Ann. N.Y. Acad. Sci.

ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: The Year in Diabetes and Obesity

Early life nutrition and metabolic programming

Denise S. Fernandez-Twinn and Susan E. Ozanne
Department of Clinical Biochemistry, University of Cambridge, Metabolic Research Laboratories, Institute of Metabolic
Sciences, Addenbrookes Hospital, Cambridge, United Kingdom

Address for correspondence: Denise S. Fernandez-Twinn, Ph.D., Department of Clinical Biochemistry, University of
Cambridge, Metabolic Research Laboratories, Institute of Metabolic Sciences, Addenbrookes Hospital, Cambridge, United
Kingdom. df220@cam.ac.uk

Research investigating the early programming of adult metabolic disease has in recent years provided much mecha-
nistic insight into how the early environment impacts on long-term health. It includes studies addressing the roles
of intrauterine nutrient availability, which is determined by maternal nutrition, maternal exposure to oxygen, toxic
events, and infection; the placental interface; and also the early postnatal environment. This review will explore
the epidemiological evidence for programming of metabolic disease and provide an overview of the various studies
using animals to model metabolic phenotypic outcome. It will also discuss evidence for the proposed molecular
mechanisms and the potential for intervention.

Keywords: metabolic syndrome; insulin resistance; early life nutritional programming; insulin signaling

The metabolic syndrome phenotype as fuel. Type 2 diabetes is an age-related disease,

Metabolic syndrome presents as a combination of a
variety of symptoms, which include fasting hyper-
glycemia, impaired fasting glucose, impaired glu-
cose tolerance or insulin resistance, systemic inflam-
mation, hypertension, central obesity (also known
as visceral, male-pattern or apple-shaped adiposity),
decreased HDL cholesterol, and elevated triglyc-
erides. While there is some disagreement regarding
causality and the relative importance of each of these
criteria in the formal definition of the metabolic syn-
drome, it is undisputed that the extent of this disease
is far-reaching, with prevalence in the US estimated
at 25% of the population.1 Equally important is the
associated risk for cardiovascular disease.
Of the many features outlined, impaired glu-
cose homeostasis is one of the major components
of metabolic syndrome. Islet beta cell dysfunction
and/or resistance to the actions of insulin in skeletal
muscle, liver, and adipose tissue are instrumental
in the development of type 2 diabetes. Insufficient
insulin production by the beta cells of the islets and
its secretion results in beta cell dysfunction while
insulin resistance is characterized by incomplete up-
take and storage of glucose and amino acids for use
generally occurring in individuals over the age of
40; however, in some ethnic groups, such as South
Asian and African-Caribbean, it often appears af-
ter the age of 25. An increasing number of children
are also being diagnosed with the condition. More
than 2.6 million people have been diagnosed with
the disease, and the number is predicted to rise to 4
million by 2025.2
The concept of programming
From the moment of conception, an organisms fu-
ture physiological and even psychological attributes
are being defined by the conditions in which it is
growing. Early blastocyst cell division, implantation
into the maternal endometrium, trophoblastic inva-
sion, and subsequent patterning are all orchestrated
by the quantity and quality of nutrition obtained
through the maternal-fetal interface, that is, the pla-
centa. Fetal demands that are not met by maternal
nutrient supply can hinder the fetal growth trajec-
tory,3 which could have consequences for long-term
health. Conversely, maternal substrate availability
that exceeds fetal requirements can also superim-
pose deviations in normal growth patterns and thus

doi: 10.1111/j.1749-6632.2010.05798.x
78 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
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Fernandez-Twinn & Ozanne
confer disease outcomes over the life course. Fetal
programming describes the in-utero phenomenon
through which these deviations are hard-wired into
every cell in the organism, and thereby determines
future disease susceptibility. Catastrophic events in
history such as the Dutch Hunger Winter and the
Leningrad Siege, during which rations were strictly
imposed on all sections of the population including
pregnant and nursing mothers, presented the op-
portunity to study associations with chronic adult
disease in babies born around the period. These
studies stress the importance of the pre- and early
postnatal environment in growth and development,
and that the timing of an insult or deviation from
the norm is as important as the insult itself in deter-
mining (a) the organ system(s) affected and (b) the
onset and severity of disease outcome.
Since these initial studies, a large number of
epidemiological studies have consistently demon-
strated the relationship between poor fetal and
early postnatal growth and the development of
adult diseases. Using proxy indices based on records
of birth weight, length, abdominal and head cir-
cumference, placental weight, and various relative
indices as markers of in-utero growth, numerous
epidemiological studies have explored the relation-
ships between early human growth and suscepti-
bility to adult disease. Since there is still no single
comprehensive test to identify if a newborn has at-
tained its full growth potential in-utero, these crude
measures are still widely used. Hence, strong as-
sociations between low birth weight and increased
risk of metabolic disorders are well documented.
Cardiovascular disease,4 type 2 diabetes,5 central
adiposity,6 abnormal lipid metabolism and hy-
pertension,7 and increased risk of death from is-
chemic heart disease8,9 have all been consistently
linked to birth weight. The associations with acute
myeloid leukemia10 and certain cancers such as
testicular cancer11 are much weaker (probably at
least in part due to low sample number and effect
size), while associations with esophageal adenocar-
cinoma12 is thought to be explained by increased
gastroesophageal reflux during infancy among in-
fants born preterm and/or small for gestational age
(SGA). Birth weight associations with breast cancer
are seemingly contradictory, as most studies show
increased risk with increasing birth weight,13,14
while some other studies regarded risk as increased
in a J-shaped association, that is, in both the low-
Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
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Early life programming
est and highest birth weight individuals.1517 An
increased risk of reproductive disorders such as
polycystic ovary syndrome at adolescence, which
is related to premature adrenarche in girls18 as
well as hypospadia and cryptorchidism in boys,19
is also linked with low birth weight. A few stud-
ies have suggested that the increased risk of end-
stage renal disease20 in low birth weight individuals
stems from impaired nephron development result-
ing in reduced glomerular number and increased
glomerular volume.21 Finally, while links between
lower neuropsychological performance22 and inci-
dence of schizophrenia23 and low birth weight have
been shown, some of these observations have been
suggested to be confounded by maternal psychi-
atric disorders and paternal age,24 with maternal
schizophrenia being associated with an increased
risk of low birth weight. Schizophrenia is also related
to increased risk of febrile seizures, which may be ge-
netically transmitted through either parent.25 Fur-
thermore, some factors relating to low birth weight
such as prenatal exposure to maternal smoking, ma-
ternal alcohol and coffee consumption, maternal
binge drinking, maternal stress, and preeclampsia
were shown to play little or no causal role in the
etiology of febrile seizures.26
Early growth and metabolic disease:
current hypotheses
The relationship particularly between poor early
growth and the development of type 2 diabetes and
the metabolic syndrome is robust, as it has been ob-
served in a wide range of populations and in many
different ethnicities.27,28 The mechanisms underly-
ing this relationship and the relative contributions
of genes and the environment and the interactions
between the two, however, are much debated.
