated by a
number of epigenetic factors that can silence or switch on
transcription through modulating access of the transcriptional
machinery to the DNA strands. DNA methylation
and histone acetylation essentially control the tightness of
the coiling of DNA around the histone proteins within the
chromosome, with methylation leading to gene silencing
and acetylation promoting transcription (Bird, 2002). DNA
methylation patterns have been shown to be stably inherited
and may therefore allow phenotypic traits, acquired as a
result of nutritional programming, to be passed on to any
offspring. Undernutrition during embryonic, fetal or even
early postnatal development may irreversibly modify DNA
methylation. In this way, even a brief period of sub-optimal
nutrition can alter gene expression over a long period and
compromise normal physiology and metabolism.
The major interest in a role for DNA methylation in
nutritional programming was largely initiated by the work
of Waterland & Jirtle (2003). Experiments using the Agouti
mouse, in which a yellow coat colour is determined by the
overexpression of a single gene locus (Avy), demonstrated
that nutritional factors in fetal life can modify expression
of genes. The over-expression of this gene is due to
hypomethylation of CpG islands in the Avy locus. When
yellow mice were supplemented with folic acid, vitamin
B12, choline chloride and betaine, the methylation of Avy
was increased in the offspring. This resulted in more mice
with an intermediate brown coat colour being born to
the supplemented mothers.
Methylation may be important in the context of disease
programming. Sinclair et al. (2007) fed sheep a diet that
was deficient in the methyl donors, folic acid, vitamin B12
and methionine for a period of 8 weeks prior to conception
and for the first 6 days of pregnancy. Male offspring
from these ewes were insulin resistant and had elevated
blood pressure. Restriction landmark genome scanning
showed that 4% of 1400 CpG islands in the fetal liver were
differentially methylated. The majority of these loci
were either hypomethylated or totally demethylated. This
finding is in keeping with studies in rats that have suggested
that exposure to maternal low protein diets also
leads to hypomethylation and therefore overexpression of
certain genes (Lillycrop et al. 2005, 2007, 2008).
Lillycrop and colleagues have reported that low protein
feeding in rat pregnancy results in the adult offspring overexpressing
the peroxisome proliferator activated receptorα
(PPARα) and glucocorticoid receptor in this manner
(Lillycrop et al. 2005). Furthermore, changes to histone
acetylation accompanying hypomethylation of the glucocorticoid
receptor (GR10) promoter further facilitate transcription.
There is some evidence that the expression of DNA
methyltransferase 1, the enzyme responsible for maintenance
of DNA methylation patterns that are set in the embryonic
period, may be down-regulated by the maternal low protein
diet (Lillycrop et al. 2007). Availability of folic acid as a
donor of methyl groups for methylation may also be of
importance in these processes. Supplementation of maternal
low protein diets with high-dose folate supplements
can prevent expression of the physiological phenotypes
and also appears to normalize the DNA methylation
patterns (Lillycrop et al. 2005, 2007; Torrens et al. 2006).
The findings from these sheep and rat studies suggest
that prenatal undernutrition can impact upon epigenetic
regulation of gene expression. These changes may be a
Fig. 6 The glucocorticoid programming
hypothesis. Overexposure of fetal tissues to
glucocorticoids of maternal origin may play a
central role in nutritional programming. 11βhydroxysteroid
dehydrogenase 2 (11βHSD2)
has a barrier role in placenta, preventing
movement of active corticosteroids from the
maternal to the fetal compartment. Nutritional
down-regulation of this enzyme can be
postulated to have a major impact on organ
development.
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
47
direct cause of the metabolic phenotype associated with
exposure to undernutrition, by modifying tissue development
and the long-term expression of key genes. In this
context PPARα provides an interesting example. Like
Lillycrop et al. (2005) we have shown in our laboratory
that the maternal low protein diet results in hepatic overexpression
of this gene during early adulthood (Erhuma
et al. 2007). The downstream target genes for this transcription
factor result in a obesity-resistant phenotype with
apparently increased insulin sensitivity. With ageing,
however, PPARα expression declines, and in older rats
exposed to low protein diets in utero it is expressed at only
half the level seen in control animals. This change is
accompanied by a dramatic change in phenotype to an
obesity-prone state, accompanied by hepatic steatosis and
insulin resistance. It is possible that this change is driven by
a phenomenon called epigenetic drift. This process occurs
during ageing, and can promote DNA hypo- and hypermethylation.
