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Gestational obesity/prediabetes and folic acid supplementation program fetal one-carbon metabolism and… Mussai, Ei-Xia 2021

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GESTATIONAL OBESITY/PREDIABETES AND FOLIC ACID SUPPLEMENTATION PROGRAM FETAL ONE-CARBON METABOLISM AND BETA CELL MASS   by  Ei-Xia Mussai  B.Sc., University of Guelph, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Reproductive and Developmental Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2021  © Ei-Xia Mussai, 2021 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Gestational obesity/prediabetes and folic acid supplementation program fetal one-carbon metabolism and beta cell mass   submitted by Ei-Xia Mussai in partial fulfillment of the requirements for the degree of Master of Science in Reproductive and Developmental Sciences  Examining Committee: Dr. Angela Devlin, UBC Pediatrics Supervisor  Dr. Alexander Beristain, UBC Obstetrics and Gynaecology Supervisory Committee Member  Dr. Julian Christians, Simon Fraser University Biological Sciences Supervisory Committee Member Dr. Bradford Hoffman, UBC Surgery Additional Examiner  Additional Supervisory Committee Members: Dr. Crystal Karakochuk, UBC Food, Nutrition, and Health Supervisory Committee Member          iii  Abstract Background: Folic acid supplementation is recommended for women of childbearing age to prevent birth defects. Women with pregestational obesity (BMI30kg/m2) and/or diabetes are recommended to take up to 12x the recommended dose of folic acid. Concerns have been raised that elevated folic acid during pregnancy may have negative impacts on the cardiometabolic health of the mother and child. The objective of my thesis was to determine the effects of folic acid supplementation in gestational obesity/prediabetes on maternal and fetal health.   Methods: Female (C57BL/6J) mice were fed from weaning a control diet (10% kcal fat; control dams) or western diet (45% kcal fat; western dams; model of obesity/prediabetes). Diets contained supplemental (10mg/kg diet) or recommended level (2mg/kg diet) folic acid. Dams (n=11-14/diet) were fed for 13 weeks prior to breeding with control males. Tissue from dams and fetal offspring were collected at embryonic day (E)18.5.   Results: Prior to breeding, western dams had greater body weight and adiposity accompanied by glucose intolerance and impaired  cell function. Folic acid supplementation reduced insulin sensitivity in control dams and improved insulin sensitivity in western dams. At E18.5, western dams had larger livers and key methyl donor, S-adenosylmethionine (SAM), and it’s demethylated product, S-adenosylhomocysteine (SAH), were lower in liver, compared to control dams. Male offspring from western dams had smaller livers and higher hepatic betaine and choline compared to offspring from control dams. Female offspring from western dams had smaller livers and higher hepatic betaine compared to those from control dams. Maternal folic acid supplementation increased hepatic SAM in female offspring, but not in male offspring. iv  Maternal diet did not affect fetal pancreas size, but male offspring from folic acid supplemented dams had greater  cell mass and  cell density compared to those from non-supplemented dams; no effect of maternal diet on  cell mass was observed in female offspring.     Conclusion: Folic acid supplementation does not exacerbate adiposity and glucose tolerance in dams with gestational obesity/prediabetes. However, maternal folic acid supplementation and gestational obesity/prediabetes has sex-specific effects on hepatic one-carbon metabolism and pancreatic  cell mass in fetal offspring.   v  Lay Summary Women of childbearing age are recommended to take a daily supplement containing 0.4mg of folic acid for the prevention of birth defects. There is concern that too much folic acid during pregnancy may be harmful for the health of the mother and offspring. My thesis research used a mouse model to investigate if folic acid supplementation during pregnancy influences changes in body fat, blood sugar regulation, and the amount of carbon-donating nutrients required for metabolic functions in the mother and fetus, particularly if the mother has obesity. I found that folic acid supplementation during pregnancy had little impact on the mother’s health, but caused an increase in cells that control sugar levels in male fetuses and increased a key carbon-donating nutrient in female fetuses. These findings provide insight into how the mother’s body responds to folic acid supplementation, and how it may affect the health of the fetal offspring.   vi  Preface This thesis is submitted in partial fulfillment of the requirements for the Master of Science degree in the Reproductive and Developmental Sciences (RDS) program in the Department of Obstetrics and Gynaecology, University of British Columbia (UBC). Experimental procedures presented in this thesis were conducted in the laboratory of Dr. Angela Devlin at the BC Children’s Hospital Research Institute (BCCHRI), except when stated otherwise. Animal work was conducted in the Animal Care Facility of BCCHRI and was approved by the UBC Animal Care Committee (certificate # A14-0246, A14-0030, and A18-0059) and the UBC Biosafety Committee (certificate # B18-0029).  Animal breeding, physiological assessments, and tissue collection of mice were performed by me. Genotyping for sex determination of fetal offspring was performed by me with the help of Rebecca Lim, Martina Stokes, and Danielle Cohen. Pancreas processing was completed by me, with some samples prepared by the Histology Core Lab at BCCHRI. Immunohistochemistry and imaging experiments were conducted by me; imaging was completed in the Imaging Suite at BCCHRI. Analysis of  cell mass was performed by me, and analysis of  cell mass was performed by me and Danielle Cohen. Quantification of liver triglyceride content was completed by me. Water soluble choline metabolites were quantified in the Analytics Core for Metabolomics and Nutrition at BCCHRI by Roger Dyer. Total S-adenosylmethionine and S-adenosylhomocysteine levels were quantified in the laboratory of Dr. Joshua Miller in the Department of Nutritional Sciences at Rutgers University, New Brunswick, NJ, USA.  Part of my initial research findings were presented as a virtual poster format at the Canadian Nutrition Society 2020 Annual Conference in May 2020; this meeting was hosted as a virtual vii  format due to the COVID-19 pandemic. My abstract from this meeting has been published: Mussai E, Boonpattrawong NP, Ramírez CY, Mehran AE, and Devlin AM. Maternal folic acid supplementation during pregnancy has sex-specific effects on pancreatic beta cell mass in fetal offspring. Appl. Physiol. Nutr. Metab. 2020;45:S36. Data from my thesis will be submitted for publication with the addition of RNA sequencing data from fetal liver and  cell mass analysis from dams; a manuscript is in preparation. viii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Developmental Origins of Health and Disease ............................................................... 1 1.2 One-Carbon Metabolism ................................................................................................. 2 1.2.1 Folate....................................................................................................................... 2 1.2.2 Folic Acid................................................................................................................ 3 1.2.3 Folates in One-Carbon Metabolism ........................................................................ 3 1.3 Folic Acid Supplementation ........................................................................................... 6 1.3.1 Neural Tube Defects ............................................................................................... 7 1.3.2 Prevention of Neural Tube Defects......................................................................... 7 1.3.3 Folic Acid Fortification and Recommendations ..................................................... 8 1.3.4 Folate Status of Canadians ...................................................................................... 9 1.4 Epidemiological Studies of Maternal Folate Status and Offspring Health ..................... 9 1.5 Rodent Studies of Maternal Folic Acid Supplementation and Offspring Health ......... 11 ix  1.5.1 Effects on Adult Offspring Adiposity and Glucose Homeostasis ........................ 11 1.5.2 Effects on Offspring One-Carbon Metabolism ..................................................... 12 1.6 Maternal Obesity and Obstetrical and Neonatal Outcomes .......................................... 13 1.7 Maternal Obesity and Offspring Cardiometabolic Health ............................................ 