Genes: fetal insulin hypothesis
Rare mutations in the glucokinase gene have been
shown to be associated with low birth weight and the
development of a rare monogenic form of diabetes,
maturity onset diabetes of the young (MODY).29
This data formed the basis of the Fetal Insulin
Hypothesis that suggests the relationship between
birth weight and type 2 diabetes arises because of
polymorphisms/mutations in genes such as those
implicated in insulin secretion causing both re-
duced fetal growth and increased risk of type two
diabetes. Since this finding, rapid advances in
79
Early life programming
sequencing technology have facilitated a revolution
in genome wide association studies and large Eu-
ropean cohort studies have identified genetic vari-
ants associated with birth weight.30 More recently,
one study demonstrated the association of Single
Nucleotide Polymorphisms within adenylate cy-
clase type 5 (ADCY5), prospero homeobox pro-
tein 1 (PROX1), glucokinase (GCK ), glucokinase
regulator (GCKR) and diacylglycerol kinase beta-
transmembrane protein 195 (DGKB-TMEM195)
with Type-2 diabetes31 and in the second, vari-
ants at the ADCY5, vacuolar protein sorting 13 ho-
molog (VPS13C), GCKR and transcription factor
7-like (TCF7L2) loci correlated with 2-h glucose.
These provide evidence that the well-described as-
sociation between low birth weight and subsequent
type 2 diabetes has a genetic component, distinct
from the proposed role of programming by maternal
nutrition.
Maternal environment and the developmental
origins of health and disease
Nutrient restriction. The in-utero environment is
extremely labile to maternal influence, which could
therefore play a crucial role in fetal growth. The ev-
idence for this comes from epidemiological studies
demonstrating associations between various differ-
ent types of maternal exposures on birth weight,
with adverse consequences for metabolic disease.
The earliest epidemiological study linking poor fetal
growth and subsequent development of type 2 dia-
betes was the observation by Hales and colleagues,5
which found that among men in their sixties, those
who had lower birth weights and weights at 1 year
were more likely to develop poor glucose tolerance
and type 2 diabetes. These findings led to the pro-
posal of the thrifty phenotype hypothesis,32 which
suggests that fetal malnutrition may induce physio-
logical and/or metabolic adaptations to ensure nu-
trient supply to the most vital organs (such as the
brain) at the expense of other organs (e.g., the en-
docrine pancreas). Furthermore, such adaptations
during critical windows of development may per-
manently reset (e.g., reduced beta cell mass) or
program fetal metabolism in order to enhance its
chances of survival in what it expects to be a poor nu-
tritional environment (Fig. 1). This program of phe-
notypic adjustment selectively maximises metabolic
thrift; however, this advantage becomes detrimen-
tal to the programmed individual/offspring when it
80 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
Fernandez-Twinn & Ozanne
meets with nutritional abundance in later life. Fur-
ther studies supporting this hypothesis followed. In
the Dutch Hunger Winter study, reduced glucose
tolerance was observed in adults who were in-utero
at the time of strict food rationing towards the end
of World War II.33 This effect was most marked in
individuals who were in their third trimester during
the period of the famine, which added a temporal di-
mension to the severity of disease. Further strong ev-
idence for the importance of the fetal environment
came from studies of twins,3436 which showed dis-
cordance for type 2 diabetes, with the diabetic twin
having a significantly lower birth weight than the
nondiabetic co-twin regardless of zygosity.36 Many
more reviews have since supported the association
between low birth weight and adult glucose and in-
sulin metabolism.37,38 In recent years, Gluckman &
Hanson proposed a related hypothesis, that is, the
predictive adaptive response hypothesis (PAR),39
which states that the fetus makes adaptive metabolic
adjustments based on its in-utero environment that
predicts the environment into which it is likely to
be born in order to gain a competitive advantage
after birth. The effects of the maternal environment
on the developmental path of the offspring are thus
to gain evolutionary advantage by epigenetic means
and transmission via soft inheritance40 to future
generations.
Hypoxia. Fetal hypoxia correlates with cardiovas-
cular disease in later adult life; evidence for this
comes from studies on the highland inhabitants
of Bolivia, when Giussani and colleagues showed
that a low oxygen tension was associated41 with low
birth weight and altered body shape at birth. Epi-
demiological studies in other populations living at
high altitudes in Saudi Arabia42 confirmed the ef-
fects on low birth weight. Adaptations in multigen-
erational high altitude populations (e.g., Andeans
and Tibetans), however, seem to permit higher uter-
ine artery blood flows and protect against altitude-
associated IUGR.43
Maternal anaemia/micronutrient deficiency. The
prevalence of anaemia in pregnant women is es-
timated at 41.8% globally,44 and is particularly a
problem in the developing world. Both maternal
anaemia45 and iron deficiency are associated with
infant and childhood mortality. Epidemiological
observations demonstrate a relationship between
maternal anemia and preterm delivery and low
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Fernandez-Twinn & Ozanne Early life programming

Figure 1. A schematic representation of the structural and molecular programming effects of a suboptimal in-utero environment
on early growth and subsequent development of the metabolic syndrome.

birth weight46 with reduced placental vasculariza-
tion and deregulation of fetal insulin-like growth
factor 1 (IGF-1) and corticotropin-releasing hor-
mone (CRH). It is suggested that effects of iron
deficiency or hypoxia on the fetus may be me-
diated by sympathetic activation through raised
norepinephrine, and this may serve as a stimulus
for cortisol and CRH release.47 The fetus is nor-
mally protected against excessive cortisol exposure
through placental 11--hydroxysteriod dehydroge-
nase (11HSD2), which converts cortisol to inactive
cortisone, however, evidence from animal studies
suggest that copper, zinc or vitamin E deficiency re-
duce the expression of this enzyme.48 At present,
associations between maternal iron deficiency and
metabolic consequences for offspring are indirect,
coming from studies of maternal supplementation.