The basis of age-related epigenetic drift is
that there is a progressive decline in expression of DNA
methyltransferase-1 (Casillas et al. 2003; Fraga & Esteller,
2007). This leads to passive demethylation of the whole
genome. In some tissues the response to this may be
up-regulation of DNA methyltransferase-3b, leading to
hypermethylation of CpG islands in gene promoters
(Casillas et al. 2003; Fraga & Esteller, 2007). This mechanism
has already been linked to the development of human
cancers. The possibility that it explains the age-related
emergence of some programmed phenotypes has not yet
been explored experimentally.
Future priorities
Work with animal models of nutritional programming has
clearly demonstrated the plausibility of the hypothesis
that nutrition-related factors in human pregnancy could
be related to disease in adulthood. The effects of even
very brief periods of under- or overnutrition during pregnancy
have been neatly demonstrated, and despite the
huge diversity in the experimental approaches that have
been used, the range of phenotypes displayed by the
offspring is remarkably narrow. With evidence firmly
pointing to cardiovascular disease, obesity and metabolic
disorders as conditions that are influenced by programming
in utero, there is no longer any need for observational
studies that characterize the effects of manipulating
maternal nutrition and all attention must focus firmly on
understanding the mechanisms that link nutritional
factors to disease processes. In addressing this priority
there are a number of key issues that should be addressed:
1 There needs to be a systematic and unbiased search for
programming mechanisms. The nutritional programming
field as a whole needs to recognize that, as yet, there is no
definitive mechanism which is known to drive programming.
Even the glucocorticoid and epigenetic hypotheses
describe processes that are likely to be secondary to other
changes induced by signals related to maternal nutrition.
Animal studies of programming tend to characterize the
downstream phenotypes that are observed in adult offspring
and hence will focus on processes that may mediate
the exact pathology or metabolic consequences of
programming. Many of these processes are likely to be
secondary phenomena and do not explain the fundamental
basis of programming. Broad assumptions have been drawn
about observed changes in the expression of single genes
or specific pathways that have been selected on the basis
of their plausible involvement in development of metabolic
or vascular phenotypes. Close association with the
phenotype actually increases the likelihood that observed
effects are secondary events. It seems likely that changes in
the expression of a small number of ‘gatekeeper’ genes
and/or their protein products at critical phases of development
will have a major impact on cell type and number
within tissues, and hence responsiveness to homeostatic
signals, gene expression and tissue function. A systematic
and unbiased search for these gatekeepers should be a
high priority for the field. The diversity of animal models
with similar phenotypes may provide a mechanistic tool to
conduct such a search. For example, the low protein model
and the iron deficiency model of rat pregnancy both
yield offspring with elevated blood pressure (Langley &
Jackson, 1994; Gambling et al. 2003). Proteomic and DNA
microarray comparisons of the two models, timed to
appropriate stages in fetal or embryonic life, would therefore
identify the genes, proteins and pathways that are
differentially expressed in both of the undernourished
groups. These would be putative gatekeepers that would
merit further study.
2 Studies need to consider the whole life-course of the
model species. Most work with large and small animal
models of programming has involved a nutritional manipulation
during pregnancy, with follow-up of the offspring
generally only in early adult life. Our experience with studies
of longevity revealed that programmed phenotypes can
change dramatically with ageing (Erhuma et al. 2007).
Work that seeks to confirm any putative mechanism must
therefore address this issue and make measurements very
early in life (e.g. during the embryonic or fetal period
when exposure is occurring) and at points in adult life
right through to senescence.
3 Studies must confirm the functional significance of gene
or protein changes. It is likely that any systematic screen
for candidate genes, proteins and pathways that are
responsible for mediating programming effects will yield
a large number of putative targets. Care will need to be
taken to demonstrate the true functional significance
of any up- or down-regulation of these targets using
appropriate pharamacological agonists and antagonists,
transgenic animals and other technologies. Where epigenetic
mechanisms are invoked it will be critical to demonstrate
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
48
that the often small changes in methylation or histone
acetylation reported for particular target gene promoters
are actually functionally significant. For example, Lillycrop
et al. (2008), reported that methylation of the PPARα
promoter was down-regulated by 26% in offspring of rats
fed a low protein diet during pregnancy. Studies in the far
more advanced cancer field suggest that differential
methylation needs to be of the order of two-fold or more
up- or down- to have a physiological impact.