15 1.7.1 Maternal Obesity and Offspring Adiposity........................................................... 15 1.7.2 Maternal Obesity and Offspring Glucose Homeostasis ........................................ 16 1.7.2.1 Insulin Signalling and Glucose Uptake............................................................. 16 1.7.2.2 Obesity and Diabetes During Pregnancy and Offspring Glucose Homeostasis 18 1.8 Thesis Hypotheses and Specific Aims .......................................................................... 20 Chapter 2: Materials and Methods ............................................................................................22 2.1 Mice and Study Design ................................................................................................. 22 2.2 Body Composition ........................................................................................................ 24 2.3 Physiological Assessments of Glucose Homeostasis ................................................... 25 2.4 Genotyping of the Sry Gene for Fetal Sex Determination ............................................ 26 2.5 Pancreas Processing ...................................................................................................... 27 2.6 Immunohistochemical Analysis of Insulin and Glucagon ............................................ 27 2.7 Quantification of Fetal Liver Triglyceride Concentrations .......................................... 28 2.8 Quantification of Fetal Liver Water Soluble Choline Metabolite Concentrations ....... 29 2.9 Quantification of Liver SAM and SAH Concentrations ............................................... 30 2.10 Statistical Analyses ....................................................................................................... 30 Chapter 3: Effects of Folic Acid Supplementation on Maternal Adiposity and Glucose Homeostasis ..................................................................................................................................31 3.1 Rationale ....................................................................................................................... 31 x  3.2 Results ........................................................................................................................... 32 3.2.1 Western diet-fed dams had increased body weight prior to pregnancy ................ 32 3.2.2 Folic acid supplementation altered insulin sensitivity prior to pregnancy ........... 33 3.2.3 Western diet feeding increased adiposity before and during pregnancy .............. 34 3.2.4 Maternal diet-induced obesity and glucose intolerance affected liver one-carbon metabolism at E18.5 of pregnancy........................................................................................ 36 3.2.5 Folic acid supplementation did not affect litter development ............................... 37 Chapter 4: Effects of Maternal Diet-Induced Obesity/Glucose Intolerance and Folic Acid Supplementation on Fetal Offspring Liver One-Carbon Metabolism ....................................38 4.1 Rationale ....................................................................................................................... 38 4.2 Results ........................................................................................................................... 39 4.2.1 Maternal diet-induced obesity and glucose intolerance reduced fetal liver weight ..   ............................................................................................................................... 39 4.2.2 Maternal diet did not influence fetal liver triglyceride content ............................ 40 4.2.3 Maternal diet-induced obesity and glucose intolerance increased hepatic water soluble choline metabolite concentrations in fetal offspring ................................................ 41 4.2.4 Maternal folic acid supplementation increased hepatic SAM in female offspring43 Chapter 5: Effects of Maternal Diet-Induced Obesity/Glucose Intolerance and Folic Acid Supplementation on Fetal Offspring Islet Morphology ...........................................................45 5.1 Rationale ....................................................................................................................... 45 5.2 Results ........................................................................................................................... 46 5.2.1 Maternal folic acid supplementation increased fetal  cell mass in male offspring .   ............................................................................................................................... 46 xi  Chapter 6: Discussion and Conclusions .....................................................................................49 6.1 Discussion ..................................................................................................................... 49 6.1.1 Folic acid supplementation before and during pregnancy does not affect adiposity, glucose tolerance, or hepatic one-carbon metabolism in dams with or without diet-induced obesity/glucose intolerance ................................................................................................... 49 6.1.2 Maternal diet-induced obesity/glucose intolerance and folic acid supplementation affect liver one-carbon metabolism in fetal offspring........................................................... 53 6.1.3 Maternal folic acid supplementation affects islet morphology in male fetal offspring ............................................................................................................................... 56 6.2 Summary ....................................................................................................................... 60 6.3 Strengths and Limitations ............................................................................................. 61 6.4 Future Directions .......................................................................................................... 63 References .....................................................................................................................................66  xii  List of Tables Table 2.1 Diet Composition .......................................................................................................... 23   xiii  List of Figures Figure 1.1 Schematic Representation of One-Carbon Metabolism ................................................ 4 Figure 2.1 Experimental Design ................................................................................................... 24 Figure 3.1 Body Weight of Dams Throughout 13 Week Feeding Period .................................... 32 Figure 3.2 Physiological Assessments of Glucose Homeostasis Before Breeding ...................... 34 Figure 3.3 Body Composition in Dams Before Breeding and at E18.5 ........................................ 35 Figure 3.4 Organ Weights in Dams at E18.5 ................................................................................ 36 Figure 3.5 Hepatic SAM and SAH Concentrations in Dams at E18.5 ......................................... 37 Figure 3.6 Litter Size and Distribution at E18.5 ........................................................................... 37 Figure 4.1 Body, Placenta, and Liver Weights of Fetal Offspring at E18.5 ................................. 40 Figure 4.2 Fetal Liver Triglyceride Concentrations at E18.5 ....................................................... 41 Figure 4.3 Fetal Liver Water Soluble Choline Metabolite Concentrations at E18.5 .................... 42 Figure 4.4 Hepatic SAM and SAH Concentrations in E18.5 Fetal Offspring .............................. 44 Figure 5.1 Fetal  and  cell mass at E18.5.................................................................................. 47 Figure 5.2 Fetal Islet Density and Distribution at E18.5 .............................................................. 48 Figure 6.1 Overarching Summary of Findings ............................................................................. 