Christian and colleagues recently reported their
findings on a randomized trial demonstrating the
effects of maternal antenatal/postnatal supplemen-
tation with iron-folic acid plus Vitamin A at allevi-
ating mortality,49 while a meta-anaylsis by Fall and
colleagues reported a small increase in birth weight
and a reduction in the prevalence of low birth weight
(LBW) of about 10% in pregnancies supplemented
with iron-folic acid.50
Maternal stress. In humans, glucocorticoids are
administered during late gestation to treat neona-
tal respiratory morbidity and maternal asthma. The
side effects of this treatment are growth retardation
and reduced birth weight.51 It is hypothesized that
prenatal glucocorticoids or stress mediates the pro-
gramming of the hypothalamopituitaryadrenal
(HPA) axis as an excess of glucocorticoids may
be associated with hypertension and glucose intol-
erance.52 Early studies by Vallee and colleagues53
showed that offspring of prentally stressed rat dams
had increased basal blood glucose levels, ate less,
and weighed less than their control counterparts.
Later studies54 reproduced this finding that is, ma-
ternal stress reduced body, adrenal, and pancreas
weight as well as plasma corticosterone and glu-
cose levels in term fetuses, whilst aged male rats

Ann. N.Y. Acad. Sci. 1212 (2010) 7896 

c 2010 New York Academy of Sciences. 81
Early life programming
(24 months of age) demonstrated hyperglycaemia,
glucose intolerance, and decreased basal leptin lev-
els. Furthermore, food intake following a fast was
increased. Thus, an adverse glucocorticoid environ-
ment in-utero induced a long-lasting disturbance in
feeding behavior and metabolic dysfunction.
The underlying mechanism directing glucocor-
ticoid excess is thought to be physiological varia-
tions in placental 11HSD2 activity. Altered pla-
cental glucocorticoid metabolism is associated with
nutrient restriction and hypoxia, which lead to both
intrauterine growth restriction (IUGR) and fetal
programming. Placental 11HSD2 expression and
activity is regulated in a cell- and gestational age
specific manner in both humans and baboons.55
In humans, mutations in the 11HSD2 gene have
been associated with low birth weight. In rodents,
reduced 11HSD2 activity, and increased fetal cor-
tisol levels are also associated with IUGR.56 In sheep,
maternal nutrient restriction for more than 45 days
significantly decreased placental 11HSD2 in ad-
dition to causing IUGR.57 This reduced 11HSD2
expression and activity in preeclampsia58 may be
due to a sustained placental hypoxia due to defi-
cient trophoblast invasion in the first trimester.
Mutations in the 11HSD2 gene cause low birth
weight, and these infants go on to have higher
plasma cortisol levels throughout their adult life.
Phillips and colleagues also observed this association
in three different populations in Adelaide, Australia,
and in Preston & Hertfordshire in the UK. They
found that higher cortisol concentrations within
the normal range were associated with raised blood
pressure, impaired glucose tolerance, insulin resis-
tance, and raised serum triglyceride levels, suggest-
ing intrauterine programming of the HPA axis as a
mechanism underlying the association between low
birth weight and the insulin resistance syndrome in
adult life.59
Mismatch between poor in-utero growth and post-
natal growth. The influences of restricted fetal
growth need to be examined with reference to the
equal importance of postnatal nutrition, another
component of the Thrifty Phenotype hypothesis.
When cases in the epidemiological studies of both
the Hertfordshire and Dutch Hunger Winter were
examined further, it was found that the individuals
with the worst glucose tolerances were those who
were born small and became obese as adults5,33 Fur-
82 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
Fernandez-Twinn & Ozanne
ther studies across many continents have confirmed
the importance of postnatal growth in the program-
ming of metabolic syndrome in South African,60
Finnish,61 and Indian62 cohorts, with the findings
that low birth weight babies that underwent rapid
postnatal weight gain demonstrated the worst glu-
cose tolerance and were therefore the most likely to
develop type 2 diabetes in adulthood. The worsen-
ing of glucose tolerance in children who were born
small but then had the highest weight gain corre-
lated to the greatest beta cell secretory response,
and those who were the most insulin resistant.63 In
contrast, children born lighter and whose weight
remained low had the lowest beta cell secretory re-
sponse. Both the immediate postnatal period and
the first few years of childhood are attributed as the
phases during which accelerated growth has added
detrimental effects for metabolic disease.64
Gestational diabetes. Gestational diabetes (GDM)
can occur due to a pre-existing diabetic state or the
development of glucose intolerance during preg-
nancy. Depending on the severity of GDM, babies
may either be macrosomic or growth restricted and
it is thought that the placenta plays an important
role since it has been shown that placental size in
women diagnosed with GDM is inversely related to
protein intake.65 Epidemiological studies on cases of
diabetes in the Netherlands,66 Malta,67 Pima Indi-
ans,68 and African-Americans69 have demonstrated
a U-shaped relationship with birth weight, so that
macrosomia poses as much a risk for metabolic dis-
ease as low birth weight. There is also a higher sus-
ceptibility for diabetes in descendants of diabetic
great grandmothers via the maternal line, with a
higher prevalence of impaired glucose tolerance,
type 2 diabetes and gestational diabetes occurring
in children whose mothers had diabetes during that
pregnancy.68 Gestational diabetes per se is an induc-
ing factor for impaired glucose tolerance and ges-
tational diabetes in the next generation; the altered
metabolic milieu of the diabetic pregnancy causing
permanent defects in glucose homeostasis in the off-
spring leading to the development of diabetes later
in life. This forms the basis of a mechanism by which
gestational diabetes passes from one generation to
the next.
