THE IMPACT OF MATERNAL OVERNUTRITION DURING THE PERICONCEPTIONAL PERIOD ON THE DEVELOPMENT OF POSTNATAL OBESITY IN THE SHEEP Leewen Rattanatray B. Biomedical Sc. (Hons) Discipline of Physiology School of Molecular and Biomedical Science The University of Adelaide A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the University of Adelaide May 2010
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THE IMPACT OF MATERNAL OVERNUTRITION
DURING THE PERICONCEPTIONAL PERIOD ON
THE DEVELOPMENT OF POSTNATAL OBESITY IN
THE SHEEP
Leewen Rattanatray B. Biomedical Sc. (Hons)
Discipline of Physiology
School of Molecular and Biomedical Science
The University of Adelaide
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy to the University of Adelaide
May 2010
Table of Contents II
TABLE OF CONTENTS
TABLE OF CONTENTS ....................................................................................... II
ABSTRACT ........................................................................................................ VII
DECLARATION .................................................................................................. IX
ACKNOWLEDGEMENTS .................................................................................... X
RELATED PUBLICATIONS ............................................................................... XII
LIST OF FIGURES AND TABLES .................................................................... XIII
COMMONLY USED ABBREVIATIONS ........................................................... XVII
CHAPTER 1: LITERATURE REVIEW ................................................................. 2
Muhlhausler BS. (2009) The Early Origins of Later Obesity: Pathways and
Mechanisms. In B Koletzko,D Molnár, T Decsi and A de la Hunty (Eds) Early
Nutrition Programming and Health Outcomes in Later Life. Obesity and Beyond,
71-82
XIII
LIST OF FIGURES AND TABLES
Figure 1.1 The intergenerational cycle of obesity 15
Figure 1.2 A schematic diagram showing the sequence of events in origin, growth and rupture of the tertiary follicle and degradation of the corpus luteum 24
Figure 2.2.1 Nutritional protocol 50
Figure 2.2.2 The major transitions in structure of the bovine embryo during early embryogenesis 51
Figure 2.2.1 Blood sampling regime in the donor and recipient ewes 57
Figure 2.3.1 Effect of periconceptional overnutrition and/or dietary restriction on the weight of donor ewes during the nutritional feeding protocol from 35 weeks before conception to 1 week after conception 58
Figure 2.3.2 Effect of periconceptional overnutrition and/or dietary restriction on the change in the weight of the non-pregnant donor ewes between 25 weeks before conception to the day of embryo transfer (day 6-7 pregnancy) 59
Table 2.3.1 Donor ewe body condition scores between 5 weeks prior to conception and conception 60 Table 2.3.2 Plasma glucose concentrations in donor ewes at 5, 4 and 1 week prior to conception and at conception 61 Figure 2.3.3 Plasma insulin concentration in donor ewes between 5 weeks before conception and conception 62 Table 2.3.3 Periconceptional nutrition and/or dietary restriction and the stage of embryo development 63 Table 2.3.4 Plasma glucose and insulin concentrations in recipient ewes at 5 weeks before and 7 weeks after conception 65 Table 2.3.5 Periconceptional nutrition and pregnancy outcomes and lamb survival 66 Table 2.3.6 Effect of periconceptional overnutrition and/or dietary restriction on growth parameters at birth 67
XIV
Figure 2.3.4 Effect of periconceptional overnutrition and/or dietary restriction on birth weight in male and female lambs 68 Table 2.3.7 Effect of periconceptional overnutrition and/or dietary restriction on plasma glucose, non-esterified free fatty acid and insulin concentrations at birth in the offspring 69 Figure 3.3.1 The growth rate of lambs from the 4 nutritional treatment groups between 1 and 16 weeks of age 86 Table 3.3.1 The effect of periconceptional overnutrition and/or dietary restriction on growth parameters at 4 months of age 87 . Figure 3.3.2 Effect of periconceptional overnutrition and/or dietary restriction on male and female lamb weight at 4 months of age 88 Figure 3.3.3 Effect of periconceptional overnutrition and/or dietary restriction on plasma glucose concentration between birth and 4 months of age in lambs 89 Figure 3.3.4 Effect of periconceptional overnutrition and dietary restriction on the plasma non-esterified free fatty acid concentration from birth to 4 months of age in lambs 90 Figure 3.3.5 Effect of periconceptional overnutrition and/or dietary restriction on the plasma insulin concentration from birth to 4 months of age in lambs 91 Figure 3.3.6 Plasma glucose and insulin concentrations responses to intravenous glucose challenge at 3 months of age in lambs 92 Figure 3.3.7 Area under the glucose and insulin response curves after the intravenous glucose challenge 93 Figure 3.3.8 Effect of periconceptional nutrition on absolute organ weights and relative to body weight of 4 month old lambs 95 Figure 3.3.9a Effect of periconceptional overnutrition and dietary restriction on the absolute brain weight in female and male lambs at 4 months of age 98 Figure 3.3.9b Effect of periconceptional overnutrition and dietary restriction on the brain weight relative to body weight in female and male lambs at 4 months of age 99 Figure 3.3.10a Effect of periconceptional overnutrition and dietary restriction on absolute heart of 4 month old male and female lambs 100