61   xiv  List of Abbreviations 10-formylTHF 10-formyltetrahydrofolate 5-10-MTHF 5,10-methylenetetrahydrofolate 5-MTHF 5-methyltetrahydrofolate ACC Acetyl-CoA carboxylase AIN American Institute of Nutrition AKT Protein kinase B ANOVA Analysis of variance AOC Area over the curve ATP Adenosine triphosphate AUC Area under the curve BHMT Betaine-homocysteine methyltransferase BMI Body mass index CBS Cystathionine--synthase CCT CTP:phosphocholine cytidylytransferase CD Control diet with adequate folic acid CDF Control diet with supplemental folic acid CDP Cytidine diphosphate ChREBP Carbohydrate-responsive element-binding protein  CK Choline kinase CPT CDP-choline:1,2-diacylglycerol cholinephophotransferase CTH Cystathionine--lyase xv  CTP Choline-phosphate cytidylytransferase CVD Cardiovascular disease DHF Dihydrofolate DHFR Dihydrofolate reductase DIO/IGT Diet-induced obesity/impaired glucose tolerance  DMG Dimethylglycine DNA Deoxyribonucleic acid  dNTP Deoxynucleoside triphosphate  DOHaD Developmental Origins of Health and Disease E Embryonic day ELISA Enzyme-linked immunosorbent assay EtOH Ethanol FAS Fatty acid synthase FPGS Folylpolyglutamate synthetase GDM Gestational diabetes mellitus GLUT2 Glucose transporter type 2 GLUT4 Glucose transporter type 4 GPC Glycerophosphocholine GTT Glucose tolerance test GWG Gestational weight gain Hcy Homocysteine HOMA-IR Homeostatic model assessment of insulin resistance xvi  HPLC High performance liquid chromatography IP Intraperitoneal IR Insulin receptor IRS-1 Insulin receptor substrate-1 IST Insulin secretion test ITT Insulin tolerance test kcal Kilocalorie LC-MS/MS High performance liquid chromatography-tandem mass spectrometry LGA Large for gestational age MAT Methionine adenosyltransferase Met Methionine MRC Medical Research Council mRNA Messenger ribonucleic acid  MTHFD1 Methylenetetrahydrofolate dehydrogenase 1 MTHFR Methylenetetrahydrofolate reductase MTR Methionine synthase MTRR Methionine synthase reductase MTs Methyltransferases NAD Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NAFLD Nonalcoholic fatty liver disease NTD Neural tube defects xvii  OGIS Oral glucose insulin sensitivity PBS Phosphate buffered saline PC Phosphatidylcholine PCFT Proton coupled folate transporter PCR Polymerase chain reaction PE Phosphatidylethanolamine PEMT Phosphatidylethanolamine N-methyltransferase PI3K Phosphoinositide 3-kinase RCF Reduced folate carrier RCT Randomized control trial RDA Recommended daily allowance rpm Revolutions per minute SAH S-adenosylhomocysteine  SAHH S-adenosylhomocysteine hydrolase SAM S-adenosylmethionine SD Standard deviation SGA Small for gestational age SREBP-1c Sterol regulatory element-binding protein 1c T2D Type 2 diabetes THF Tetrahydrofolate UL Tolerable upper limit UV Ultraviolet xviii  VLDL Very-low-density-lipoprotein WD Western diet with adequate folic acid WDF Western diet with supplemental folic acid   xix  Acknowledgements I would like to begin by thanking my supervisor, Dr. Angela Devlin, for her continuous guidance and support over the course of my graduate studies. I am grateful for the skills I have developed and the opportunities I have had as a member of her research team. I am especially grateful to have been introduced to the field of dietetics through Dr. Devlin’s lab and for her encouragement and support as I begin to pursue my aspiration of becoming a registered dietitian. I would like to extend my appreciation to my supervisory committee, Dr. Alex Beristain, Dr. Julian Christians, Dr. Crystal Karakochuk, and Dr. Peter Leung, for their valuable insight and guidance throughout my program.  I would like to thank the past and current members of the Devlin Lab, Amanda Henderson, Dr. Arya Mehran, Dr. Abeer Aljaadi, Cynthia Ramírez, and Dr. Alejandra Wiedeman for their support, mentorship, help with experiments, and friendship. A special thank you to my esteemed friend and colleague, Dr. Nicha Boonpattrawong, for being my rock through all things personal and professional. I would also like to extend my thanks to my friends and colleagues in the RDS program and the Canucks for Kids Fund Childhood Diabetes Laboratories at BCCHRI for their friendship and peer support.  Finally, I would like to thank all my friends and family, near and far, for their endless love and support throughout my graduate school journey. Whether it be through long hours of video or phone calling, surprise visits and treats, or a warm embrace, I am so grateful for each person who has encouraged me throughout this process. To my parents and sisters, thank you for believing in me and always holding me accountable for being the best that I can be.   1  Chapter 1: Introduction 1.1 Developmental Origins of Health and Disease The theory of developmental programing suggests that exposure to environmental factors during fetal and early postnatal development may contribute to the susceptibility of developing chronic disease later in life 1. This theory, better known as the Developmental Origins of Health and Disease (DOHaD), was proposed by Dr. David Barker following epidemiological studies that revealed a high correlation between low birth weight and greater rates of mortality, as well as higher risk for coronary heart disease, type 2 diabetes, and hypertension 2–4. The biological basis for this theory stems from the idea that organisms experience a plastic, adaptable period during their development in utero in which they are sensitive to environmental factors. This plastic state allows for short-term adaptations to its immediate environment with the goal of preparing for the long-term environment it anticipates living in 5. Although this strategy may be advantageous in situations where the environment remains static, consequences arise when the postnatal environment changes, such as transitioning to an energy-rich setting following an initially energy-poor environment. A key illustration of the DOHaD theory is observed in the well-documented Dutch Famine Birth Cohort, a group of individuals conceived in the Netherlands between 1944 and 1945 during World War II when many experienced starvation and above-average stress. Studies from this cohort have shown that those who were exposed to famine and stress at any point during gestation were more likely to present with hypertension, glucose intolerance, and impaired insulin secretion during their middle-aged years 6–8. The impairments in glucose homeostasis were further associated with famine if the exposure occurred during mid-to late gestation 7,8. Furthermore, exposure during early gestation was associated with obesity, coronary heart disease, and an atherogenic lipid profile later in life 9–11. It should be 2  noted that some studies from this cohort have reported no association with exposure to famine, including no association with offspring coronary heart disease or cognitive performance in middle-aged male and female offspring 12,13. This suggests that the relationship between disease susceptibility and environmental exposures during gestation are not linear and may be influenced by other factors. Nevertheless, studies from this cohort provide evidence for the programming of offspring health by maternal health during gestation.  Studies of malnutrition have paved the way to understanding the DOHaD theory, however other aspects of maternal nutrition can affect the developmental progression of the offspring and cannot be excluded. Maternal obesity and folate status are two other nutritional environments that are of particular interest, especially in western countries where the rates of obesity [body mass index (BMI)  30 kg/m2] are high 14 and folic acid consumption through supplement use, in addition to mandatory food fortification, are common. 1.2 One-Carbon Metabolism 1.2.1 Folate Folates refer to a family of structurally and functionally similar compounds that are collectively known as vitamin B9 15. Folate is present in the food supply as naturally occurring folate or in fortified grain products as folic acid. Natural occurring folate is a reduced polyglutamylated form and sources of food with appreciable amounts include beef liver, leafy green vegetables, asparagus, and oranges 16. Prior to intestinal absorption, the glutamate residues must be cleaved by the brush border membrane glutamylhydrolase, folate hydrolase (also known as glutamate carboxypeptidase 2), that is present on the proximal small intestine 17. This monoglutamyl form can then be absorbed by the mucosal cells of the proximal small intestine via the transmembrane proton coupled folate transporter (PCFT) 15,17. As it travels through the 3  mucosal cell, the monoglutamyl form is converted into 5-methyltetrahydrofolate (5-MTHF) before it is released into the portal vein, followed by entry into the peripheral circulation 15,17. Once 5-MTHF is transported to peripheral tissues, it is taken up into the tissue via reduced folate carrier-1 (RCF), a transporter specific for reduced folates 17,18. Some tissues, such as the liver, can additionally take up reduced and oxidized folate via PCFT 17,18. Once in the cell, 5-MTHF can either be metabolized to tetrahydrofolate (THF) prior to polyglutamylation by folylpolyglutamate synthetase (FPGS) for tissue retention, or be directly used for one-carbon metabolism requirements 17.  