Maternal obesity. Pregnancies during which
mothers are obese or overeat70 can also result
in increased infant birth weight and increased
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Fernandez-Twinn & Ozanne
susceptibility to metabolic and related diseases in
adult life. Maternal obesity and gestational weight
gain significantly impact on maternal metabolism
with effects on maternal insulin sensitivity, glu-
cose tolerance and lipid metabolism, and the de-
livery of either a SGA or a large for gestational age
baby.70 Pregravid BMI > 30 was shown to be a very
strong predictor of childhood obesity body mass in-
dex (BMI).71 In addition, pregnancy weight gain72
and consumption of six glasses of cows milk per
day during mid-pregnancy73 were also shown to be
independent determinants of macrosomia. In the
latter study, increased birth weight was related to
the increased protein intake in the milk rather than
fat. BMI and pregnancy weight gain prior to preg-
nancy are also strong predictors of gestational dia-
betes, which could be a mechanism linking maternal
obesity and over-nutrition to high birth weight and
metabolic disease consequences for offspring.74
Healthy pregnancy is associated with lipid mo-
bilization (increases in plasma cholesterol and
triacylglycerols), changes in insulin sensitivity (a
gestational increase in insulin resistance) and im-
provement in endothelial function.75 In mater-
nal obesity, there is an exaggerated lipid response,
greater degree of insulin resistance76 and less
improvement in endothelium-dependent microvas-
cular function.77 The relative endothelial dysfunc-
tion in obese pregnancy is in part explained by al-
tered levels of plasma cytokines (interleukin-6 and
-10) with obesity.78 Lipid peroxides are increased
in healthy pregnancy,79 but few findings are avail-
able on oxidative stress in obese pregnancy. An as-
sociation between maternal BMI and also mater-
nal cholesterol levels and the oxidizability of low
density lipoprotein (LDL) has been observed.80 In
nonpregnant type 2 diabetics, high plasma oxys-
terol levels are particularly observed when plasma
LDL levels are above approximately 3.6 mmol/l, a
level exceeded in overweight and obese women.81
Although the literature on oxysterols during preg-
nancy is extremely limited, gestational increases and
further increases with maternal diabetes have been
reported.82
Role of the placenta
Placental dysfunction is implicated as a major cause
of human IUGR and programming of adult car-
diovascular and metabolic disease.83 The placenta
regulates fetal growth and development by facilitat-
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Early life programming
ing nutrient and gas transport, and by the synthesis
and secretion of steroid and peptide hormones. How
well it does this is dependent on vascular develop-
ment and blood flow and by growth and differenti-
ation of the trophoblast, which contains receptors,
transporters, and enzymes.84,85 Epidemiological ev-
idence has linked low birth weight and low pla-
cental weight to fetal programming. Alterations in
expression of glucose and amino acid transporters
in the placenta have been reported in pregnancies
complicated by diabetes, IUGR, and preeclampsia86
with hyperglycaemia in the first trimester result-
ing in up-regulation of glucose transporters, lead-
ing to accelerated fetal growth in late gestation. Nu-
merous excellent studies by Fowden and colleagues
have meticulously detailed the morphological and
functional adaptations of the placenta in adverse in-
trauterine conditions.87 Depending on the model of
IUGR and the severity of the insult, the consensus
from the literature is that placental weight is gener-
ally reduced in parallel with fetal weight; however,
placental efficiency, as assessed by grams fetus per
gram placenta, is largely variable. This suggests that
placental efficiency is determined in part by genet-
ics, and in part, by its response to the pregnancy
environment. The response to maternal under nu-
trition for example, is an increase in placental ef-
ficiency during the early growth spurt (gestational
days 1516), and then a decline in efficiency closer
to term.88,89 However, the severity of the insult also
affects placental efficiency as seen in cases of more
severe restriction that is, a 4% protein diet during
pregnancy, where placental efficiency was reduced
at day 21 gestation86 compared to intake of a 8%
protein diet, which resulted in increased placental
efficiency.90,91 Morphologial adaptations to surface
area, thickness of the barrier between the maternal
and fetal circulations, and the density and archi-
tecture of the vasculature contribute to improved
efficiency and these are species dependent. How-
ever, in addition to this are transporter-mediated
processes which are determined by abundance, ac-
tivity and localization of transporters as well as sim-
ple and facilitated diffusion which is affected by the
materno-fetal concentration gradient across the pla-
centa.92 The expression of a range of glucose (Slc2a1
and 3, GLUT1, GLUT3) and amino-acid trans-
porters (Slc38a2 and 4) have been found to be up-
regulated in various models of IUGR (as reviewed by
Fowden et al.87 ). What drives these morphological
83
Early life programming
and functional modifications in the placenta is im-
portant as these could potentially act as biomarkers
of IUGR, and may also serve as a route to interven-
tion. Concentrations of maternal glucocorticoids,
IGF-1, leptin, and insulin have all been found to
be associated with IUGR.91,93,94 In the mouse, dele-
tion of the Igf2 gene leads to placental and fetal
growth retardation, and conversely, overexpression
causes overgrowth.95 All these are likely candidates
for modifying placental efficiency.
Martin and Sutherland96 showed that embryos
cultured in medium lacking amino acids were un-
able to form trophoblast cell outgrowths necessary
for implantation and that mTOR is required for the
initiation of this trophectoderm protrusive activity.
Wen and colleagues later showed that mTOR func-
tions as a placental growth signaling sensor.97 Hu-
man studies are in agreement as mammalian target
of rapamycin (mTOR) protein is expressed in the
transporting epithelium of the human placenta. In
these studies,98 the protein expression of placen-
tal phospho-S6K1 (Thr-389), a measure of active
mTOR signaling was markedly reduced in IUGR
placentas.
One other means of regulating placental func-
tion is through oxidative stress. As the placenta is a
mitochondria-rich tissue, it generates reactive oxy-
gen species (ROS), even in a normal pregnancy,
which consigns it to a state of continuous oxida-
tive stress.99 This is increased in pregnancies com-
plicated by preeclampsia, IUGR, and pregestational
diabetes as measured by increased markers of pro-
duction of ROS and decreases in antioxidant de-
fences.100 The defective trophoblast invasion seen
in preeclampsia or IUGR is thought to lead to a
relative hypoxia,101 which then leads to a height-
ened inflammatory response and thereby increases
oxidative and nitrative stress. This is hypothesized
to alter placental function via covalent modification
of protein structure and function102 and hence fetal
growth and development.