4 Studies need to have a robust statistical design and
power. In stark contrast to the epidemiological literature,
studies of the animal models of programming often report
findings from relatively small numbers of animals and
have been criticized on the basis of poorly conducted
statistical analyses (Walters & Edwards, 2004). Pooling of
datasets and expertise between different groups using
similar animal models would be of huge benefit in driving
understanding of nutritional programming forward.
5 Can we intervene? Having identified the contribution
that nutritional programming effects make to the
development of disease, and with work to discover the
underpinning mechanisms in progress, the development
of novel therapeutic and disease prevention strategies is a
key priority. Interventions to offset programming of
disease might include improvements in the quality of diets
for pregnant women, identification of individuals at risk
of adult disease based upon screening of maternal and
birth characteristics, or administration of novel therapeutics
during childhood to target gatekeeper or related processes.
There are already examples of these kinds of interventions
in animal models. For example, folate or glycine supplementation
of a low protein diet during rat pregnancy can
offset the programming effect of the protein undernutrition
(Jackson et al. 2003, Torrens et al. 2006). Administration
of angiotensin II receptor antagonists in the suckling period
ameliorates the programming effect of undernutrition,
but has harmful effects on unaffected animals (Sherman &
Langley-Evans, 2000).
6 Can programming effects persist over multiple generations?
This issue also relates to the importance of being
able to intervene to offset the impact of programming.
If maternal undernutrition can exert major effects upon
fetal development and disease risk, the whole field is of
major significance to public health. The impact of this
magnifies exponentially if there is any chance that
programmed effects could be transmitted from one
generation to another. In populations such as India and
China, where for generations the quality of the diet for
most of the population has been poor, intergenerational
programming would mean that the nutritional/economic
transition associated with greater affluence may produce a
sharp decline in malnutrition-related disease to be followed
by a century (two to three generations) of unavoidable
metabolic disease. There is some evidence that immune
function can be programmed for up to three generations
in mice (Beach et al. 1982), and our own studies of low protein
diets in rat pregnancy indicate transmission of high
blood pressure to a second generation via both maternal
and paternal lines (Harrison & Langley-Evans, 2008).
Conclusion
The prevailing nutritional environment during fetal
development exerts powerful and long-lasting effects
upon physiology and metabolism. Adaptive responses to
less-than-optimal nutrition may play an important role in
the aetiology of human disease. Understanding of the
mechanisms through which this nutritional programming
occurs is still at an early stage. However, the key questions
to be addressed have been largely identified and progress
towards identifying strong candidates for future preventative
and therapeutic strategies is likely to be rapid.
References
Aihie Sayer A, Dunn R, Langley-Evans S, Cooper C (2001) Prenatal
exposure to a maternal low protein diet shortens life span in
rats. Gerontology 47, 9–14.
Andersen HS, Gambling L, Holtrop G, McArdle HJ (2006) Maternal
iron deficiency identifies critical windows for growth and
cardiovascular development in the rat postimplantation
embryo. J Nutr 136, 1171–1177.
Arai Y, Gorski RA (1968) Critical exposure time for androgenization
of the developing hypothalamus in the female rat. Endocrinology
82, 1010–1014.
Arenz S, Rückerl R, Koletzko B, von Kries R (2004) Breast-feeding
and childhood obesity – a systematic review. Int J Obes Relat
Metab Disord 28, 1247–1256.
Barker DJ, Winter PD, Osmond C, Margetts B, Simmonds SJ (1989)
Weight in infancy and death from ischaemic heart disease.
Lancet ii, 577–580.
Barker DJ, Bull AR, Osmond C, Simmonds SJ (1990) Fetal and
placental size and risk of hypertension in adult life. BMJ 301,
259–262.
Barker DJ, Godfrey KM, Osmond C, Bull A (1992) The relation of
fetal length, ponderal index and head circumference to blood
pressure and the risk of hypertension in adult life. Paediatr
Perinat Epidemiol 6, 35–44.