1.2.2 Folic Acid  Folic acid is the synthetic, oxidized form of folate. It is more easily absorbed compared to naturally occurring folates because it is found in the monoglutamyl form and does not need to undergo glutamate hydrolysis 18. Folic acid is the form that is added to fortified foods and supplements 15. Similar to natural folates, folic acid is absorbed through the mucosal cells of the proximal small intestine. As it passes through the mucosal cell, folic acid is reduced to dihydrofolate (DHF) and then to THF by dihydrofolate reductase (DHFR), followed by further methylation to 5-MTHF before it is released into the portal vein and peripheral circulation (Figure 1.1) 18,19. This sequential reduction is necessary for folic acid to become metabolically useful as oxidized folic acid lacks coenzyme activity 18,19.  1.2.3 Folates in One-Carbon Metabolism One-carbon metabolism refers to a collective of interlinking metabolic pathways that generate methyl groups to be used for various cellular reactions including the synthesis of DNA, amino acids, and phospholipids 19. One-carbon metabolism is active throughout the body, but a 4  major site is in the liver. Key pathways include the folate cycle, methionine cycle, and the transsulfuration pathway. These pathways are summarized in Figure 1.1.   Figure 1.1. Schematic Representation of One-Carbon Metabolism. One-carbon metabolism is primarily formed by the folate cycle, methionine cycle, and the transsulfuration pathway. DHFR, dihydrofolate reductase; DHF, dihydrofolate; THF, tetrahydrofolate; 5-10-MTHF, 5,10-methylenetetrahydrofolate; 10-formylTHF, 10-formyltetrahydrofolate; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; MTHFR, methylenetetrahydrofolate reductase; 5-MTHF, 5-methyltetrahydrofolate; MTR, methionine synthase; MTRR, methionine synthase reductase; Hcy, homocysteine; Met, methionine; MAT, methionine adenosyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; MTs, methyltransferases; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PEMT, phosphatidylethanolamine N-methyltransferase; BHMT, betaine-homocysteine methyltransferase; DMG, dimethylglycine; CBS, cystathionine--synthase; CTH, cystathionine--lyase. Some enzymatic activities and pathways are tissue specific: DHFR reduction of folic acid occurs predominantly in the small intestine and to a lesser amount in the liver 19. BHMT is limited to the liver and kidneys 19 while PEMT is specific to the liver only 20. CBS and the transsulfuration pathway occur in the liver, brain, and pancreas 19.   Folate is one of many methyl nutrients that feed into one-carbon metabolism. Folate and folic acid are metabolized to 5-MTHF before entering the folate cycle where it will serve as the 5  methyl donor for the remethylation of homocysteine to methionine, a reaction catalyzed by methionine synthase (MTR) and cofactors methionine synthase reductase (MTRR) and vitamin B12 19.  Following methyl donation to homocysteine, 5-MTHF produces THF which can subsequently be converted to 10-formylTHF or 5,10-methyleneTHF for the synthesis of purines or pyrimidines, respectively 19. In addition, 5-MTHF is regenerated from 5,10-methyleneTHF through methylene-tetrahydrofolate reductase (MTHFR) to continue the folate cycle 19. Folate metabolism occurs in several compartments of the cell including the cytosol and mitochondria. In order to conserve cytosolic NADPH and uncouple one-carbon metabolism from glycolysis as both pathways require electron acceptors (NAD+/NADP+), folate metabolism is compartmentalized in the cytosol and the mitochondria 19. To enable mitochondrial one-carbon metabolism reactions to ensue, a mitochondrial folate transporter (SLC25A32) facilitates translocation of THF from the cytosol to the mitochondria 19.   The generation of methionine through 5-MTHF is the connection between the folate and methionine cycles. Methionine is converted into S-adenosylmethionine (SAM) through the enzymatic activity of methionine adenosyltransferase (MAT) 21. S-adenosylmethionine subsequently serves as the primary methyl donor in the body for several cellular reactions 20. Following its methyl donation, S-adenosylhomocysteine (SAH) is produced and can be hydrolyzed back to homocysteine via SAH hydrolase (SAHH) to continue the methionine cycle, or it can enter the transsulfuration pathway where it will be transsulfurated to cystathionine followed by cysteine through the catalytic activities of cystathionine--synthase (CBS) and cystathionine--lyase (CTH), respectively 15,20. A folate-independent, alternative source of methyl groups for the remethylation of homocysteine to methionine is betaine. This methylation reaction is catalyzed by betaine-6  homocysteine S-methyltransferase (BHMT) and predominantly occurs in the liver and kidneys 20. Betaine is an intermediate of choline oxidation 20. Choline is an essential nutrient predominantly obtained through the diet in which it is found as both water-soluble forms (free choline, phosphocholine, and glycerophosphocholine) and lipid-soluble forms (phosphatidylcholine and sphingomyelin) 22. Choline may also be synthesized in the liver as phosphatidylcholine (PC) through the methylation of phosphatidylethanolamine (PE) via phosphatidylethanolamine N-methyltransferase (PEMT) or through the cytidine diphosphate (CDP)-choline pathway 23. This pathway utilizes pre-existing dietary choline to produce PC through a three step process 24. First, choline is phosphorylated by choline kinase (CK) into phosphocholine. This is followed by CTP:phosphocholine cytidylytransferase (CCT) converting phosphocholine into the high-energy donor, CDP-choline; this is a rate limiting reaction. In the final step, CDP-choline is converted to PC by CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT).  1.3 Folic Acid Supplementation Folate is essential for proper fetal growth and development. To support this healthy development, folic acid is recommended to women prior to, and during pregnancy due to its role in preventing birth defects such as neural tube defects (NTDs) 25. During pregnancy, there is a greater need for folate due to many physiological changes that are occurring in the mother’s body, particularly rapid cell division for the growth of the fetus and uteroplacental organs 26. Other factors may contribute to the greater need for folate to a lesser extent, these include increased dilution of folate concentration due to blood volume expansion, increased urinary folate excretion, and increased folate catabolism 26.  7  1.3.1 Neural Tube Defects Birth defects affect between 1-3% of births around the world and are considered one of the major causes of infant mortality 27. Neural tube defects are defined as a defect in the brain, spine, or spinal cord, and occur due to failed closure of the neural tube during embryogenesis. This closure occurs within the first 28 days of gestation, and initiates the development of the central and peripheral nervous systems 27,28. The most common type of NTD is spina bifida. Spina bifida refers to an incomplete closure of the spinal column, resulting in a partial exposure of the spinal cord through a hernia 27. Neural tube defects may lead to death of the offspring, however there is a significantly higher chance of survival in those with spina bifida (7%) as compared to other types of NTDs 27.  1.3.2 Prevention of Neural Tube Defects In 1976, Smithells and colleagues reported that NTDs were associated with lower serum levels of several micronutrients, including folate, in women during pregnancy 29. This lead to a series of small trials of periconceptional supplementation during pregnancy and a foundation for investigation into the role of folate in the prevention of NTDs 28,30,31. The British Medical Research Council (MRC) and Hungarian randomized control trials (RCTs) were launched in 1983 and 1984, respectively. The MRC study (n=1817 women) sought to study the prevention of NTD recurrence in women with a previous NTD-affected pregnancy by using a 4.0 mg supplement of folic acid 25. A 72% reduction in the risk of a recurring NTD was observed, suggesting that if a second NTD-affected pregnancy could be prevented using folic acid, supplementation must likely prevent a first occurrence as well. In the Hungarian RCT (n=4704 women), prevention of a first NTD occurrence was assessed using a vitamin mix that included 0.8 mg of folic acid 32. This study found that 93% of NTDs were prevented in first time 8  pregnancies. In both studies, women took supplements during the periconceptional period and continued throughout pregnancy. Another similar study was conducted in China between 1993 and 1995 in which they supplemented women (n=247,283) with 0.4 mg of folic acid in two regions of China; the northern region known to have high rates of NTDs, and the southern region, an area with lower rates 33. Findings from this study were consistent with the RCTs such that supplementation with folic acid resulted in a reduction in the risk of NTD-affected pregnancies, particularly in the northern region of China. Taken together, these studies highlighted the importance of folic acid in the prevention of NTDs for all women of child-bearing age and recommendations were made for