Animal models of programming
The following section provides a snapshot of re-
cent and current lines of research that have aimed
to dissect the physiological and molecular mech-
anisms underlying the programming of metabolic
disease. The major rodent animal models used to
identify mechanisms of metabolic programming
and observed offspring phenotypes are outlined in a
84 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
Fernandez-Twinn & Ozanne
schematic in Figure 1. Large animal models of IUGR
have also been studied in depth,103 and although
these will not be discussed here, it is noteworthy
that they share many similarities with the rodent
models that will be deliberated in greater detail.
Rodent models and underlying mechanisms
Maternal calorie restriction. Total maternal calo-
rie restriction to 50% of ad libitum intake during the
last week of pregnancy results in impairment of beta
cell development.104 Continued calorie restriction
during lactation results in a permanent reduction
in beta-cell mass and number, and impaired glucose
tolerance in the offspring.105 A more severe food re-
striction to 30% of ad libitum intake resulted in
systolic hypertension and increased fasting insulin
concentrations, hyperphagic behavior, and obesity
in the offspring.106 When treated with IGF-I, this
phenotype was ameliorated107 while neonatal lep-
tin administration from day 3 to day 13 of postnatal
life normalized caloric intake, locomotor activity,
body weight, fat mass, and fasting plasma glucose,
insulin, and leptin concentrations in adult life.108
Postnatal consequences for the HPA axis include re-
duced adrenal weight, reduced basal corticosterone
as well as reduced glucocorticoid receptor (GR) and
CRH mRNA levels. Similar findings were observed
by Dumortier and colleagues109 with fetuses of dams
fed a low energy diet demonstrating 3356% reduc-
tion in beta cell mass and increased corticosterone
levels. Furthermore, the number of cells producing
neurogenin 3 (NEUROG3) or pancreatic and duo-
denal homeobox gene 1 (PDX-1) was also reduced.
Maternal protein restriction. The low protein (LP)
model of programming is the best characterized
of all developmental programming models. The
Hoet group first implemented the model,110 which
corresponds with the levels of protein deprivation
observed in countries with poor socio-economic
conditions. The LP diet, which is fed only to the
rat mothers, consists of 8% casein protein and is
isocaloric with the control diet, which contains 20%
casein protein by comparison. Offspring suckle on
their respective mothers milk and then are weaned
onto standard chow, which contains 20% protein.
The offspring are growth restricted, as characterized
by low birth weight and reduced placental weights,
immediately demonstrating its relevance for study-
ing human growth restriction. Others have since
replicated this gross morphological phenotype.91,111
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Fernandez-Twinn & Ozanne
At the tissue level, neonate beta cell proliferation
and islet size was reduced,112 and islet vasculariza-
tion was also reduced.109 Our group and others have
extensively studied the adult phenotype also. LP off-
spring at first show an improvement in their glucose
tolerance up to 3 months of age. They undergo an
age-dependent loss of glucose intolerance such that
by 15 months of age, they have impaired glucose
tolerance and insulin resistance and finally, frank
diabetes by 17 months of age.113 The insulin resis-
tance is associated with a reduced expression of key
insulin signaling proteins, including the p110 cat-
alytic subunit of phosphatidyl inositol 3-kinase in
adipose tissue114 and protein kinase C- in skele-
tal muscle.115 The further relevance of this model
to humans at the molecular level is the strikingly
similar profile of insulin signaling protein expres-
sion in muscle and adipose tissue from LP offspring
compared to those in tissues from humans with a
low birth weight.116,117 That the differences in pro-
tein expression occur prior to development of in-
sulin resistance, suggests that they are not conse-
quences of hyperinsulinemia or hyperglycemia but
play a role in determining future susceptibility to in-
sulin resistance, cardiovascular disease, and type 2
diabetes.
Catch-up growth. The respective roles of the preg-
nancy and lactation period have been dissected by
imposing the LP diet only during either of these pe-
riods through crossing-over techniques in rodents.
The offspring of rat dams fed a LP diet (8%) dur-
ing pregnancy have a 15% reduction in birth weight
compared to control offspring of dams fed a 20%
diet. If these offspring are then cross-fostered to 20%
protein fed dams from birth, they undergo rapid
catch-up growth during the lactation period. The
most startling consequence was a 25% reduction in
life span in the recuperated male rats.118,119 The
major cause of death in male rats is reported to
be kidney failure and consistent with this, reduced
longevity in the recuperated animals was associated
with shorter kidney telomeres,119 although shorter
islet telomeres and increased markers of cell senes-
cence have also been observed in these animals.120
Similar findings were observed in mice121,122 and
these studies also revealed an interaction with the
postweaning diet such that an obesity-inducing diet
further reduced the longevity of both control and
recuperated mice. Conversely, mice offspring that
Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
c 2010 New York Academy of Sciences.
Early life programming
received 20% protein in-utero and were suckled by
LP mothers, lived longer and contrary to the recu-
perated and control mice, were protected from the
detrimental effects of the obesogenic diet to reduce
longevity.123
The metabolic effects of catch-up growth have
also been studied in different models. In mice off-
spring that were exposed to 50% food restriction
in-utero followed by catch-up growth, increased
adiposity was primarily associated with lipogene-
sis rather than adipogenesis.124 However, in a study
of forced catch-up growth following fetal protein re-
striction that showed similar increases in relative fat
mass and adipocyte hypertrophy, along with hyper-
glycemia, hypercholesterolemia, and hyperleptine-
mia, Bol and colleagues125 reported a contradictory
down-regulation in mRNA expression of enzymes
involved in lipogenesis, although there was a con-
comitant up-regulation of proteins involved in fatty
acid uptake that could contribute to hypertrophy of
fat cells.
In a similar model, Sutton and colleagues126
found abnormal daily rhythms in food intake and
metabolism, increased lipogenesis, and inflamma-
tion prior to increased obesity. At the molecular
level, arrhythmic expression of circadian oscillator
genes in brain, liver, and muscle was evident as early
as 8 weeks of age (preobese); in particular the expres-
sion of the clock-associated nuclear receptor and
transcription repressor Rev-erb was reduced, with
concordant increased expression of its downstream
targets Bmal1 and Per2 in liver and muscle. This
disruption in circadian rhythm is a potential mech-
anism for altered feeding behavior and substrate
metabolism, which may contribute to an increased
diet-induced obesity and insulin resistance by 20
weeks of age. It remains unclear, however, due to the
lack of a group that was not cross-fostered at birth,
whether this disruption is programmed in-utero or
during catch-up growth.