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XV
Figure 3.3.10b Effect of periconceptional overnutrition and dietary restriction on heart weight relative to body weight of 4 month old male and female lambs 101 Figure 3.3.11 Effect of periconceptional overnutrition and/or dietary restriction on absolute adrenal weight and relative to body weight of 4 month old lambs 102 Figure 3.3.12 Effect of periconceptional overnutrition and/or dietary restriction on total fat mass of male and female lambs at 4 months of age 103 Figure 3.3.13 Relationship between total fat mass in 4 month old female offspring and the weight of the donor ewe at conception 104 Figure 3.3.14 Effect of periconceptional overnutrition and/or dietary restriction on omental adipose tissue depot weight of 4 months old male and female lambs 105 Figure 3.3.15 Effect of periconceptional overnutrition and/or dietary restriction on perirenal adipose tissue depot weight of 4 months old male and female lambs 106 Figure 3.3.16 Effect of periconceptional overnutrition and/or dietary restriction on subcutaneous adipose tissue depot weight of 4 months old male and female lambs 107 Figure 3.3.17 The proportion of the total fat mass in the different adipose depots in female lambs at 4months of age 108 Figure 3.3.18 The proportion of the total fat mass in the different adipose depots in male lambs at 4 months of age 109 Table 3.3.2 Differences between the proportion of the total adipose tissue in each fat depot between male and female lambs at 4 months of age 110 Table 4.2.1 Sequences of real time PCR primers for adipogenic, lipogenic and reference genes 130 Figure 4.3.1 Normalised expression of adipogenic and lipogenic genes in the subcutaneous adipose tissue depot of male and female lambs at 4 months of age 135 Figure 4.3.2 Normalised expression of adipogenic and lipogenic genes in the perirenal adipose tissue depot of male and female lambs at 4 months of age 137 Figure 4.3.3 Normalised expression of adipogenic and lipogenic genes in the omental adipose tissue depot of male and female lambs at 4 months of age 139
XVI
Figure 4.3.4 Correlation between donor ewe weight at conception and perirenal G3PDH mRNA expression in female lambs at 4 months of age 141 Table 4.3.1 Correlations between the relative mRNA expression of PPARγ and other adipogenic and lipogenic genes in the subcutaneous, perirenal and omental adipose tissue depots of lambs at 4 months of age 142 Figure 4.3.5 Relative adipose tissue depot specific expression of PPARγ mRNA of male and female lambs at 4 months of age 143 Figure 4.3.6 Relative adipose tissue depot specific expression of G3PDH mRNA of male and female lambs at 4 months of age 144 Figure 4.3.7 Relative adipose tissue depot specific expression of LPL mRNA of male and female lambs at 4 months of age 145 Figure 4.3.8 Relative adipose tissue depot specific expression of leptin mRNA of male and female lambs at 4 months of age 146 Figure 4.3.9 Relative adipose tissue depot specific expression of adiponectin mRNA of male and female lambs at 4 months of age 147 Figure 5.1 Proposed mechanism for the impact of periconceptional overnutrition on the intergenerational cycle of obesity 159 Figure 5.2 Proposed mechanism for the impact of late gestational overnutrition on the intergenerational cycle of obesity 161 Figure 5.3 Proposed mechanism for the impact of periconceptional overnutrition and late gestational overnutrition on the intergenerational cycle of obesity 162
XVII
COMMONLY USED ABBREVIATIONS
A B C
ACE angiotensin-converting enzyme ACTH adrenocorticotrophic hormone Ad libitum to any desired extent ANOVA analysis of variance BAT brown adipose tissue BCS body condition score BMI body mass index bp base pairs cAMP cyclic adenosine monophosphate CC control-control C/EBP CCAAT enhancer binding protein CR control-restricted D E F G
d days DNA deoxyribonucleic acid FFA free fatty acids FSH follicle stimulating hormone GDM gestational diabetes mellitus GLUT glucose transporter G3PDH glycerol-3-phoshate dehydrogenase H I J K L
h hours HFD high fat diet hGh human growth hormone HH high-high HPA hypothalamic-pituitary-adrenal axis HR high-restricted ICM inner cell mass IGF insulin-like growth factor IGFR insulin-like growth factor receptor I.V intravenous LGA large for gestational age LH luteinizing hormone LPL lipoprotein lipase
XVIII
M N O
MER maintenance energy requirements min minute(s) mRNA messenger ribonucleic acid n number NEFA non-esterified free fatty acids P Q R S
polymerase chain reaction RAS renin-angiotensin system RNA ribonucleic acid RPLP0 ribosomal protein large subunit P0 RT-PCR reverse transcription polymerase chain reaction RxR retinoid x receptor SEM standard error of the mean SPSS statistical package for the social sciences
T U V W X Y Z
TZD thiazolidinediones WAT white adipose tissue
I
CHAPTER 1
2
CHAPTER 1: LITERATURE REVIEW
1.1 INTRODUCTION
There is a current global increase in the prevalence of obesity (body mass index
(BMI) ≥ 30kg/m2), with a disproportionate number of young women being classed
as obese. Studies suggest that more women are now entering pregnancy with a
higher BMI than a decade ago, with deleterious consequences not only for the
health of the mother, but for the long-term health of her child. Furthermore,
obesity during pregnancy is also associated with adverse health risks for the
mother, pregnancy complications and long-term health implications for the