Barker DJ, Hales CN, Fall CH, Osmond C, Phipps K, Clark PM (1993a)
Type 2 (non-insulin-dependent) diabetes mellitus, hypertension
and hyperlipidaemia (syndrome X): relation to reduced fetal
growth. Diabetologia 36, 62–67.
Barker DJ, Osmond C, Simmonds SJ, Wield GA (1993b) The relation
of small head circumference and thinness at birth to death from
cardiovascular disease in adult life. BMJ 306, 422–426.
Bayol SA, Farrington SJ, Stickland NC (2007) A maternal ‘junk
food’ diet in pregnancy and lactation promotes an exacerbated
taste for ‘junk food’ and a greater propensity for obesity in rat
offspring. Br J Nutr 98, 843–851.
Beach RS, Gershwin ME, Hurley LS (1982) Gestational zinc deprivation
in mice: persistence of immunodeficiency for three
generations. Science 218, 469–471.
Bellinger L, Lilley C, Langley-Evans SC (2004) Prenatal exposure to
a maternal low-protein diet programmes a preference for highfat
foods in the young adult rat. Br J Nutr 92, 513–520.
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
49
Bellinger L, Sculley DV, Langley-Evans SC (2006) Exposure to
undernutrition in fetal life determines fat distribution, locomotor
activity and food intake in ageing rats. Int J Obes (Lond) 30,
729–738.
Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR (1993)
Glucocorticoid exposure in utero: new model for adult hypertension.
Lancet 341, 339–341.
Bennis-Taleb N, Remacle C, Hoet JJ, Reusens B (1999) A lowprotein
isocaloric diet during gestation affects brain development
and alters permanently cerebral cortex blood vessels in rat
offspring. J Nutr 129, 1613–1619.
Bibby E, Stewart A (2004) The epidemiology of preterm birth.
Neuro Endocrinol Lett 25 (Suppl. 1), 43–47.
Bird A (2002) DNA methylation patterns and epigenetic memory.
Genes Dev 16, 6–21.
Brameld JM, Buttery PJ, Dawson JM, Harper JM (1998) Nutritional
and hormonal control of skeletal-muscle cell growth and
differentiation. Proc Nutr Soc 57, 207–217.
Brown JE, Carlson M (2000) Nutrition and multifetal pregnancy.
J Am Diet Assoc 100, 343–348.
Calder PC, Yaqoob P (2000) The level of protein and type of fat in
the diet of pregnant rats both affect lymphocyte function in the
offspring. Nutr Res 20, 995–1005.
Campbell DM, Hall MH, Barker DJ, Cross J, Shiell AW, Godfrey KM
(1996) Diet in pregnancy and the offspring’s blood pressure 40
years later. Br J Obstet Gynaecol 103, 273–280.
Casillas MA, Lopatina N, Andrews LG, Tollefsbol TO (2003)
Transcriptional control of the DNA methyltransferases is altered
in aging and neoplastically-transformed human fibroblasts. Mol
Cell Biochem 252, 33–43.
Celsi G, Kistner A, Aizman R, et al. (1998) Prenatal dexamethasone
causes oligonephronia, sodium retention, and higher blood
pressure in the offspring. Pediatr Res 44, 317–322.
Cheema KK, Dent MR, Saini HK, Aroutiounova N, Tappia PS (2005)
Prenatal exposure to maternal undernutrition induces adult
cardiac dysfunction. Br J Nutr 93, 471–477.
Cleal JK, Lewis RM (2008) The mechanisms and regulation of
placental amino acid transport to the human foetus. J Neuroendocrinol
20, 419–426.
Daenzer M, Ortmann S, Klaus S, Metges CC (2002) Prenatal high
protein exposure decreases energy expenditure and increases
adiposity in young rats. J Nutr 132, 142–144.
Dahri S, Snoeck A, Reusens-Billen B, Remacle C, Hoet JJ (1991)
Islet function in offspring of mothers on low-protein diet during
gestation. Diabetes 40 (Suppl. 2), 115–120.
Dejana E (1996) Endothelial adherens junctions: implications in
the control of vascular permeability and angiogenesis, J Clin
Invest 98 1949–1953.
Desai M, Byrne CD, Zhang J, Petry CJ, Lucas A, Hales CN (1997) Programming
of hepatic insulin-sensitive enzymes in offspring of rat
dams fed a protein-restricted diet. Am J Physiol 272, G1083–G1090.