Maternal micro-nutrient restriction. Early studies
in rats showed that insulin and glucagon levels in the
fetus were affected by maternal zinc deficiency.127
More recent studies have shown offspring of zinc
restricted dams weighed less than control offspring
at birth and weaning and up till 6 months of age.128
Their percentage of body fat was increased with a
concomitant decrease in lean mass. Fasting plasma
insulin levels in both male and female offspring at
85
Early life programming
6 months of age were lower compared to controls,
which was related to a decreased glucose-induced
insulin secretion in females.
Maternal iron restriction in a rat model has been
shown to reduce birth weight and to elevate off-
spring blood pressure. This was suggested to be
due to a deficit in nephron number.129 As with
the protein restriction model, glucose tolerance was
improved at 3 months of age, however, an age-
related loss of glucose tolerance was observed in
14-months-old rats, and it was suggested that this
may deteriorate with advancing age.130
Maternal hypoxia. Illsley and colleagues have re-
cently hypothesized that hypoxic conditions induce
metabolic reprogramming in the placenta such that
mitochondrial oxygen consumption is reduced and
anaerobic glucose consumption increases, thus al-
tering the mix of fuel substrates available for trans-
fer to the fetus.131 Placental oxygen has been shown
to be preserved under maternal hypoxia;132 how-
ever, this may be at the cost of less glucose availabil-
ity, leading to hypoglycemia-mediated fetal growth
restriction.
Intauterine artery ligation. It is now well estab-
lished that a large proportion of human intrauter-
ine growth retardation is due to impaired nutrient
perfusion through the placenta.133 Both unilateral
and bilateral uterine artery ligation models of pla-
cental insufficiency replicate the phenotype of re-
duced offspring birthweight and observations have
included an altered development of the pancreas,
with offspring at birth having a reduced beta cell
mass that persists into adulthood.134 This leads to
the development of type 2 diabetes at 26 weeks
of age.135 Other effects of this model include re-
duced nephron number and impaired renal func-
tion136 and a 20% decrease in glomerular number
with compensatory glomerular enlargement associ-
ating with an increased proteinuria.137 The effects of
uterine ligation are transmitted to the second gen-
eration as seen in female offspring of uterine ligated
dams, which have reduced beta cell mass and be-
come diabetic during pregnancy, whereupon their
offspring, the second generation, are heavier at birth
and remain heavy throughout life, develop insulin
resistance very early in life, demonstrate defects in
insulin secretion from as early as 5 weeks of age and
by 26 weeks of age are overtly diabetic.138
86 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
Fernandez-Twinn & Ozanne
Maternal glucocorticoid exposure. Prenatal glu-
cocorticoids or stress are hypothesized as a mech-
anism linking reduced fetal growth with adult
metabolic syndrome disease. This has been tested in
rats where the treatment of pregnant rats with dex-
amethasone resulted in reduced birth weight, with
subsequent catch up in weight compared to controls
by weaning.139 In adulthood, offspring were hyper-
tensive and hyperglycemic, concurrent with an in-
creased HPA axis activity and behavior reminiscent
of anxiety.140 The underlying mechanism directing
glucocorticoid excess is thought to be physiological
variations in placental 11HSD2 activity.141 In hu-
mans, 11HSD2 gene mutations result in low birth
weight with consequences for higher plasma cortisol
levels in adult life, a clear programming effect on the
HPA axis. The effects are apparently gender-specific
such that males exhibit a metabolic phenotype139
whereas females are hypertensive and have an acti-
vated reninangiotensin system.142
Gestational diabetes. Experimental gestational di-
abetes may be induced by streptozotocin treatment
of pregnant dams. This chemical destruction of
islet beta cells of the pancreatic islets can be reg-
ulated such that a low dose induces mild gesta-
tional diabetes resulting in fetal hyperinsulinemia
and macrosomia. Conversely, a high dose causes
severe maternal hyperglycemia resulting in hyper-
stimulation of fetal islets followed by beta cell de-
granulation, disorganization, and finally secretory
failure. The result is a low birth weight pup.143 One
significant finding was that both extremes of birth
weight converged on insulin secretory function and
impaired glucose metabolism. Offspring of both
the mild and severely diabetic streptozotocin mod-
els subsequently develop gestational diabetes them-
selves,144 which is further transmissible to the third
generation.
Maternal high fat feeding/over-nutrition. With
the prevalence of obesity rising to over a third
of the population in some developed nations, it
has become increasingly relevant and important
to study models of obesity and particularly the
consequences of obesity on the health of future gen-
erations. Recent years have seen much study dedi-
cated to the characterization of the phenotype of ro-
dent models of maternal obesity. One of the first of
these demonstrated the development of hyperpha-
gia, adiposity, hypertension, and insulin resistance
c 2010 New York Academy of Sciences.
Fernandez-Twinn & Ozanne
in the offspring between 3 months and 6 months
of age.145 Further work showed an attenuation
of leptin-induced appetite suppression and phos-
phorylation of signal transducer of transcription 3
(STAT3) in the arcuate nucleus (ARC) and a re-
duction in AgRP-immunoreactivity in the hypotha-
lamic paraventricular nucleus (PVH) at postna-
tal Day 30. These differences are therefore present
prior to the hyperphagic response and suggests lep-
tin resistance as a potential mechanism.146 Tissue
specific insulin resistance and non-alcoholic fatty
liver disease (NAFLD) in liver has been observed
in offspring challenged with a high fat diet post-
weaning.147 In skeletal muscle, the expression of
molecules involved in insulin signaling were altered
and mitochondrial-linked complex II-III activity
was decreased, which could contribute to a state
of insulin resistance.148
Other mechanisms
Permanent structural changes
Environmental factors at critical periods of devel-
opment could have long-term phenotypic conse-
quences through permanent structural changes in
key organs. Thus, inappropriate concentrations or
a temporal shift in a particular nutrient or hor-
mone necessary for the development for growth
and differentiation of a tissue may have permanent
structural consequences. There are several exam-
ples in rodent models of developmental program-
ming to suggest that the perinatal environment can
have large and lasting effects on brain development.