offspring (Garbaciak 1985; Cnattingius 1998; Cogswell, Perry et al. 2001; Jolly
2003; Kristensen 2005; McMillen, Rattanatray et al. 2009). More importantly, a
high maternal BMI and gestational diabetes are each associated with an
increase in fetal birth weight and adiposity. It has also been shown that a high
birth weight is also associated with an increased risk of being obese throughout
childhood to adulthood. Many attempts have been made to investigate the
mechanisms underlying these associations in animal models in order to
understand how an increase in early nutritional supply results in the emergence
of an increased risk of metabolic disease in the offspring. Previous studies
however, have not been able to ascertain whether maternal obesity before
pregnancy is directly involved in the programming of obesity in the offspring or
whether maternal weight gain and obesity during pregnancy plays the more
critical role in the programming of obesity. It is important to differentiate between
the effects of a nutrient rich embryonic environment and a nutrient rich fetal
3
environment on the programming of offspring obesity in later life. The timing of
maternal obesity and its consequences on the development of offspring obesity
will inform novel approaches to implementing interventions for overweight
women of child bearing age wishing to conceive, and potentially reduce the
incidence of the “intergenerational cycle of obesity”.
This review will begin by briefly summarizing human epidemiological studies
focusing on maternal obesity, diabetes and weight gain before and during
pregnancy and the association with later onset of adult obesity. The embryonic
stages of development and the impact of early nutrition on the biology of the
developing adipocyte will be addressed. In addition the effects of maternal
overnutrition at different stages of gestation, as well as the little that is known
about the effect of nutrition during the periconceptional period will be discussed,
highlighting areas which are novel and require further investigation.
1.2 FETAL ORIGINS OF ADULT DISEASE
The developmental origins of adult health hypothesis proposed that
environmental factors particularly suboptimal intra-uterine environments such as
poor maternal nutrition act in early life to result in altered fetal growth and
development. A suboptimal intra-uterine environment subsequently predisposes
the fetus to an increased risk for developing adverse health outcomes in later
adult life, including cardiovascular disease, obesity and the metabolic syndrome
(Barker, Hales et al. 1993; Barker 2007). A number of factors may induce a
4
suboptimal intra-uterine environment including maternal nutrient restriction, poor
maternal-fetal nutrient transfer by the placenta and maternal drug use, including
smoking. A suboptimal intra-uterine environment results in adaptions by the
embryo and fetus to the environment, including morphological changes to the
development of fetal tissues, changes to hormone concentrations and/or
changes to the sensitivity to the action of these hormones on target tissues. The
concept of adaptive responses in embryonic and fetal development to a
suboptimal environment, leading to an altered “setting” of a physiological system
occurring during a sensitive period of development is coined “programming”.
Whilst earlier adaptions may confer early advantages for fetal survival in utero it
is hypothesized that these “trade-offs” may incur delayed adverse health
outcomes leading to the development of diseases in later life (Lucas 1998;
Mcmillen and Robinson 2005).
In 1992, Hales and Barker coined the “thrifty phenotype” hypothesis (see (Hales
and Barker 2001) to explain the relationship between poor fetal environment,
infant growth and the increased risk of developing diseases in later life, including
impaired glucose tolerance (Hales and Barker 1992) the metabolic syndrome
(Barker, Hales et al. 1993) and cardiovascular disease (Barker and Osmond
1986). The pivotal elements of this hypothesis suggest that under the conditions
of a suboptimal fetal environment an adaptive response occurs which favours the
optimal growth of key body organs including the brain and heart to the detriment
of other organs and tissues including the musculoskeletal system. These
adaptive responses subsequently lead to a postnatal metabolism which is
designed to improve postnatal survival during critical periods of intermittent or
5
poor nutrient availability. However it is proposed that these altered physiological
adaptations become deleterious when the nutritional abundance in the postnatal
environment exceeds that of the prenatal environment. The “thrifty phenotype”
proposal and the “predictive adaptive response” concept hypothesize that the
disease manifests only when the postnatal nutrient environment is considerably
different from the predicted nutritional environment in utero (Hales and Barker
2001; Gluckman and Hanson 2004).
1.3 DEVELOPMENTAL PROGRAMMING OF THE METABOLIC
SYNDROME: EPIDEMIOLOGICAL EVIDENCE
The “fetal origins of adult disease” hypothesis was first derived from
epidemiological studies of fetal programming which focused on the relationship
between infant birth weight and the incidence of adult disease including
metabolic abnormalities associated with the metabolic syndrome including
hypertension (Barker, Dull et al. 1990), insulin resistance (Phillips, Barker et al.