Dodic M, Abouantoun T, O’Connor A, Wintour EM, Moritz KM
(2002) Programming effects of short prenatal exposure to
dexamethasone in sheep. Hypertension 40, 729–734.
Edwards CR, Benediktsson R, Lindsay RS, Seckl JR (1996)11 betaHydroxysteroid
dehydrogenases: key enzymes in determining
tissue-specific glucocorticoid effects. Steroids 61, 263–269.
Elmes MJ, Gardner DS, Langley-Evans SC (2007) Fetal exposure to
a maternal low-protein diet is associated with altered left
ventricular pressure response to ischaemia-reperfusion injury.
Br J Nutr 98, 93–100.
Erhuma A, Salter AM, Sculley DV, Langley-Evans SC, Bennett AJ
(2007) Prenatal exposure to a low-protein diet programs disordered
regulation of lipid metabolism in the aging rat. Am
J Physiol 292, E1702–E1714.
Ericsson A, Säljö K, Sjöstrand E, et al. (2007) Brief hyperglycaemia
in the early pregnant rat increases fetal weight at term by
stimulating placental growth and affecting placental nutrient
transport. J Physiol 581, 1323–1332.
Eriksson JG, Lindi V, Uusitupa M, et al. (2002a) The effects of the
Pro12Ala polymorphism of the peroxisome proliferator-activated
receptor-gamma2 gene on insulin sensitivity and insulin
metabolism interact with size at birth. Diabetes 51, 2321–2324.
Eriksson JG, Forsen T, Tuomilehto J, Jaddoe VW, Osmond C, Barker
DJ (2002b) Effects of size at birth and childhood growth on the
insulin resistance syndrome in elderly individuals. Diabetologia
45, 342–348.
Fall CH, Pandit AN, Law CM, et al. (1995) Size at birth and plasma
insulin-like growth factor-1 concentrations. Arch Dis Child 73,
287–293.
Fernandez-Twinn DS, Wayman A, Ekizoglou S, Martin MS, Hales
CN, Ozanne SE (2005) Maternal protein restriction leads to
hyperinsulinemia and reduced insulin-signaling protein expression
in 21-mo-old female rat offspring. Am J Physiol. 288, R368–R373.
Fraga MF, Esteller M (2007) Epigenetics and aging: the targets and
the marks. Trends Genet 23, 413–418.
Gambling L, Dunford S, Wallace DI, et al. (2003) Iron deficiency
during pregnancy affects postnatal blood pressure in the rat.
J Physiol 552, 603–610.
Gillman MW, Rifas-Shiman SL, Kleinman KP, Rich-Edwards JW,
Lipshultz SE (2004) Maternal calcium intake and offspring blood
pressure. Circulation 110, 1990–1995.
Gluckman PD, Hanson MA (2004) Living with the past: evolution,
development, and patterns of disease. Science 305, 1733–1736.
Gopalakrishnan GS, Gardner DS, Dandrea J, et al. (2005) Influence
of maternal pre-pregnancy body composition and diet during
early-mid pregnancy on cardiovascular function and nephron
number in juvenile sheep. Br J Nutr 94, 938–947.
Groot PH, van Vlijmen BJ, Benson GM, et al. (1996) Quantitative
assessment of aortic atherosclerosis in APOE*3 Leiden transgenic
mice and its relationship to serum cholesterol exposure. Arterioscler
Thromb Vasc Biol 16, 926–933.
Hales CN, Barker DJ, Clark PM, et al. (1991) Fetal and infant
growth and impaired glucose tolerance at age 64. BMJ 303,
1019–1022.
Harding J (2004) Nutritional basis for the fetal origins of adult
disease. In Fetal Nutrition and Adult Disease (eds Langley-Evans
SC), pp. 21–54. CABI, Wallingford, UK.
Harrison M, Langley-Evans SC (2008) Intergenerational programming
of impaired nephrogenesis and hypertension in rats
following maternal protein restriction during pregnancy. Br J
Nutr in press.
Hughson M, Farris AB, Douglas-Denton R, Hoy WE, Bertram JF
(2003) Glomerular number and size in autopsy kidneys: the
relationship to birth weight. Kidney Int 63, 2113–2122.