Neonatal treatment of rat pups during the second
week of life with high levels of insulin induces per-
manent changes in hypothalamic morphology, with
a reduced neuron density within the ventromedial
hypothalamus,149 a region implicated in regulation
of satiety. Structural alterations in this area of the
brain could therefore contribute to the excess weight
gain and glucose intolerance in these rats neonatally
treated with insulin. Leptin derangements during
perinatal life have been implicated as an important
programming factor.150 The early neonatal plasma
leptin surge has been very well characterized in both
rats and mice; however, the functional significance
of this surge is little known, with effects distinct from
those observed for adults in which leptin affects food
intake and energy expenditure. Instead, neonatal
leptin has a neurotrophic role as evidenced by its
ability to stimulate neurite outgrowth from arcu-
Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
c 2010 New York Academy of Sciences.
Early life programming
ate nucleus explants from d4-old neonatal mice.151
In-vivo, daily leptin administration to d4 leptin defi-
cient ob/ob mice was able to restore arcuate nucleus
projections to the paraventricular nucleus to wild-
type levels.
Molecular mechanisms
Oxidative stress and mitochondrial mechanisms.
It is now widely known that oxidative damage
accelerates telomere shortening and thus cellular
aging or senescence. Mitochondria, as the principal
energy generators (ATP) of the cell, generate large
amounts of ROS, such as superoxide anions and
hydroxyl radicals and are therefore the primary me-
diators of cellular oxidative damage. These oxygen
radicals are capable of damage to protein, lipids,
and DNA. Disruption in mitochondrial electron
transport chain (ETC) enzymes can lead to further
ROS generation,152 which in turn can contribute to
accelerated telomere length shortening and cellular
senescence under physiological conditions.153 Islets
isolated from aged rat offspring (15 months) of
dams protein restricted during pregnancy and
lactation (LP model),154 demonstrate increased
xanthine oxidase expression, which is indicative of
increased oxidative-stress. Furthermore, islet
antioxidant defence capacity was also impaired as
evidenced by a reduced expression of antioxidant
enzymes manganese superoxide-dismutase, copper-
zinc superoxide-dismutase, and heme oxygenase-1.
Recent work by Theys and colleagues155 showed
that male LP offspring demonstrated increases in
ROS production by islet mitochondria and this was
associated with higher expression of mitochon-
drial subunits of the ETC NADH-ubiquinone
oxireductase subunit 4L and peroxisome
proliferator-activated receptor-gamma and uncou-
pling protein-2 over-expression. It is suggested
that this could explain the earlier development
of glucose intolerance in male LP offspring com-
pared to females156 since this was only seen in
males.
In the rat, uterine artery ligation induces de-
creased pyruvate oxidation in muscle tissue of
young offspring, which leads to a chronic reduc-
tion of ATP derived from oxidative phosphoryla-
tion. Glucose transporter 4 (GLUT4) recruitment,
glucose transport, and glycogen synthesis are com-
promised, which contributes to insulin resistance
and hyperglycemia.157 Mitochondrial oxidative
87
Early life programming
phosphorylation was also found to be impaired in
the liver158 and in fetal islet cells, mitochondrial
dysfunction leads to increased ROS production, re-
duced ATP production and impaired mitochon-
drial ETC complex I and III activities. This in turn
damages mitochondrial DNA leading to progressive
deterioration of beta cell function.159 The similar-
ity in mitochondrial dysfunction between the LP
and uterine ligation models strongly suggests that
mitochondrial dysfunction plays a key role in the
metabolic phenotype of the offspring.
In a maternal diet-induced obesity model, mi-
tochondrial complex II-III linked activity was re-
duced in skeletal muscle of male offspring.148 A re-
cent study showed that mouse oocytes and zygotes of
dams exposed to diet-induced obesity demonstrated
increased mitochondrial potential, mitochondrial
DNA content, and biogenesis,160 with evidence for
raised ROS generation and oxidative stress, leading
to compromised mitochondrial metabolism very
early in development.
Epigenetics of programming
Epigenetic mechanisms provide a tantalizing means
of explaining how the early life environment may
affect long-term chronic disease susceptibility. Epi-
genetic dysregulation is increasingly implicated in
cancer and many cancer types display hallmark epi-
genetic changes including histone modifications,
gene specific hypermethylation, and genome-wide
hypomethylation. Of these, global hypomethylation
is suggested as occurring early in transformation
and also important in tumor progression. The evi-
dence for this comes from several studies showing
global hypomethylation in the adjacent healthy tis-
sue suggesting this change is an early event in the
transformation process, resulting in karyotypic
instability and further oncogenic processes.161,162
Controversially, several other investigations have
failed to detect reduced global methylcytosine lev-
els in benign lesions or during malignant tumor
progression,163165 bringing into question the causal
nexus of genome-wide hypomethylation in carcino-
genesis, and suggesting instead that it is a passive
inconsequential side effect of carcinogenesis.166 Hy-
pomethylation has also been observed in neuro-
developmental disorders, for example, a reduction
in methyl CpG binding protein 2 (MeCP2) expres-
sion compared to age-matched controls was found
in 11/14 autism (79%), 9/9 Rett syndrome (100%),
88 Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
Fernandez-Twinn & Ozanne
4/4 Angelman syndrome (100%), 3/4 Prader-Willi
syndrome (75%), 3/5 Down syndrome (60%), and
2/2 attention deficit hyperactivity disorder (100%)
frontal cortex samples.167 One of the earliest re-
ports that demonstrated methylation as mediat-
ing the effects of early nutrition on adult disease
susceptibility was provided by Waterland and Jir-
tle.168 More recently, Waterland and Jirtle showed
that maternal obesity accumulated over successive
generations shifted the population distribution to-
ward increased adult body weight, and suggested
that epigenetic mechanisms are involved in this pro-
cess.169 Spurred on by the concept that epigenet-
ics would provide the fundamental mechanisms of
early-life programming170 and therefore a means
of intervention, many groups have invested in high-
throughput methylation analyses. Global changes in
DNA methylation have been observed in sheep that
experienced alterations in vitamin B and methion-
ine during the periconceptional period.171 There is
also evidence to suggest that the postnatal period is a
critical window in mammalian developmental epi-
genetics as demonstrated by developmental changes
in promoter methylation, which are associated with
expression changes in liver tissue from embryonic
day 17.5 to postnatal day 21.172
Maternal protein restriction has been shown to
alter the methylation status of the promoters of the
GR,173 PPAR,174 and the angiotensin receptor,175
which parallelled changes in gene expression. More
recent studies have shown that histone modifica-
tions may also be influenced by the early environ-
ment. Both DNA methylation and histone acetyla-
tion changes in the PDX-1 promoter are observed
in pancreatic beta cells of offspring of intrauter-
ine artery ligation pregnancies.176 These epigenetic
changes in the neonatal period were reversible by
histone deacetylase inhibition. However, after dia-
betes onset, the PDX-1 locus was permanently si-
lenced through the methylation of the CpG island
in the proximal promoter. Using a genome-wide
approach and whole islets isolated from male IUGR
rat offspring at 7 weeks of age (preceding diabetes),
Thompson and colleagues found dysregulated cy-
tosine methylation at 1400 loci, preferentially at
conserved intergenic sequences, and frequently near
genes regulating islet vascularization, beta cell pro-
liferation, insulin secretion, and cell death. They
also showed these to be associated with concor-
dant changes in mRNA expression thus representing
c 2010 New York Academy of Sciences.