1994), obesity (Yajnik 2002) and dyslipidemia (Barker, Hales et al. 1993).
Epidemiological data found that in localised areas of Britain there were high
infant mortality rates between 1921-1925. These localised areas were similar to
those which experienced high incidences of ischemic heart disease between
1968-1978 (Barker and Osmond 1986). It has been well established that a low
birth weight is a reflection of a suboptimal intra-uterine environment and
therefore poor fetal growth and is associated with an increased incidence of fetal
morbidity and mortality. The importance of maternal nutrient deprivation on birth
6
weight and subsequent adulthood disease has been investigated in a series of
epidemiological studies most notably, the Dutch Hunger Winter Famine study, in
which pregnant women experienced 5 months of famine during the winter of
1944-1945 in Amsterdam, the Netherlands. Exposure to a restricted caloric
intake during early gestation, as an embryo or fetus in the first trimester, resulted
in individuals with an increased incidence of coronary heart disease,
hypertension, increased body mass index and glucose intolerance and these
associations were independent of size at birth (Ravelli, Stein et al. 1976; Ravelli,
van der Meulen et al. 1999; Roseboom, van der Meulen et al. 2000; Roseboom,
van der Meulen et al. 2001). Exposure to the famine in late gestation, however,
was associated with an increased incidence of adulthood obesity and glucose
intolerance (Ravelli, Stein et al. 1976; Ravelli, van der Meulen et al. 1999).
Furthermore babies born small after exposure to the famine at any time in
gestation, particularly female babies were more susceptible to developing adult
onset diabetes (Roseboom, van der Meulen et al. 2001). However, great
controversy surrounds the relationship between birth weight and adulthood
metabolic or cardiovascular disease (Huxley, Neil et al. 2002). The siege of
Leningrad, in Russia from 1941-1943 (in which caloric intake was restricted)
found no apparent relationship between birth weight and adult glucose
homeostasis (Stanner, Bulmer et al. 1997). The “thrifty phenotype” and
“predictive adaptive response” hypotheses may provide an explanation for this
observation, since a poor nutritional environment preceded and followed the
Leningrad siege. Therefore the adaptive fetal response may have been suitable
for the nutrient-poor postnatal environment. In contrast, nutrient availability
following the Dutch winter famine was relatively abundant and therefore the
7
postnatal plane of nutrition was greater than that was predicted by the adaptive
response and therefore an increase incidence of adult disease was the result.
1.3.1 MATERNAL OVERNUTRITION AND THE DEVELOPMENTAL PROGRAMMING OF
OBESITY
Previous studies have focused on the impact of maternal nutrient detriment in
the context of “early origins of adult disease” over the past decade, however
maternal nutrient abundance and an increase in maternal body mass is
becoming more prevalent in both developed and developing countries. The effect
of maternal overnutrition and thus embryonic and/or fetal overnutrition on the
developmental origins of adult health and disease is an emerging area of
research interest. Early exposure to overnutrition during critical developmental
windows alters the development of body systems including central and peripheral
neuroendocrine responses which affect the developmental programming of fat
cells and the appetite regulatory system, altering whole body metabolism.
Subsequently maternal overnutrition in early fetal life results in the emergence of
poor health for the offspring in childhood and adulthood.
1.3.2 THE OBESITY EPIDEMIC
There has been a marked increase in the global prevalence of obesity in the past
two decades. Currently more than 50% of all American, British and Australian
adults are classed as overweight (i.e. have a body mass index (BMI) ≥ 25 kg/m2)
and more than 24% of adults in the United States (U.S) are classified as obese
(BMI ≥ 30 kg/m2) (LaCoursiere, Bloebaum et al. 2005). There has also been a
8
marked increase in the proportion of children who are overweight or obese, with
more than 15% of U.S children aged 6-19 being classed as overweight (Ogden,
Flegal et al. 2002). The prevalence of both adulthood and childhood obesity
continues to increase. Despite the increase in overweight and obesity trends in
adults particularly women of child-bearing age, who are at a higher risk of being
overweight or obese (Cogswell, Perry et al. 2001), there is less attention paid to
the trends of increased BMI specifically among pregnant women (LaCoursiere,
Bloebaum et al. 2005).
1.3.3 MATERNAL OBESITY
More women are now entering pregnancy with a higher BMI than a decade ago.
A recent study by La Coursiere and colleagues (LaCoursiere, Bloebaum et al.
2005) showed that pre-pregnancy overweight and obesity increased from 25.1%
in 1991 to 35.2% in 2001, whereas maternal obesity at delivery rose from 28.7%
to 39.1% during the same period. This increased incidence in the proportion of
women entering pregnancy with a high BMI is concerning, since there are many
adverse risks associated with being overweight or obese entering pregnancy or
during pregnancy.