Huxley R, Neil A, Collins R (2002) Unravelling the fetal origins
hypothesis: is there really an inverse association between birthweight
and subsequent blood pressure? Lancet 360, 659–665.
Jackson AA, Dunn RL, Marchand MC, Langley-Evans SC (2003)
Increased systolic blood pressure in rats induced by a maternal
low-protein diet is reversed by dietary supplementation
with glycine. Clin Sci (Lond) 103, 633–639.
Jansson N, Pettersson J, Haafiz A, et al. (2006) Down-regulation of
placental transport of amino acids precedes the development of
intrauterine growth restriction in rats fed a low protein diet. J
Physiol 576, 935–946.
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
50
Jensen DM, Damm P, Sørensen B, et al. (2003) Pregnancy outcome
and prepregnancy body mass index in 2459 glucose-tolerant
Danish women. Am J Obs Gynecol 189, 239–244.
Joseph KS, Kramer MS (1996) Review of the evidence on fetal and
early childhood antecedents of adult chronic disease. Epidemiol
Rev 18, 158–174.
Khan IY, Taylor PD, Dekou V, et al. (2003) Gender-linked hypertension
in offspring of lard-fed pregnant rats. Hypertension 41,
168–175.
Khan IY, Dekou V, Douglas G, et al. (2005) A high-fat diet during
rat pregnancy or suckling induces cardiovascular dysfunction in
adult offspring. Am J Physiol 288, R127–R133.
Klerk M, Verhoef P, Clarke R, Blom HJ, Kok FJ, Schouten EG;
MTHFR Studies Collaboration Group (2002) MTHFR 677C→T
polymorphism and risk of coronary heart disease: a meta-analysis.
JAMA 288, 2023–2031.
Langley-Evans SC (1997a) Intrauterine programming of hypertension
by glucocorticoids. Life Sci 60, 1213–1221.
Langley-Evans SC (1997b) Maternal carbenoxolone treatment
lowers birthweight and induces hypertension in the offspring of
rats fed a protein-replete diet. Clin Sci (Lond) 93, 423–429.
Langley-Evans SC (2006) Developmental programming of health
and disease. Proc Nutr Soc 65, 97–105.
Langley SC, Jackson AA (1994) Increased systolic blood pressure in
adult rats induced by fetal exposure to maternal low protein
diets. Clin Sci (Lond) 86, 217–222.
Langley-Evans SC, Jackson AA (1995) Captopril normalises systolic
blood pressure in rats with hypertension induced by fetal exposure
to maternal low protein diets. Comp Biochem Physiol 110, 223–
228.
Langley-Evans AJ, Langley-Evans SC (2003) Relationship between
maternal nutrient intakes in early and late pregnancy and
infants weight and proportions at birth: prospective cohort
study. J R Soc Health 123, 210–216.
Langley-Evans SC, Nwagwu M (1998) Impaired growth and increased
glucocorticoid-sensitive enzyme activities in tissues of rat fetuses
exposed to maternal low protein diets. Life Sci 63, 605–615.
Langley-Evans SC, Sculley DV (2005) Programming of hepatic antioxidant
capacity and oxidative injury in the ageing rat. Mech
Ageing Dev 126, 804–812.
Langley-Evans SC, Phillips GJ, Jackson AA (1994) In utero exposure to
maternal low protein diets induces hypertension in weanling
rats, independently of maternal blood pressure changes. Clinical
Nutrition 13, 319–324.
Langley-Evans SC, Gardner DS, Jackson AA (1996a) Association of
disproportionate growth of fetal rats in late gestation with
raised systolic blood pressure in later life. J Reprod Fertil 106,
307–312.
Langley-Evans SC, Phillips GJ, Benediktsson R, et al. (1996b) Protein
intake in pregnancy, placental glucocorticoid metabolism
and the programming of hypertension in the rat. Placenta 17,
169–172.
Langley-Evans SC, Gardner DS, Jackson AA (1996c) Maternal
protein restriction influences the programming of the rat
hypothalamic-pituitary-adrenal axis. J Nutr 126, 1578–1585.
Langley-Evans SC, Welham SJ, Jackson AA (1999) Fetal exposure
to a maternal low protein diet impairs nephrogenesis and
promotes hypertension in the rat. Life Sci 64, 965–974.