women to take 0.4 mg/day of folic acid. 1.3.3 Folic Acid Fortification and Recommendations Ensuring adequate folic acid intake prior to conception is critical as the neural tube closes very early in the first trimester; a period in which a women may be unaware that she is pregnant. Compliance towards folic acid supplementation was difficult to achieve in women of child-bearing age despite public campaigns. This therefore prompted the proposal for fortification of folic acid in staple foods in many countries 25,32. Fortification of grain products became mandated in Canada on November 11, 1998 34. This included the fortification of all types of white flour, enriched pasta, and cornmeal. Another form of obtaining adequate folic acid intake is through oral supplement capsules. Currently, the recommended dietary allowance (RDA) for folic acid, determined by the National Academy of Medicine (formerly the Institute of Medicine), is 0.4 mg/ day for all women of child-bearing age to be consumed before and during pregnancy and lactation; this amount is also recommended by the Society of Obstetricians and Gynaecologists of Canada (SOGC) Clinical Practice Guidelines 35. Daily prenatal supplements in Canada however, typically contain between 0.6-1.0 mg of folic acid, in which 1.0 mg is the 9  tolerable upper limit (UL) 36. Furthermore, the SOGC Clinical Practice Guidelines recommend that women who are at a higher risk of having a NTD-affected pregnancy, such as those with pregestational obesity or diabetes, take up to 5.0 mg of folic acid per day; this is 12.5X the recommended RDA, and 5X the UL 35. To meet the RDA and SOGC Clinical Practice Guidelines, folates should be obtained from fortified foods and supplements, in addition to folate obtained from natural dietary sources 37,38.  1.3.4 Folate Status of Canadians Between food fortification and supplement use, women are consuming higher-than-recommended doses of folic acid. Fortification alone in the general Canadian population has resulted in an almost complete lack of folate deficiency (red blood cell folate < 305 nmol/L) 39. A study from Alberta (n= 599 women) reported that over 40% of pregnant women are considered to have high red blood cell folate concentrations (> 1360 nmol/L) 40. Folic acid must be reduced by DHFR to DHF, followed by THF before being sequentially methylated into 5-MTHF in order to become metabolically useful 19. The activity of DHFR in the intestinal mucosal cells however, has limited ability to reduce folic acid, therefore resulting in circulating unmetabolized folic acid following folic acid intakes > 0.2 mg 41,42. Unmetabolized folic acid has been detected in populations with 43,44 and without fortification 45, as well as in the serum and cord blood, and breast milk of women taking supplements during pregnancy and lactation 46,47. The potential adverse effects of unmetabolized folic acid are not understood.  1.4 Epidemiological Studies of Maternal Folate Status and Offspring Health The successful implementation of folic acid supplementation in Canada and several other countries has resulted in elevated folate status, particularly during pregnancy 48. There is epidemiological evidence to suggest that high maternal folate status can lead to adverse 10  metabolic outcomes for offspring. The Pune Maternal Nutrition Study in India is a longitudinal prospective cohort study that was established in 1994 to assess maternal dietary intakes and micronutrient status during pregnancy and continuously assess body composition and cardiometabolic risk factors in offspring every 6 months. In a study from this cohort (n=653 mother-child dyads) investigating maternal folate status and offspring adiposity and insulin resistance at 6 years of age, authors reported that high maternal erythrocyte folate (> 1144 nmol/L) at 28 weeks of pregnancy was associated with greater adiposity and indicators of insulin resistance (calculated by homeostatic model assessment of insulin resistance, HOMA-IR), in children at age 6 years 49. Children had the highest HOMA-IR when their mothers had high erythrocyte folate but low plasma vitamin B12 (<114 pmol/L) at 18 weeks of pregnancy 49. Another study conducted in India, the Parthenon Study (n=533-539 mother-child dyads), aimed to replicate findings of the Pune study. This study reported that maternal folate concentrations around 30 weeks of pregnancy were positively associated with insulin resistance (calculated by HOMA-IR) in the children at ages 9.5 and 13.5 years 50. In contrast to the Pune study, there was no interaction between folate and vitamin B12 in relation to childhood insulin resistance as both vitamin B12 deficient mothers, with or without high folate concentrations, had children that presented with insulin resistance 50.  The majority of pregnant and non-pregnant women of reproductive age residing in countries with mandatory folic acid fortification have improved folate status and a significant decline in the prevalence of folate deficiency (erythrocyte folate <305 nmol/L) 40,51–53. However, there remain some women who are considered to have low maternal folate status in which their offspring can display negative metabolic outcomes 54. The Boston Birth Cohort study (n=1517 mother-child dyads) reported an L-shaped relationship between maternal plasma folate at 48-72 11  hours post-delivery and child BMI z-scores at age 9 years 54. Children from mothers in the lowest quartile of plasma folate (6.6- <20.4 nmol/L) had the highest BMI z-scores and greatest incidence of overweight or obesity (BMI ≥ 85th percentile for sex and age) compared to children from women with plasma folate in the 2nd- 4th  quartiles 54. These studies investigating maternal folate status provide evidence of a role for folate in the development of metabolic risk in offspring.  1.5 Rodent Studies of Maternal Folic Acid Supplementation and Offspring Health 1.5.1 Effects on Adult Offspring Adiposity and Glucose Homeostasis Rodent models of maternal folic acid supplementation provide further insight into the mechanisms underlying the effects of maternal folate status on offspring metabolism. Rodent models are advantageous due to their short gestational period and life span, as well as the ability to control various aspects of the pre- and postnatal environment, including diet. Several studies have sought to confirm and further understand the impact of maternal high folic acid supplementation on offspring adiposity and glucose homeostasis 55–60. The Devlin lab previously established a mouse model of maternal folate and vitamin B12 imbalance to further understand the molecular and physiological effects on adult offspring 55,56. Female C57BL/6J mice were fed a folic acid supplemented diet [10 mg/kg diet; 5X American Institute of Nutrition (AIN) recommendations for mice] with, or without vitamin B12 before and during pregnancy and lactation. Sex-specific differences in adiposity and glucose homeostasis were observed. Male offspring from folic acid supplemented dams gained less weight, had smaller retroperitoneal and subcutaneous fat pads, and had no changes in glucose tolerance (as assessed by an intraperitoneal glucose tolerance test, IPGTT) 55. In contrast, female offspring from folic acid supplemented 12  dams had greater fat mass, larger gonadal fat pads, fasting hyperglycemia, and increased glucose intolerance [increased IPGTT area under the curve (AUC)] 56.  Similar to observations made in the Devlin Lab, others have investigated differences in offspring adiposity and glucose homeostasis from mothers fed a diet supplemented with folic acid. A study of Sprague-Dawley rat offspring from dams supplemented with folic acid (5 mg/kg; 2.5X AIN recommendations) reported that male offspring gained more body weight and female offspring gained less body weight compared to male and female offspring from un-supplemented dams, respectively 57. Similar to reports from the Devlin Lab, Hoile et al. reported sex-specific differences in fasting plasma glucose in offspring from Wistar rat dams supplemented with folic acid (5 mg/kg diet; 2.5X AIN recommendations) in which female, but not male offspring, displayed elevated fasting blood glucose 58. In another study, investigators fed female Sprague-Dawley rats a diet supplemented with folic acid (40 mg/kg diet; 20X AIN recommendations) and reported reduced glucose tolerance (as assessed by oral GTT) in both male and female adult offspring 59. Greater glucose intolerance was also reported in high fat diet-fed male C57BL/6 mouse offspring from folic acid supplemented dams (20 mg/kg diet; 20X AIN recommendations); females were not assessed in this study 60. Despite the variable findings observed in rodent models, it is clear that the key findings suggest a role for maternal folate status on the development of adiposity and impaired glucose homeostasis in adult offspring.  