Entering pregnancy obese is associated with an increased risk of impaired
reproductive function and infertility, which may be attributed to the increase in
insulin resistance (Clark, Thornley et al. 1998; Bellver, Rossal et al. 2003; Bellver
and Pellicer 2004; Rhind 2004). Being obese during pregnancy increases the risk
of health complications for the mother, leading to a higher risk of developing
hypertension, preeclampsia and gestational diabetes mellitus (GDM) during
9
pregnancy (Cogswell, Perry et al. 2001). There are also health consequences for
the children born to obese mothers. There is an increased risk for the
development of congenital abnormalities and perinatal mortality (Garbaciak
1985; Cnattingius 1998; Cogswell, Perry et al. 2001; Kristensen 2005). Maternal
obesity also increases the risk of giving birth to a large for gestational age (LGA)
infant (i.e. birth weight ≥ 95th percentile of infants of the same age) (Garbaciak
1985; Frisancho 2000; Laitinen 2001; Parsons, Power et al. 2001; Pietiläinen
Figure 4.3.9 Relative adipose tissue depot specific expression of
adiponectin mRNA of 4 month old lambs
There was a significant increase in the relative expression of adiponectin in the
omental adipose tissue depot compared with the subcutaneous and perirenal
depots. Different superscripts (e.g. a, b) denotes a significant difference
between relative expression of adiponectin mRNA in the adipose depots at 4
months of age, P<0.05.
a a
b
148
4.4 DISCUSSION
We have demonstrated that periconceptional overnutrition and/or dietary
restriction does not alter the expression of key adipogenic, lipogenic and
adipokine gene expression in the major adipose tissue depots of 4 month old
lambs. There was however a positive relationship between donor weight at
conception and fat mass (Chapter 3) and the expression of perirenal G3PDH in
female lambs. This may suggest that the increase in total fat mass in the female
lambs at 4 months of age may be attributed to an increase in triglyceride storage
in the fat, rather than hyperplasia of the fat cells. Further work however, is
required to investigate the impact of periconceptional nutrition on adipocyte cell
size and number.
Many studies have attempted to elucidate the importance of timing of nutritional
perturbations in order to determine critical windows during development which
are important in the programming of obesity in later life. Studies by Khan and
colleagues (Khan, Dekou et al. 2004; Khan, Dekou et al. 2005) have shown that
maternal consumption of high fat diets of lard for 10 days prior to conception
through to weaning lead to increased hypertension, hyperinsulinemia and
adiposity in the offspring at 6 months of age. It is difficult however, to elucidate
whether the conditions observed in the offspring are due to the early exposure of
a high fat diet during critical stages of embryonic development or whether they
are due to the exposure of a high fat diet throughout pregnancy through to
weaning or the cumulative effect of both. Dams fed the high fat diet, however,
gained significantly more weight during pregnancy than the control fed rats,
suggesting that this model may mimic the effects of maternal weight gain during
149
pregnancy rather than the specific effects of an increased body fat mass at
conception.
A study by Shankar and colleagues (Shankar, Harrell et al. 2008) determined the
impact of maternal obesity, by feeding female rats with 15% excess calories/day
for 3 weeks prior to mating and resulted in increased body weight, adiposity,
insulin, leptin and insulin resistance. Exposure to maternal obesity was limited to
the in utero environment by cross-fostering pups to lean dams with ad libitum
diets throughout lactation. They showed that there was no effect of maternal
obesity on body weight or size at birth; however offspring from obese dams
gained remarkably greater body weight and higher percentage body fat when fed
a high-fat diet. Their results suggest that maternal body composition at
conception is important in programming offspring adiposity in later life.
Furthermore in this study there was no difference in the gestational weight gain
in the obese or lean dams.
These studies highlight the importance of the nutritional environment in early
pregnancy particularly at critical stages of implantation (~16d gestation in the
sheep) and placentation (~24-30d gestation in the sheep) on the development of
postnatal adiposity in the offspring and their role in predicting adversity and
producing adaptive responses to the current environment. These studies suggest
that offspring adiposity may be determined by exposure to nutritional influences
and is susceptible during several critical windows during early development.
150
It has been suggested by previous studies that increased maternal nutrition late
gestation stimulates the transcriptional co activator PPARγ in the adipose tissue
before birth and subsequently activates a suite of adipogenic, lipogenic and
adipokine genes in the adipose tissue which may be involved in the
pathogenesis of obesity in later life (Muhlhausler, Duffield et al. 2007). The
current study however, shows that periconceptional overnutrition did not affect
the expression of PPARγ, G3PDH, LPL, leptin nor adiponectin, despite the
increase in fat mass in female lambs. We propose that periconceptional
overnutrition may have produced changes to the expression of key adipogenic
and lipogenic genes earlier in development however the postnatal development
of adipose tissues at 4 months of age in the lambs may no longer show
increased expression of adipogenic and lipogenic genes. This suggests that
other mechanisms may be involved in the development of postnatal obesity in
lambs exposed to periconceptional overnutrition, such as the increase in insulin
sensitivity in the female lambs which could alter the threshold of activation of
PPARγ, the transcriptional co activator of adipogenic, lipogenic and adipokine
genes. This change in activation may suggest that minute changes in gene
expression may produce an enhanced downstream response and hence
increased PPARγ activity may contribute to an increase in fat mass in the female
offspring. Further work is required to investigate the impact of periconceptional
overnutrition on the insulin signalling pathways in the major adipose tissue
depots.