Fernandez-Twinn & Ozanne
candidate loci for mediating the pathogenesis and
progression of metabolic disease in later life.177 Al-
terations in histone modifications are also impli-
cated in mediating the effect of caloric restriction
in the second trimester, with a programmed reduc-
tion of GLUT4 expression in the offspring.178 Hu-
man studies have also yielded evidence for the effect
of maternal diet on epigenetic marks in individ-
uals who were exposed to famine in-utero during
the Dutch Hunger Winter. White blood cells taken
from these individuals in their sixties, were found
to have less DNA methylation of the maternally im-
printed IGF2 gene compared with their same-sex
siblings, who were not exposed.179 The association
was specific for exposure during the periconcep-
tional period, reinforcing this period as a critical
window for the establishment and maintenance of
epigenetic marks. In a more recent study, cord blood
taken from IUGR neonates showed changes in cy-
tosine methylation, particularly in the hepatocyte
nuclear factor 4alpha (HNF4A) gene, a well-known
diabetes candidate gene, and in other loci encoding
HNF4A-interacting proteins.180 While both these
studies used peripheral blood, one main difference
between these studies is the timing of insult. In the
Heijman study, this was specific to the periconcep-
tal period; this was not ascertainable in the Einstein
study. It should also be noted that individuals in the
Dutch Hunger cohort were already insulin resistant
at the time of sampling, which could have affected
methylation status.
The role of methylation in complex chronic dis-
eases, such as cardiovascular disease and obesity
is beginning to emerge. One recent study demon-
strated cell-specific histone-methylation modi-
fications and expression of accompanying ly-
sine methyltransferases in the carotid arteries
of apoE/+ mice born to hypercholesterolemic
apoE/ mothers.181 Furthermore, offspring from
apoE/ mothers demonstrated differences in hi-
stone triple-methylation modifications in vascular
endothelial and smooth muscle cells in response to a
high cholesterol diet when compared with offspring
from wild-type mothers. Thus, both in-utero pro-
gramming and postnatal hypercholesterolemia alter
epigenetic events in the vasculature.
All these studies suggest that critical windows in
mammalian developmental epigenetics extend well
beyond early embryonic development. The com-
plicated interplay between the dynamics, positions,
Ann. N.Y. Acad. Sci. 1212 (2010) 7896 
c 2010 New York Academy of Sciences.
Early life programming
and functions of epigenetic marks will continue to
puzzle for years to come.
Intervention strategies
Nutritionists, clinicians, and researchers agree that
optimal nutrition during early growth is vital to
health outcome. Current advice and public health
initiatives promote healthy eating and these have
reduced the incidence of low birth weight in most
developing countries. Obesity and higher levels of
fat intake during pregnancy has emerged as a larger
problem in recent years and is potentially attributed
with the rise in cases of high birth weight, correlat-
ing with an increase in preeclampsia and obstetric
complications as well as an increased risk of fu-
ture diabetes in the mother. It has vitally important
consequences for obesity and metabolic disease in
children.
The need for therapeutic solutions to improve
glycemic control has seen the incretin Exendin-
4, a long lasting analogue glucagon-like peptide-1
emerge as an interesting candidate. Not only does it
improve insulin action through increasing beta cell
mass and function via induction of PDX-1 expres-
sion in beta cells,182 it also has effects on reducing
appetite and food intake. In the restricted placental
growth model of IUGR, a 6-day neonatal course of
exendin-4 prevented the development of diabetes in
the placentally restricted rat and normalised glucose
tolerance after birth and into adulthood.183 beta cell
mass and PDX-1 expression were also normalized
and remarkably, this effect of neonatal exendin-4
also restored the epigenetic state of the PDX-1 pro-
moter to control levels.184 The fetal rat pancreas,
however, unlike the human and sheep, does not de-
velop insulin-containing cells until the second half
of gestation, and pancreatic remodeling with peaks
in replication and apoptosis occur in the early post-
natal period at 12 weeks of age.185,186 Sheep, how-
ever, as with humans, have nearly complete pancre-
atic development before birth (reviewed By Gatford
et al.187 ). Exendin-4 treatment in neonatal lambs
from 1 day to 16 days after birth (1 nmol kg1 d1 )
was able to increase glucose-stimulated insulin se-
cretion by 155% in the twin IUGR lamb188 suggest-
ing that this period of development is amenable to
either or both of its effects that is, increasing beta
cell mass and induction of PDX-1 expression. Such
intervention may be useful in preventing future ad-
verse outcomes of the growth-restricted baby.
89
Early life programming
While recent animal studies suggest that mater-
nal dietary supplementation with methyl donors
and cofactors in late pregnancy can ameliorate the
metabolic effects of growth restriction,187 we are still
a long way from any kind of human intervention.
Summary
Taking a global perspective of all the various ani-
mal models of suboptimal early life, it is remark-
able that without exception, all of these mod-
els, sooner or later, converge on an offspring
phenotype of metabolic disease. Our understand-
ing of disease mechanisms has been enhanced by
each of these models, which meet in a crossroads
of metabolic dysregulation, with progression to as-
sociated pathologies such as cardiovascular disease,
hypertension, and cancer radiating from this point.
With recent technological advances, such as the de-
velopment of deep sequencing, answers to some of
the remaining mechanistic questions should start to
emerge.
Conflicts of interest
The authors declare no conflicts of interest.
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