1.5.2 Effects on Offspring One-Carbon Metabolism Maternal folic acid supplementation during pregnancy may alter one-carbon metabolism in the offspring. Few studies have investigated changes in methyl metabolism in offspring from dams supplemented with folic acid. The Devlin Lab previously reported differential mRNA expression of enzymes involved in methyl nutrient metabolism in the liver of adult male and 13  female offspring from folic acid supplemented dams (10 mg/kg diet; 5X AIN recommendations) 55,56. Male offspring expressed higher Mthfr mRNA 55 whereas female offspring expressed lower Mtr mRNA and had increased liver SAH 56. In another study, Bahous et al. quantified several hepatic one carbon enzymes and choline metabolites in fetal (embryonic day 17) and 3 week old weanling male C57BL/6 mouse offspring from dams fed a diet supplemented with folic acid (20 mg/kg diet; 10X AIN recommendations). Fetal offspring liver had reduced MTHFR protein and mRNA, decreased Pemt and Mat1a mRNA, a trend for reduced Bhmt mRNA, and decreased betaine, choline, and phosphocholine 61. At 3 weeks of age, male offspring continued to have reduced MTHFR protein and phosphocholine in the liver 61. Although observations vary between studies, these findings suggest that maternal folic acid supplementation disturbs offspring one-carbon metabolism, beginning during embryonic development and continuing into adulthood.  1.6 Maternal Obesity and Obstetrical and Neonatal Outcomes Obesity is a human condition defined by a BMI of 30 kg/m2 and is characterized as an accumulation of excess body fat 62. This condition is associated with an increased risk of several adverse health conditions including cardiovascular disease (CVD) and type 2 diabetes (T2D) 62. Obesity is becoming increasingly prevalent around the world 62. This is particularly of concern for women of childbearing age, due to the effects on pregnancy and offspring health. In 2016, the World Health Organization reported that 40% of adult women (aged 18 years and older) had overweight (BMI 25-29.9 kg/m2) and 15% had obesity 62. Women with overweight or obesity before pregnancy are at an increased risk of having an obstetrical complication such as gestational hypertension, preeclampsia, or gestational diabetes mellitus (GDM) 63. A secondary analysis of a prospective study conducted in Canada (n=1996 singleton pregnancies) reported that women with increasing prepregnancy BMI had increasing risk of developing GDM 64. 14  Furthermore, pregnancy is accompanied by gestational weight gain (GWG) which is necessary to ensure proper fetal growth. It encompasses multiple characteristics, including maternal fat accumulation, fluid expansion, and the growth of the fetus, placenta, and uterus 65,66. Guidelines from The US National Academy of Medicine (formerly the Institute of Medicine) are in place for GWG and take into consideration maternal prepregnancy BMI; women who weigh less prior to pregnancy should gain more weight during gestation compared to those who have prepregnancy overweight or obesity 65. Similar to maternal prepregnancy BMI, excessive GWG has also been associated with adverse outcomes for both the mother and offspring 63,67. The risk for adverse obstetrical complications increases with increasing prepregnancy BMI and GWG across the full range of BMI and weight gain, with the highest risk for women with obesity who experience excessive GWG 63,67.  Maternal prepregnancy BMI and GWG contribute to adverse neonatal outcomes including preterm birth (<32 weeks of gestation), stillbirth, or large for gestational age (LGA) at birth 63,68. A cross-sectional study from the Canadian Maternity Experience Survey (n=71,200 women) investigated the risk of prepregnancy BMI and GWG on preterm birth and LGA from Canadian pregnancies and reported that maternal weight, and GWG in particular, significantly increased the risk of these adverse neonatal outcomes 68. Similarly in the United States of America (USA), a retrospective study of pregnant women (n=112,309 deliveries) reported an increase in preterm birth, LGA, and intensive care unit admission as maternal BMI increased 69. An individual participant data meta-analysis of European, North American, and Australian cohorts investigated the impact of maternal prepregnancy BMI and GWG on obstetrical and neonatal outcomes and reported that both maternal parameters were associated with increased risk of preterm birth and LGA 63. Findings from these reports investigating obstetrical and 15  neonatal outcomes identify the importance of promoting and maintaining a healthy weight prior to, and during pregnancy, in order to have a successful pregnancy and birth for both mother and child.  1.7 Maternal Obesity and Offspring Cardiometabolic Health Cardiometabolic disease is a cluster of metabolic derangements such as obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension that promote the development of CVD and/or T2D 70. Aside from lifestyle choices and genetic predisposition, research has provided ample evidence to support the theory of developmental programming as a factor driving the development of cardiometabolic disease. This evidence will be discussed in the following sub-sections.  1.7.1 Maternal Obesity and Offspring Adiposity Maternal obesity increases the likelihood that the offspring will also present with obesity beginning in childhood and often carrying into adulthood. A prospective, longitudinal study by Fraser et al. (n=5154 mother-child dyads) sought to investigate the association between maternal prepregnancy BMI and GWG, and offspring health in 9-year-old children 71. They reported that children whose mothers gained more than the recommended amount of weight during pregnancy had greater BMI, waist circumference, and fat mass. Additionally, GWG before 14 weeks was positively associated with offspring adiposity. Many studies have observed the association between maternal adiposity and offspring health not only in childhood, but in adulthood as well, indicating that exposure in utero can persist much later into life. A prospective study (n=1400 adults) investigated adiposity and cardiometabolic outcomes in 32-year-old offspring and correlated them to maternal prepregnancy BMI and GWG 72. Maternal prepregnancy BMI and GWG were both independently positively associated with offspring BMI, waist circumference, 16  and several other cardiometabolic health risks. These studies and others 73,74 indicate that maternal overweight and obesity are influential factors in the progression of cardiometabolic disease. Furthermore, disease progression is associated with greater all-cause premature mortality and cardiovascular events 75, adding to the evidence that maternal BMI status can influence offspring health during development and have lasting consequences in adulthood.  Several rodent studies have replicated similar findings of offspring adiposity from mothers with diet-induced obesity. Samuelsson et al. fed female C57BL/6J mice either a standard chow or an obesogenic diet [20% kcal fat as lard with ad libitum access to sweetened condensed milk (8% kcal fat)] for 6 weeks before breeding and assessing male and female offspring 76. At 3 and 6 months of age, offspring from dams fed the obesogenic diet displayed increased body weight, larger fat pads, and had elevated fasting plasma triglycerides. Another study fed female C57BL/6J mice either a control or high fat diet (62.2% kcal fat as soybean oil + lard) during gestation and male and female offspring were assessed 77. Body weight in both male and female offspring from dams fed the high fat diet were greater at birth and continued from weaning until 6 weeks of age. Male offspring additionally had increased liver triglyceride concentrations. Other studies in C57BL/6J male mouse offspring from dams fed obesogenic diets have similarly reported increased body weight at 3 weeks and at 3 months of age 78,79.  