Langley-Evans SC, Lilley C, McMullen S (2006) Maternal protein
restriction and fetal growth: lack of evidence of a role for homocysteine
in fetal programming. Br J Nutr 96, 578–586.
Lillycrop KA, Phillips ES, Jackson AA, Hanson MA, Burdge GC
(2005) Dietary protein restriction of pregnant rats induces and
folic acid supplementation prevents epigenetic modification of
hepatic gene expression in the offspring. J Nutr 135, 1382–1386.
Lillycrop KA, Slater-Jefferies JL, Hanson MA, Godfrey KM,
Jackson AA, Burdge GC (2007) Induction of altered epigenetic
regulation of the hepatic glucocorticoid receptor in the offspring
of rats fed a protein-restricted diet during pregnancy
suggests that reduced DNA methyltransferase-1 expression is
involved in impaired DNA methylation and changes in histone
modifications. Br J Nutr 97, 1064–1073.
Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA,
Burdge GC (2008) Feeding pregnant rats a protein-restricted
diet persistently alters the methylation of specific cytosines in the
hepatic PPARalpha promoter of the offspring. Br J Nutr, in press.
Mackenzie HS, Brenner BM (1995) Fewer nephrons at birth: a missing
link in the etiology of essential hypertension? Am J Kidney Dis
26, 91–98.
Martin RM, Ben-Shlomo Y, Gunnell D, Elwood P, Yarnell JW,
Davey Smith G (2005) Breast feeding and cardiovascular disease
risk factors, incidence, and mortality: the Caerphilly study. J
Epidemiol Commun Health 59, 121–129.
Martyn CN, Barker DJ, Jespersen S, Greenwald S, Osmond C, Berry
C (1995) Growth in utero, adult blood pressure, and arterial
compliance. Br Heart J 73, 116–121.
Mathews F, Yudkin P, Neil A (1999) Influence of maternal nutrition
on outcome of pregnancy: prospective cohort study. BMJ
319, 339–343.
McMullen S, Langley-Evans SC (2005) Maternal low-protein diet in
rat pregnancy programs blood pressure through sex-specific
mechanisms. Am J Physiol 288, R85–R90.
McMullen S, Gardner DS, Langley-Evans SC (2004) Prenatal programming
of angiotensin II type 2 receptor expression in the rat.
Br J Nutr 91, 133–140.
Milnes MR, Roberts RN, Guillette LJ (2002) Effects of incubation
temperature and estrogen exposure on aromatase activity in
the brain and gonads of embryonic alligators. Environ Health
Perspect 110, 393–396.
Nüsken KD, Dötsch J, Rauh M, Rascher W, Schneider H (2008)
Uteroplacental insufficiency after bilateral uterine artery
ligation in the rat: impact on postnatal glucose and lipid metabolism
and evidence for metabolic programming of the offspring
by sham operation. Endocrinology 149, 1056–1063.
Ozanne SE, Olsen GS, Hansen LL, et al. (2003) Early growth
restriction leads to down regulation of protein kinase C zeta
and insulin resistance in skeletal muscle. J Endocrinol 177, 235–
241.
Paneth N, Ahmed F, Stein AD (1996) Early nutritional origins of
hypertension: a hypothesis still lacking support. J Hypertens 14,
S121–S129.
Plagemann A, Harder T, Rake A, Melchior K, Rohde W, Dorner G
(2001) Hypothalamic nuclei are malformed in weanling offspring
of low protein malnourished rat dams. J Nutr 130, 2582–
2589.
Poston L (2006) Endothelial dysfunction in pre-eclampsia. Pharmacol
Reports 58 Suppl, 69–74.
Roseboom TJ, van der Meulen JH, Ravelli AC, Osmond C, Barker DJ,
Bleker OP (2001) Effects of prenatal exposure to the Dutch famine
on adult disease in later life: an overview. Mol Cell Endocrinol
185, 93–98.
Rutland CS, Latunde-Dada AO, Thorpe A, Plant R, Langley-Evans
S, Leach L (2007) Effect of gestational nutrition on vascular
integrity in the murine placenta. Placenta 28, 734–742.
Samuelsson AM, Matthews PA, Argenton M, et al. (2008) Dietinduced
obesity in female mice leads to offspring hyperphagia,
Nutritional programming, S. C. Langley-Evans
© 2008 The Author
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
51
adiposity, hypertension, and insulin resistance: a novel murine
model of developmental programming. Hypertension 51, 383–
392.