We demonstrated that PPAR mRNA expression was directly related to LPL
mRNA expression in subcutaneous, perirenal and omental adipose tissue and to
151
G3PDH in the perirenal adipose tissue which is consistent with studies in the
adult rodent in which the activation of PPAR promotes lipogenesis within mature
adipocytes by increasing the expression of genes, which regulate the
incorporation of circulating free fatty acids (LPL) or nutrient-derived factors
(G3PDH) into adipose cells (Semple 2006). Interestingly we found that maternal
donor weight at conception was positively associated with perirenal G3PDH
mRNA expression in the female lambs at 4 months of age which suggests that
the increase in fat mass observed in the female lambs may be attributed to an
increase in fat storage. Furthermore we propose that PPARγ activation may be
dependent on the timing of exposure to maternal overnutrition, and may occur as
a consequence of exposure to excess nutrients during gestation rather than
when exposure occurs during critical stages of embryo development.
In Chapter 2 I showed that maternal periconceptional overnutrition results in
increased insulin resistance in the ewes. It has been clearly demonstrated by
Catalano and colleagues that an increase in insulin resistance entering
pregnancy is associated with an increased risk of developing increased adiposity
in the offspring (Catalano 2003).
It has been shown that alterations in the nutrient environment of the developing
embryo in vivo and in vitro can alter the allocation of cells within the inner cell
mass and trophectoderm, therefore altering the growth and development of the
embryo (Kwong, Wild et al. 2000; Minge, Bennett et al. 2008). In a recent study
by Minge and colleagues (Minge, Bennett et al. 2008) murine oocytes exposed to
a high fat diet for 16 weeks prior to conception resulted in poor quality oocytes. It
152
was observed that there was a decline in the number of embryos undergoing
further differentiation into 4 to 8 cell blastocysts, as well as the abnormal
localization of blastomeres in the inner cell mass and an increase in the
proportion of cells in the trophectoderm. Further evidence from other animal
models has shown that maternal periconceptional undernutrition alters the
proportion of cells between the inner cell mass and trophectoderm (Erwich and
Robinson 1997; Kwong, Wild et al. 2000).
It has been previously shown that maternal nutrient restriction early in
development may alter the development of adiposity in the offspring. Nutrient
restriction in ewes from 8d post conception throughout gestation showed an
increase in fetal adiposity in twin fetuses when compared to periconceptional
nutrient restriction from 60d prior to conception to 7d post conception (Edwards,
McFarlane et al. 2005). Bispham and colleagues (Bispham, Gopalakrishnan et
al. 2003) also showed an increase in relative adiposity in fetuses of ewes which
underwent nutrient restriction between 28 to 80d gestation (at a time when
maximum growth of the placenta is occurring) and then fed to appetite (~150%
MER) until 140d gestation compared to fetuses exposed to an ad libitum diet
from 28 to 140d gestation.
We found that maternal periconceptional overnutrition increases maternal insulin
concentrations and it is possible maternal insulin resistance may affect the
developing blastocyst, consequently increasing adiposity in susceptible female
offspring in later life. Periconceptional overnutrition and/or dietary restriction may
alter the proportion of cells allocated to the inner cell mass or trophectoderm in
153
the developing blastocyst, as well as alter the embryonic environment affecting
the differentiation and developmental potential of adipocytes. These alterations
may be important mechanisms by which periconceptional overnutrition and/or
dietary restriction may alter the growth and development of the offspring. It is
clear that further work is required to investigate the impact of the nutrient
environment during the periconceptional period on the developing blastocyst.
4.5 SUMMARY
We have shown that periconceptional overnutrition does not affect the gene
expression of PPARγ, G3PDH, LPL, leptin and adiponectin of 4 month old
lambs. There was a greater expression of genes however, involved in the
storage of fatty acids in mature adipocytes across all major depots in female
offspring. Furthermore we found that there was a positive relationship between
maternal body weight at conception and the expression of perirenal G3PDH in
the female offspring. This suggests that the increase in total fat mass observed
in the female lambs compared to the male lambs may be due to an increase in
adipocyte hypertrophy postnatally. We proposed that the development of
maternal insulin resistance in the periconceptionally overnourished ewe
subsequently alters the embryonic environment resulting in the development of
insulin sensitivity in the offspring which could alter the threshold of activation of
PPARγ, the transcriptional co-activator of adipogenic, lipogenic and adipokine
genes.
154
CHAPTER 5
155
CHAPTER 5: SUMMARY AND CONCLUSIONS
Maternal obesity and/or glucose intolerance is a major determinant of the
relationship between birth weight and BMI in adulthood and it has been proposed
that an increase in fetal nutrient supply, as a consequence of these conditions, is
an important determinant of fetal body condition both in utero and in later life.
