Martino, Jole (2013) Metabolic alterations induced by high maternal BMI and gestational diabetes ...

331 Pages·2017·7.71 MB·English

by  Christine Middleton

Metabolic alterations induced by
high maternal BMI and
gestational diabetes in
maternal, placental and
neonatal outcomes
by Jole Martino B.Sc., M.Sc.
Thesis submitted to the University of Nottingham
for the degree of Doctor of Philosophy
December 2013
i
Abstract
Maternal obesity and diabetes increase the risk of delivering large for
gestational age infants (LGA), who have higher risk of long term obesity or
metabolic syndrome [1]. As the underpinning mechanisms of how fetal growth
is regulated by the placenta remain unclear [2], this thesis has investigated
placental responses to high maternal BMI and gestational diabetes.
Spanish pregnant women recruited at 20 gestational weeks were classified
according to pre-pregnancy BMI as control (BMI<25kg/m2; n=59), overweight
(BMI=25-30kg/m2; n=29) or obese (BMI>30kg/m2; n=22), and gestational
diabetes status (GDM) classified at 28 weeks. Maternal anthropometry and
gestational weight gain (GWG) were measured during pregnancy. Placenta,
cord blood, newborn antrophometry and infant weight were sampled or
measured. Expression of genes involved in placental energy sensing
pathways, folate transporters and DNA methylation was determined using
real-time PCR, and placental triglyceride concentrations, lipid peroxidation and
genomic DNA methylation patterns measured. Data were analysed according
to their parametric distribution by Kruskal-Wallis or 1-way ANOVA.
Despite lower GWG, a greater proportion of obese women exceeded
recommended weight gain [3], had higher placental weight and increased
numbers of LGA infants. Maternal hyperinsulinaemia and hyperglycaemia with
obesity were accompanied by unchanged placental IGFR1 and ISR1
expression, similar cord blood glucose and triglyceride concentrations.
Placental mTOR was halved with obesity, whilst SIRT1 and UCP2 gene
expression were 1.8 and 1.6 fold upregulated respectively with no differences
in TBARS concentrations. Hyperleptinaemia in obese women resulted in
unchanged placental leptin and leptin receptor expression, but higher cord
blood leptin and monocyte concentrations with placental hypermethylation of
genes involved in the immune response. Lower folate concentrations in obese
mothers led to similar cord blood folate, and decreased placental FRα, but
raised DNMT1, mRNA expression. No major differences were observed with
GDM, probably due to small sample size.
In conclusion, it appears that the placenta can protect the fetus of obese
women by increasing antioxidant capacity, compensating for maternal
hyperglycaemia and lower folate. However, maternal obesity was associated
with enhanced cord blood leptin and monocyte concentrations, increased
placental weight and more LGA delivery, leaving infants at ongoing risk of
increased adiposity and inflammation. Therefore, ongoing studies are
currently exploring these interacting aspects.
i
Acknowledgements
Firstly, I would like to thank my funding organisations, Abbott Laboratories for
funding the sample collection in Granada, the Nottingham Respiratory
Biomedical Research Unit, the Nottingham University Hospitals Charity and
the Rowett Research Institute for funding my PhD in Nottingham. I am
grateful to Dr Cristina Campoy for giving me the opportunity to take part in
this project and further investigations in Nottingham. My sincerest thanks go
to Professor Michael Symonds and Dr Helen Budge for providing excellent
supervision and direction throughout this period, enabling me to interpret my
research within a professional and clinical setting. Special thanks go to
Professor Harry McArdle for his advice and scientific support.
I am sincerely thankful to Sylvain Sebert for his directions during the first part
of my PhD, and for his friendship and genuine dedication to science. I really
appreciated the support and friendship of all the staff within the Clinical
Hospital in Granada, who shared with me the Preobe experience, and the
Academic Division of Child Health in Nottingham. Special thanks go to Dr
Andrew Prayle for being always helpful with my statistical enquiries, and to
Vicky Wilson and Mark Pope for their excellent technical support and
expertise. I am truly grateful to Dr Ian Bloor, Dr Shalini Ojha, Dr Neele
Dellschaft, Stuart Astbury and Dr Mathias Droescher for their support to reach
the end of this thesis. I would like to thank all my friends in Nottingham,
especially Dr Marta Larraona, Marta Cigna, Angeles Muñoz and Bruno
Rodriguez, for their friendship and moral support both on the good and hard
days.
Finally, sincere thanks go to my family for their continuous encouragement
and for being always there.