Sherman RC, Langley-Evans SC (1998) Early administration of
angiotensin-converting enzyme inhibitor captopril, prevents
the development of hypertension programmed by intrauterine
exposure to a maternal low-protein diet in the rat. Clin Sci
(Lond) 94, 373–381.
Sherman RC, Langley-Evans SC (2000) Antihypertensive treatment
in early postnatal life modulates prenatal dietary influences
upon blood pressure in the rat. Clin Sci 98, 269–275.
Sinclair KD, Allegrucci C, Singh R, et al. (2007) DNA methylation,
insulin resistance, and blood pressure in offspring determined
by maternal periconceptional B vitamin and methionine status.
Proc Natl Acad Sci USA 104, 19351–19356.
Singh GR, Hoy WE (2004) Kidney volume, blood pressure, and
albuminuria: findings in an Australian aboriginal community.
Am J Kidney Dis 43, 254–259.
Smits L, Krabbendam L, de Bie R, Essed G, van Os J (2006) Lower
birth weight of Dutch neonates who were in utero at the time
of the 9/11 attacks. J Psychosom Res 61, 715–717.
Snoeck A, Remacle C, Reusens B, Hoet JJ (1990) Effect of a low protein
diet during pregnancy on the fetal rat endocrine pancreas.
Biol Neonate 57, 107–118.
Stein AD (2004) Birthweight and the development of overweight
and obesity. In Fetal Nutrition and Adult Disease (eds LangleyEvans
SC), pp. 195–210. CABI, Wallingford, UK.
Stoltzfus RJ (2003) Iron deficiency: global prevalence and consequences.
Food Nutr Bull 24, S99–S103.
Tappia PS, Nijjar MS, Mahay A, Aroutiounova N, Dhalla NS (2005)
Phospholipid profile of developing heart of rats exposed to lowprotein
diet in pregnancy. Am J Physiol 289, R1400–R1406.
Torrens C, Brawley L, Anthony FW, et al. (2006) Folate supplementation
during pregnancy improves offspring cardiovascular
dysfunction induced by protein restriction. Hypertension 47,
982–987.
Vehaskari VM, Aviles DH, Manning J (2001) Prenatal programming
of adult hypertension in the rat. Kidney Int 59, 238–245.
Vickers MH, Breier BH, McCarthy D, Gluckman PD (2003) Sedentary
behavior during postnatal life is determined by the prenatal
environment and exacerbated by postnatal hypercaloric nutrition.
Am J Physiol 285, R271–R273.
Walters E, Edwards RG (2004) Further thoughts regarding evidence
offered in support of the ‘Barker hypothesis’. Reprod
Biomed Online 9, 129–131.
Walton A, Hammond J (1938) The maternal effects on growth and
conformation in Shire horse-Shetland pony crosses. Proc R Soc
Lond B Biol Sci 125, 311–335.
Waterland RA, Jirtle RL (2003) Transposable elements: targets for
early nutritional effects on epigenetic gene regulation. Mol Cell
Biol 23, 5293–5300.
Willett WC (2006) Trans fatty acids and cardiovascular disease –
epidemiological data. Atherosclerosis 7, 5–8.
Woodall SM, Johnston BM, Breier BH, Gluckman PD (1996) Chronic
maternal undernutrition in the rat leads to delayed postnatal
growth and elevated blood pressure of offspring. Ped Res 40,
438–443.
World Cancer Research Fund (2007) Food, Nutrition, Physical
Activity and the Prevention of Cancer. London: WCRF.
Yajnik CS, Fall CH, Vaidya U, et al. (1995) Fetal growth and glucose
and insulin metabolism in four-year-old Indian children. Diabet
Med 12, 330–336.
Yates Z, Tarling EJ, Langley-Evans SC, Salter AM (2008) Maternal
undernutrition programmes atherosclerosis in the Apo E*3
Leiden mouse. Br J Nutr, in press.
Zhang J, Lewis RM, Wang C, Hales N, Byrne CD (2005) Maternal
dietary iron restriction modulates hepatic lipid metabolism in
the fetuses. Am J Physiol 288, R104–R111.