Interestingly a study by Catalano and colleagues (Catalano and Ehrenberg 2006)
showed that maternal weight and insulin resistance before pregnancy is strongly
correlated with an infant‟s fat mass, whereas the development of insulin
resistance before pregnancy is associated with infants fat free mass (e.g.
skeletal muscle). Few studies have attempted to distinguish between the
contribution of maternal obesity and the development of maternal glucose
intolerance at later stages of pregnancy. In this study I was able to determine the
contribution of a high maternal BMI and maternal insulin resistance entering
pregnancy on the development of the oocyte/embryo and on the development of
adiposity in the offspring in later life.
In the current study we have successfully developed a novel model of maternal
periconceptional overnutrition in the ewe. We have shown that periconceptional
overnutrition for at least 5 months prior to conception to one week post
conception resulted in a significant increase in weight gain and body condition
scores in the HH ewes. A short period of dietary restriction for one month prior to
conception to one week post conception in the periconceptionally overnourished
ewes resulted in lower weight gain than the HH group, although the HR ewes
remained significantly heavier and fatter than the control fed, CC and
156
periconceptionally nutrient restricted, CR ewes. Our data suggests that
overnourishing non-pregnant ewes for at least 5 months prior to conception, as
in the HH and HR ewes leads to an obese body condition accompanied by
increased plasma insulin concentrations and unaltered plasma glucose
concentrations, indicative of the pathogenesis of insulin resistance prior to
conception in these ewes.
There was no effect of periconceptional overnutrition or restriction on the growth
parameters at birth, including birth weight. Recent studies have debated whether
birth weight is in fact an appropriate end point for determining developmental
origins of adult disease. It has long been recognised that in the „developmental
origins of health and disease‟ (DOHAD) hypothesis was coined to explain the
associations between low and high birth weights and the development of a range
of diseases later in life, including obesity. Over time the hypothesis has
generated some controversy, since birth weight is a non-specific marker of a
number of conditions including premature birth, maternal health and fetal
genetics. Furthermore it is argued that postnatal growth may be a more
important risk factor for the subsequent pathogenesis of disease than growth in
utero (Cole 2004; Singhal and Lucas 2004). Therefore birth weight may be one
of many important markers for the increased risk of the development of diseases
later in life, but this may not always be the case.
It has been recently shown that periconceptional undernutrition from 45 days
prior to conception to 7 days post conception in the sheep led to an increase in
placental and fetal weights in twin fetuses (MacLaughlin, Walker et al. 2005).
157
This suggests that a compensatory response by the placenta occurs as a
consequence of the exposure of periconceptional undernutrition, therefore
altering the nutrient supply to the fetus. It has been suggested that PCUN alters
the allocation of cells within the blastocyst, which may restrict early embryonic
proliferation and differentiation of appropriately sized stem cell lineages,
therefore affecting the fetoplacental growth trajectory in early pregnancy. This
suggests PCUN alters the placental nutrient supply to the fetus which may
further impede the development of vital organ systems including the
cardiovascular system during gestation. In the CR group in the present study,
however there was no effect of periconceptional nutrient restriction on the birth
weight of the lambs which may suggest appropriate placental development or
compensatory growth of the placenta has occurred early in development.
At 4 months of age female lambs had significantly more body fat than male
lambs. Female lambs exposed to periconceptional overnutrition (HH) had higher
total fat mass than lambs from the CC and CR groups despite no change in
plasma insulin, glucose and non-esterified free fatty acid concentrations and this
was positively correlated with donor ewe weight at conception. Interestingly a
dietary intervention period of one month prior to conception to one week post
conception ablated this effect. In studies not reported in this thesis, we have also
shown that in the HH female lambs there was a decreased expression of hepatic
PEPCK mRNA, a key rate limiting enzyme involved in the gluconeogenic
pathway (personal communication Nichols, Rattanatray 2009). This suggests
that the liver of the HH lambs may be relatively more sensitive to the actions of
insulin.
158
I propose that exposure of the developing oocyte/embryo to maternal
overnutrition during the periconceptional period which results in an increase in
maternal weight and fat mass, may program adipose tissue and hepatic
metabolism to prepare for a future of predicted nutrient abundance. An increase
in total fat mass in the female offspring and a decrease in hepatic PEPCK mRNA
in the periconceptionally overnourished lambs suggests that the adipose tissue
and liver may be more sensitive to the actions of insulin in these lambs. Female
lambs are apparently more vulnerable to the effects of periconceptional
overnutrition and may also be more sensitive to the actions of insulin on the
adipose tissue (Figure 5.1). This enhanced insulin action in the adipose tissue in
the female lambs may explain the increase in fat mass in the female lambs,
whereas male lambs may be more sensitive to the actions of insulin in the
skeletal muscle. This would explain why the increase in fat mass in the postnatal
lambs exposed to periconceptional overnutrition was not associated with an
increase in PPARγ or other adipogenic gene expression.