1.7.2 Maternal Obesity and Offspring Glucose Homeostasis 1.7.2.1 Insulin Signalling and Glucose Uptake Other cardiometabolic factors leading to CVD and/or T2D are related to derangements in glucose homeostasis. Glucose homeostasis refers to the tight regulation of blood glucose levels by the pancreatic hormones insulin and glucagon. Insulin is secreted by β cells, and glucagon is secreted by  cells. These two cells, along with other cell types make up a cluster of cells known 17  as an islet of Langerhans 80. The β cell is responsible for producing, storing, and releasing insulin to maintain glucose homeostasis. When plasma glucose levels rise, glucose enters the β cells through insulin-independent glucose transporter type 2 (GLUT2) where it will be metabolized, resulting in the generation of ATP 81. This will lead to the closure of ATP-dependent potassium channels, depolarization, and an influx of calcium into the β cells 81. Calcium will subsequently initiate the exocytosis of insulin-secreting vesicles which will release insulin into circulation, proportional to the concentration of blood glucose 81. Insulin promotes the uptake of glucose in peripheral tissues including skeletal muscle and adipose tissue. When insulin binds to insulin receptor (IR) on the surface of these tissues, a signaling cascade occurs to phosphorylate insulin receptor substrate-1 (IRS-1) and subsequently activate phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) 82. Activation of these kinases initiates the translocation of glucose transporter 4 (GLUT4) to the plasma membrane for glucose uptake 83. When insulin signalling is impaired and cells no longer adequately respond to insulin, this is referred to as insulin resistance; a condition that is associated with obesity and the development of T2D 84. Insulin resistance can cause hyperglycemia, which promotes increased demand on pancreatic β cells to produce and secrete more insulin. In the presence of obesity where β cells are chronically exposed to excess nutrients including glucose and free fatty acids, insulin release is augmented 81. This hyperinsulinemia can lead to β cell compensation, followed by peripheral insulin resistance, or vice versa 80. β cell compensation includes further secretion of insulin and increased β cell mass. Ultimately, these events will result in β cell dysfunction if hyperinsulinemia is prolonged and β cell compensation is unable to be sustained 80.  18  1.7.2.2 Obesity and Diabetes During Pregnancy and Offspring Glucose Homeostasis Impairments in offspring glucose homeostasis can stem from maternal obesity and/or GDM. Gestational diabetes mellitus refers to the development of any degree of hyperglycemia recognized for the first time during pregnancy 85. There is controversy over the definition of GDM based on diagnostic criteria and screening method 85. Two types of screening methods exist, universal and selective screening 86. Selective screening is centered on the presence of specific risk factors based on ethnicity 87, whereas universal screening, the approach used in Canada, screens all pregnant women for GDM between 24-28 weeks of gestation 86. There are several risk factors that can lead to the development of GDM, including the presence of pregestational overweight or obesity 63,67,88. A healthy pregnancy involves a change in insulin sensitivity, usually resulting in a mild state of insulin resistance that is reversed following birth 89. This change in insulin sensitivity is due to fetal growth, GWG, greater food intake, and placental hormone production 88. Maternal pancreatic islets respond to this demand by enhancing insulin secretion by  cells, augmenting  cell proliferation, and  cell mass expansion 90. In the case of GDM, impaired  cell function is a result of inadequate secretion of, and responsiveness to insulin, therefore leading to insulin resistance and glucose intolerance 88. Exposure to elevated glucose concentrations during pregnancy can program the offspring to have adverse health risks such as obesity, impaired glucose tolerance, and T2DM in childhood and adulthood 91–94. For example, a prospective study investigated adiposity and insulin resistance in offspring of mothers who had GDM (n=232 mother-child dyads) and reported that at 11 years of age, children exposed to hyperglycemia during gestation were overweight and had insulin resistance (calculated by HOMA-IR) 94. This insulin resistance was further associated with the child’s BMI. Studies have similarly investigated changes in offspring glucose 19  homeostasis in relation to maternal obesity. A family-based study of adult offspring from mothers with obesity during pregnancy (n=67 individuals) assessed insulin sensitivity and secretion from 1-3 siblings of each family 95. All offspring from mothers with obesity had a greater BMI, waist circumference, and fat mass in adulthood. Following an oral GTT, offspring were glucose intolerant and insulin resistant [calculated using an oral glucose insulin sensitivity (OGIS) index 96]. Interestingly, only male offspring exposed to maternal obesity were able to compensate for the insulin resistance by increasing insulin secretion (calculated using a method developed by Mari et al. 97), whereas females were not able to do the same.    It is clear that maternal obesity and hyperglycemia are capable of impairing offspring glucose homeostasis. Rodent studies have further identified the sex-specific differences in offspring glucose homeostasis. Reports from these studies have been variable largely due to differences in fat source and fat content in the obesogenic diets employed in each study. This is in addition to species and study design differences. Nevertheless, information from these studies continue to provide understanding into the programming of offspring metabolic health following maternal obesity. A study fed female C57BL/6J mice either a control or high fat diet (62.2% kcal fat as soybean oil + lard) during gestation and male and female offspring were assessed 77. Glucose tolerance was assessed by IPGTT in which male offspring were glucose intolerant at 6, 14, and 20 weeks of age whereas female offspring were glucose intolerant at 6 weeks only. Furthermore, insulin tolerance tests conducted at 20 weeks revealed that both males and females were unable to clear insulin after 60 minutes following an insulin bolus. Another study of adult female Sprague-Dawley rat offspring from dams fed a fat-rich diet (20% kcal fat as lard) reported that female offspring were more insulin resistant as assessed by a euglycemic-hyperinsulinemic clamp 98. Moreover, ex vivo glucose-stimulated insulin secretion from isolated 20  islets from this same study was reduced in offspring from dams fed the fat-rich diet compared to offspring from chow-fed dams. In a study of male C57BL/6 mouse offspring from dams fed a high fat diet (49% kcal fat as soybean oil + lard), male offspring were glucose intolerant (as assessed by oral GTT AUC), had elevated fasting plasma insulin concentrations, and had increased β and  cell mass 79. Other studies in male rodent offspring from high fat diet-fed dams have reported similar findings in glucose intolerance following an IPGTT 99,100.  1.8 Thesis Hypotheses and Specific Aims Folic acid is required for proper development and NTD prevention. However, little is known about the metabolic effects of high folic acid intakes, particularly in women with gestational obesity and/or prediabetes, on the consequences for the developing fetus. Individually, maternal gestational obesity and prediabetes have several negative consequences for offspring health 71,76,79. Many studies have examined the individual effects of maternal gestational obesity/prediabetes or folic acid supplementation in adult offspring 55,56,77, however what remains unclear is the timing at which the adverse effects manifest and how both maternal environments work together to influence offspring health. The objective of my MSc research was therefore to use a mouse model to determine the effect of maternal folic acid supplementation in a healthy pregnancy and a pregnancy complicated by obesity and glucose intolerance on fetal offspring liver and pancreas, and on the metabolic health of the mother.  I hypothesize that supplemental folic acid during pregnancy will exacerbate i) the adverse metabolic health outcomes associated with gestational obesity and prediabetes in the mother; and ii) the adverse effects of maternal gestational obesity and prediabetes on fetal offspring liver and pancreas. These hypotheses were addressed by the following three specific aims: 21  AIM 1: To determine if maternal folic acid supplementation before and during pregnancy affect maternal adiposity, glucose and insulin tolerance, and β cell function in dams with or without diet-induced obesity (Chapter 3). AIM 2: To determine if maternal diet-induced obesity/glucose intolerance and folic acid supplementation affect liver one-carbon metabolism in fetal offspring (Chapter 4). AIM 3: To determine if maternal diet-induced obesity/glucose intolerance and folic acid supplementation affect β and  cell mass in fetal offspring (Chapter 5).   