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Table of Contents
Abstract...............................................................................................i
Acknowledgements.............................................................................ii
Table of Contents...............................................................................iii
Declaration....................................................................................... vii
Abbreviations .................................................................................. viii
List of Figures....................................................................................xi
List of Tables ................................................................................... xvi
List of Equations.............................................................................. xxi
Chapter 1 - Introduction...............................................................1
1.1. General study overview.......................................................2
1.2. Early life programming........................................................3
1.3. The obesity pandemic..........................................................5
1.4. Obesity in pregnancy...........................................................6
1.4.1. Maternal diet and nutrient intake...........................................8
1.4.2. Maternal weight gain ...........................................................9
1.5. Gestational diabetes mellitus............................................11
1.6. Metabolic alterations with obesity and diabetes................13
1.7. Insulin signalling in obese and GDM pregnancies..............15
1.8. Regulation of human fetal growth.....................................17
1.8.1. Maternal metabolic changes in pregnancy..............................18
1.8.2. The placenta: the programming agent ..................................21
1.8.3. Placental blood flow and vascular development......................23
1.8.4. Placental size and morphology.............................................26
1.8.5. Placental nutrient transport .................................................28
1.8.6. Placental hormonal regulation..............................................34
1.9. Placental metabolic regulation..........................................42
1.9.1. Placental insulin signalling...................................................42
1.9.2. Placental nutrient sensing....................................................44
1.9.3. Placental inflammation........................................................45
1.9.4. Placental oxidative stress ....................................................47
1.9.5. Folate metabolism and placental transport.............................50
1.10. Epigenetics and imprinted genes.......................................55
1.11. Hypothesis and Aims:........................................................59
Chapter 2 - General materials and methods................................61
2.1. Ethical Approval ................................................................62
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2.2. Participants.......................................................................62
2.2.1. Pre-pregnancy BMI classification..........................................65
2.2.2. Gestational diabetes mellitus...............................................67
2.3. Maternal questionnaires....................................................68
2.4. Collection of biological samples.........................................69
2.4.1. Blood sampling ..................................................................70
2.4.2. Tissue sampling .................................................................71
2.5. Tissue analysis..................................................................72
2.6. Ribonucleic acid analysis...................................................73
2.6.1. RNA extraction...................................................................73
2.6.2. RNA integrity.....................................................................76
2.6.3. Reverse transcription polymerase chain reaction....................77
2.6.4. Classical polymerase chain reaction......................................78
2.6.5. Design of primers for RT-PCR...............................................81
2.6.6. Agarose gel electrophoresis and DNA extraction.....................83
2.6.7. Real-Time polymerase chain reaction....................................85
2.6.8. Housekeeping genes...........................................................91
2.7. Triglycerides extraction.....................................................94
2.7.1. Triglycerides extraction procedure........................................95
2.8. Thiobarbituric acid reactive substances analysis...............97
2.8.1. TBARS procedure ...............................................................98
2.8.2. Bicinchoninic acid total protein determination.......................100
2.9. Histological analysis........................................................101
2.9.1. Histological tissue processing.............................................101
2.9.2. Haematoxylin and eosin staining........................................102
2.10. Immunohistochemistry ...................................................104
2.10.1. IHC procedure ..............................................................104
2.11. Western blot analysis......................................................108
2.11.1. Protein extraction procedure...........................................110
2.11.2. Sample preparation procedure........................................112
2.11.3. Protein detection procedure............................................112
2.11.4. Gel electrophoresis procedure.........................................113
2.11.5. Immunoblotting procedure.............................................114
2.11.6. Immunodetection procedure...........................................115
2.11.7. Western blot troubleshooting..........................................116
2.11.8. Protein interactions and urea treatment ...........................117
2.12. Methylation analysis........................................................118
2.12.1. DNA isolation................................................................119
2.12.2. Quantification of DNA methylation levels..........................124
2.12.3. Analysis of methylation data...........................................125
2.13. Statistical analysis...........................................................127
Chapter 3 - Nutritional and biochemical influence of maternal BMI