Consistent with previous studies, male lambs were born heavier, with higher
plasma insulin levels than female lambs irrespective of treatment group (Duffield,
Vuocolo et al. 2009). Skeletal myogenesis occurs during early embryo
development involving the differentiation of mesodermal stem cells to myogenic
precursor cells. The majority of muscle fibre formation however, occurs mid-
gestation in the sheep. Studies have shown that the development of skeletal
muscle in the fetal sheep is susceptible to maternal nutrient availability during
pregnancy (Tong, Yan et al. 2009), the peri-implantation environment (Quigley,
159
Periconcep-tional
overnutrition/ insulin
resistance
↔Birth weight
↑ Susceptibility of obesity in
females in later life
↔ In utero environment
↑ insulin sensitivity in
adipose tissue
↑ Fat mass in female lambs at
4 months
Kleemann et al. 2005) and gender specific hormones (Galluzzo, Rastelli et al.
2009). I suggest that the skeletal muscle of male lambs is more insulin sensitive
than the skeletal muscle of female lambs and therefore is more inclined to take
up glucose, increasing the growth of muscle fibres in the male lambs before
birth. Further studies are required to determine the impact of periconceptional
overnutrition and gender on the insulin signalling pathways in key organs and
tissues regulating whole body glucose homeostasis, such as the liver, skeletal
muscle and adipose tissue.
Figure 5.1 Proposed mechanisms for the impact of periconceptional
overnutrition on the intergenerational cycle of obesity
160
In the current study I have shown that a dietary restriction period of one month
prior to conception to one week post conception in the periconceptionally
overnourished ewe, ameliorates the impact of periconceptional overnutrition on
the development of increased adiposity in the female lambs at 4 months of age.
This suggests that a dietary intervention period restores normal insulin action in
the adipose tissue in the postnatal lamb. The dietary restriction period however,
was also associated with changes which have implications for the development
of the hypothalamic pituitary adrenal (HPA) axis (increased adrenal weights in
the CR and HR groups) brain development (increase in brain weight in the CR
group) and the cardiovascular system (increase in heart weight in the CR group)
in these lambs postnatally.
In contrast to previous work which has investigated late gestational overnutrition
in the sheep (Muhlhausler 2002), periconceptional overnutrition did not alter the
expression of key adipogenic, lipogenic and adipokine genes. This suggests that
periconceptional overnutrition alone may act through an enhanced insulin action
in the adipose tissue and/or early programming of preferential differentiation of
mesenchymal stem cells into the adipocyte cell lineage, whereas late gestational
overnutrition increases the expression of the key adipogenic (PPARγ) and
lipogenic genes (Figure 5.2). The periconceptional and late gestational periods of
development may both independently increase fat mass in the offspring in later
life and the susceptibility to develop obesity. The cumulative effect of
periconceptional obesity and late gestational overnutrition may further amplify
the risk of developing obesity in later life by programming an enhanced insulin
signalling action in the adipose tissue early in development and an increase
161
Maternal late gestational
overnutrition
↑Birth weight ↑PPARγ mRNA expression in adipose tissue
↑ Susceptibility of obesity in
later life
↑ Fetal nutrient
supply
↑ Fat mass ↑glucose ↑insulin
concentrations postnatally
expression of PPARγ mRNA later in development, increasing fat mass in the
offspring (Figure 5.3).
Figure 5.2 Proposed mechanisms for the impact of late gestational
overnutrition on the intergenerational cycle of obesity
162
Periconception and late
gestational overnutrition
↑Birth weight ↑insulin
sensitivity in adipose tissue ↑PPARγ mRNA
↑ Susceptibility of obesity in
later life
Maternal insulin resistance
↑Fetal nutrient supply
↑ Fat mass ↑glucose ↑insulin
concentrations postnatally
Figure 5.3 Proposed mechanisms for the impact of periconceptional
overnutrition and late gestational overnutrition on the intergenerational
cycle of obesity
Therefore from the findings from this study, it would not be recommended to
acutely restrict dietary intake in pregnant women who are overweight or obese
immediately before conception. Further research is required to determine the
time period and level of dietary restriction which if imposed may ablate the
“intergenerational cycle of obesity”.
163
In conclusion, the results of this thesis therefore highlight the complex interaction
between the early exposure to excess nutrition during early embryo development
and the subsequent development of the offspring. Maternal periconceptional
overnutrition has been found to alter the development of the lamb. Female lambs
were found to be particularly vulnerable to the development of obesity in later
life, which may be attributed to an increase in lipogenesis and/or insulin
sensitivity in the adipose tissue, when exposed to an excess nutrient
environment during critical stages of embryo development. Restricted
periconceptional nutrition from either a high plane or control plane of nutrition
resulted in increased adrenal growth in the postnatal lambs. This may suggest
that dietary restriction before conception and early stages of embryo
development may alter the development of the HPA axis. Periconceptional
dietary restriction also increased heart growth in female lambs which may
indicate an altered developmental trajectory of the cardiovascular system and
program a susceptibility to developing cardio vascular related diseases in later
life. Although dietary restriction in the overnourished ewes ablated the increase
in adiposity in female lambs postnatally, implications for the development of the
HPA axis and cardiovascular system suggest caution is required in providing
advice to obese/overweight mothers to lose weight rapidly in the weeks
immediately before conception.
164
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