and diabetes on maternal health and newborn growth...................129
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3.1. Introduction and hypotheses...........................................130
3.2. Methods...........................................................................133
3.3. Results............................................................................135
3.3.1. Maternal socio-economical outcomes..................................135
3.3.2. Maternal dietary intake.....................................................137
3.3.3. Maternal clinical characteristics and anthropometry..............141
3.3.4. Maternal plasma metabolites .............................................147
3.3.5. Fetal and placental outcomes.............................................151
3.3.6. Newborn outcomes and postnatal growth............................154
3.4. Discussion.......................................................................159
3.4.1. Maternal BMI and diabetes influences maternal socio-economic
status and dietary choices .............................................................159
3.4.2. Obesity, diabetes and maternal weight gain in pregnancy......162
3.4.3. Placental and neonatal anthropometry related to maternal BMI,
GDM and GWG.............................................................................165
3.4.4. Insulin resistance in pregnancy influences maternal but not
neonatal glycaemia: protective role of the placenta?.........................169
Chapter 4 - Effect of BMI and gestational diabetes on placental
metabolism 173
4.1. Introduction and hypotheses...........................................174
4.2. Materials and methods....................................................177
4.3. Results............................................................................179
4.3.1. Maternal and cord blood physiological outcomes...................179
4.3.2. Placental glucose metabolism and gene expression...............183
4.3.3. Placental antioxidant and inflammatory response .................190
4.4. Discussion.......................................................................194
4.4.1. Influence of high maternal BMI and diabetes in pregnancy on
placental insulin signalling.............................................................194
4.4.2. Placental anti-oxidant and anti-inflammatory response..........204
Chapter 5 - Effects of maternal BMI and diabetes on folate
metabolism and placental methylation...........................................208
5.1. Introduction and hypotheses...........................................209
5.2. Methods...........................................................................213
5.3. Results............................................................................216
5.3.1. Folate intake and physiological outcomes............................216
5.3.2. Placental gene expression of folate transporters and metabolism
220
5.3.3. Methylation analysis .........................................................227
5.4. Discussion.......................................................................233
5.4.1. Effect of high BMI.............................................................233
5.4.2. Effect of gestational diabetes.............................................238
Chapter 6 - Conclusions............................................................243
v
6.1. General aims ...................................................................244
6.2. Summary of findings .......................................................245
6.2.1. Maternal diet and weight gain in women with high BMI and GDM
245
6.2.2. Influence of obesity and GDM on placental and newborn
anthropometry and metabolism......................................................246
6.2.3. Folate availability and DNA methylation...............................248
6.3. Limitations of the study...................................................250
6.3.1. Sampling and data collection .............................................250
6.3.2. Measurements and self-reported data.................................251
6.4. Future work.....................................................................252
6.4.1. Future directions for the study of offspring obesity ...............252
6.4.2. Future directions for the study of offspring neurodevelopment..253
6.5. Final remarks ..................................................................253
Bibliography ...................................................................................254
Appendix A: Conference abstracts..................................................302
Appendix B: Suppliers.....................................................................307
vi
Declaration
The work in this thesis was performed at the Granada Clinical Hospital,
University of Granada between 2008 and 2009 for the collection of samples
and within the Academic Child Health Division, School of Clinical Sciences,
University of Nottingham between November 2009 and October 2012.
This thesis illustrates my own work, completed under the supervision of
Professor Michael Symonds and Dr Helen Budge in Nottingham and Dr Cristina
Campoy and Professor Harry McArdle in Granada and Aberdeen respectively.
This report is an accurate representation of the work performed and no other
study reproducing this work, to my knowledge, has been carried out within
the the University of Nottingham or University of Granada.
Jole Martino
May 2013
vii
Abbreviations
11βHSD 11-beta hydroxysteroid dehydrogenase
11βHSD1 11-beta hydroxysteroid dehydrogenase type-1
11βHSD2 11-beta hydroxysteroid dehydrogenase type-2
5-MTHF 5-methyl tetrahydrofolate
Akt v-akt murine thymoma viral oncogene homologue
AMPK AMP-activated protein kinase
BHMT betaine–homocysteine methyltransferase
BPM basal plasma membrane
BMI body mass index
cAMP cyclic adenosine monophosphate
COX2 cyclooxygenase-2
CpG cytosine-phosphate-guanine
CRP C-reactive protein
CVD cardiovascular disease
CβS cystathionine beta-synthase
DMG dimethylglycine
DMRs differentially methylated regions
DNMT1 DNA methyltransferase-1
DNMT3α DNA methyltransferases-3-alpha
DNMT3β DNA methyltransferases -3-beta
DNMTs DNA methyltransferases
FABP fatty acid binding protein
FATB fatty acid transport protein
FFA free fatty acids
FRα folate receptor-alpha
GC glucocorticoids
GDM gestational diabetes mellitus
GH growth hormone
GLUT1 glucose transporter-1
GLUT4 glucose transporter-4
GLUTs glucose transporters
GR glucocorticoid receptor
GRα glucocorticoid receptor-alpha
GWG gestational weight gain
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