22  Chapter 2: Materials and Methods 2.1 Mice and Study Design Female and male C57BL/6J mice were gifted by Dr. Francis Lynn (Associate Professor, Department of Surgery, UBC). All mice were housed in cages of 2-4 animals per cage in the Animal Care Facility at the BC Children’s Hospital Research Institute (BCCHRI) under a standard 12-hour light-dark cycle with ad libitum access to food and water. Female mice were fed from weaning (3 weeks of age) one of four diets formulated by Research Diets Inc. (New Brunswick, NJ, USA); details on the composition of the diets are provided in Table 2.1. The four diets were as follows: control diet with recommended level folic acid (CD), control diet with supplemental folic acid (CDF), western diet with recommended level folic acid (WD), and western diet with supplemental folic acid (WDF). The control diets (CD, CDF; control dams) contained 10% energy from fat, and the western diets (WD, WDF; western dams) contained 45% energy from fat to induce excess adiposity and glucose intolerance in the mice as a model of gestational obesity and prediabetes. The western diet was chosen to mimic the fat content commonly observed in westernized human populations. The control and western diets with recommended folic acid (CD, WD) contained approximately 2 mg/kg diet of folic acid, as recommended for rodents by the AIN-93G diet 101,102. Diets with supplemental folic acid (CDF, WDF) contained approximately 10 mg/kg diet of folic acid which is 5X the AIN-93G recommendations. All diets met the nutrition requirements for rodents set by the AIN and the National Research Council 101–103.     23  Table 2.1. Diet Composition Ingredients  (g/kg diet)  Control Diet (CD) D12450K Western Diet (WD) D12451 Control Diet + Folic Acid (CDF) D17032103 Western Diet + Folic Acid (WDF) D17032104 Fat (% kcal) 10 45 10 45      Lard 20 177.5 20 177.5      Soybean Oil 25 25 25 25 Protein (% kcal) 20 20 20 20      Casein, 30 Mesh 200 200 200 200      L-Cystine 3 3 3 3 Carbohydrate (% kcal) 70 35 70 35      Cornstarch 550 72.8 550 72.8      Sucrose 0 172.8 0 172.8      Maltodextrin 10 150 100 150 100      Cellulose, BW200 50 50 50 50 Vitamin Mix V10001* 10 10 10 10 Choline Bitartrate 2 2 2 2 Folic Acid 0 0 0.008 0.008 Folic Acid (mg/kg diet) 1.97 2.42 9.55 11.75 Total Energy  (kcal/kg diet) 3800 4700 3800 4700 * Contains 2 mg folic acid per 10 g V10001. Female mice were assigned to a diet group at weaning and fed for 13 weeks with body weight measured weekly. At 13 weeks of feeding, female mice were singly-housed and bred with age-matched male mice fed the control diet. A male was placed into each female cage in the evening and separated the following morning; this was repeated until pregnancy was confirmed by the presence of a vaginal plug, indicating embryonic day (E) 0.5. On E18.5 of pregnancy, dams were anesthetized with isoflurane for blood collection by cardiac puncture, followed by immediate sacrifice by cervical dislocation and collection of maternal and fetal tissue. To collect fetal tissues, the uterine horn was dissected and placed in ice cold 1X phosphate buffered saline (PBS) before individually dissecting each fetus on a petri dish filled with ice. The total number of viable and non-viable (fetal resorption) pups were counted to calculate embryonic loss as the 24  percent of resorptions per total litter size. All maternal and fetal tissues were either flash frozen in liquid nitrogen to be stored in -80C, or fixed in 4% paraformaldehyde overnight. Blood was allowed to clot on ice for 30 mins before centrifugation at 8000 rpm for 10 mins at 4C. Serum was separated into 100 L aliquots and stored at -80C. The experimental design is illustrated in Figure 2.1.   Figure 2.1. Experimental Design. Female C57BL/6J mice (dams) were fed either a control or western diet (CD, WD), with or without supplemental folic acid (CDF, WDF) from weaning (3 weeks of age). At 13 weeks of feeding, dams were bred and pregnancy was confirmed by presence of a vaginal plug to mark embryonic day (E) 0.5. Pregnancy was carried out for 18 days before collecting maternal and fetal tissue.   2.2 Body Composition Body composition was quantified in conscious female mice by quantitative magnetic resonance (EchoMRI-100; Echo Medical Systems, Houston, TX, USA) after 10 weeks on diet; 2-3 days before undergoing physiological assessments of glucose homeostasis. For each mouse, 2-3 scans were taken and averaged to calculate the percentage of fat and lean mass.   CD WD CDF WDFBreedingFemale C57BL/6J MiceTissue Collection (E18.5)13 weeks18 daysPregnancy25  2.3 Physiological Assessments of Glucose Homeostasis Glucose homeostasis was assessed in females prior to breeding. A glucose stimulated insulin secretion test (IST) was conducted as a physiological indicator of  cell function at 11 weeks on diet, an intraperitoneal insulin tolerance test (IPITT) was conducted as a physiological indicator of insulin sensitivity at 12 weeks on diet, and an IPGTT was carried out to assess glucose excursion at 13 weeks on diet. Assessments were conducted with one week between tests to provide sufficient recovery time.   For ISTs, D-dextrose (Sigma-Aldrich) was dissolved in 0.9% NaCl and filtered through a 0.2 m syringe filter to a final concentration of 0.15 g/mL. Animals were fasted for 5 hrs prior to injection, fasting blood glucose was measured from a tail prick with a glucometer (OneTouch Verio Meter; LifeScan, Malvern, PA, USA), and 70 L of blood from the tail vein was collected. Mice were subsequently injected with 0.75 g D-dextrose/kg lean mass and 35 L of blood from the saphenous vein was collected at 2, 15, and 30 mins post-injection; blood glucose concentrations were also measured at 15 and 30 mins by glucometer. Blood samples were allowed to clot on ice for 30 mins, centrifuged at 8000 rpm at 4C for 10 mins, and serum was collected and stored at -80C. Fasting serum insulin concentrations were quantified by the Mouse Ultrasensitive Insulin ELISA kit (ALPCO) as per manufacturer’s instructions.  For IPITTs, insulin (Novolin ge Toronto; Novo Nordisk Canada Inc., Mississauga, ON, CAN) was diluted in filtered 1X PBS to 0.1 U/mL. Mice were fasted for 5 hrs and fasting blood glucose was measured by glucometer. Mice were then injected with 0.75 U insulin/kg lean mass and blood glucose concentrations were measured by tail prick at 15, 30, 60, 90, and 120 mins post-injection. Similarly, for IPGTTs, animals were fasted for 5 hrs and fasting blood glucose 26  measured. Mice were injected with 0.75 g D-dextrose/kg lean mass and blood glucose concentrations were measured by tail prick at 15, 30, 60, 90, and 120 mins post-injection.  2.4 Genotyping of the Sry Gene for Fetal Sex Determination  Genomic DNA was extracted from fetal tail snips using the Gentra Puregene Tissue Kit (Qiagen) following the manufacturer’s protocol. The purity and concentration of extracted DNA was assessed with a NanoDrop spectrophotometer (NanoDrop 2000c; NanoDrop Technologies, Wilmington, DE, USA) in which a 260/280 nm absorbance ratio of approximately 2.0 was considered pure DNA. Multiplex polymerase chain reaction (PCR) was used to simultaneously amplify a 273 base pair sequence of the Sry gene (encoding sex-determining region of the Y chromosome) and a 203 base pair sequence of the Actb gene (encoding -Actin), which was used as an internal control. The forward and reverse primers for the Sry and Actb sequences were as follows: Sry forward, 5'-TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA-3'; Sry reverse, 5'-CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA-3'; Actb forward, 5'-AGC TCA GTA ACA GTC CGC CTA-3'; and Actb reverse, 5'-CAG AGA GCT CAC CAT TCA CCA T-3' (Integrated DNA Technologies). Extracted liver DNA from a known male C57BL/6N mouse was used as a positive control. Preparation of the PCR reaction comprised of 2 L of extracted DNA and 23 L of a master mix [10X PCR buffer (Qiagen), Taq DNA polymerase (Invitrogen), 10 mM dNTPs, 10 mM each of forward and reverse Sry and Actb primers, dH2O]. Thermal cycler conditions for the PCR were as follows: denaturation at 95C for 2 mins; amplification (35 cycles) at 95C for 1 min, 62C for 1 min, and 72C for 1 min; extension at 72C for 4 mins; and hold at 4C. Reactions were subsequently run on a 2.5% 27  agarose gel to visualize bands. The presence of both Sry and Actb bands indicated a male offspring, whereas the presence of only the Actb band indicated a female offspring. 2.5 Pancreas Processing Dissected pancreata were immediately fixed in 4% paraformaldehyde at 4C overnight, washed with 1X PBS, and stored in 70% EtOH at 4C until embedding in paraffin. Embedding consisted of dehydrating the tissue at room temperature through a series of graded ethanol baths (2x 95% EtOH, 3x 100% EtOH; 30 mins each) to displace water, clearing the ethanol with xylene (2x 30 mins each), and infiltrating with paraffin wax (2x 1 hr each) before embedding the tissue in wax blocks for long term storage.  Sections of pancreas were cut from paraffin blocks using a microtome to prepare 5 m thick slices. Tissue sections were floated in a 40C distilled water bath, mounted on glass microscope slides (VWR), air-dried overnight, and stored at room temperature.  2.6 Immunohistochemical Analysis of Insulin and Glucagon   For each fetal pup, 3 pancreatic sections separated by 30 m each were stained for insulin and glucagon. Slides containing pancreatic sections were deparaffinized in xylene (3x 10 mins each) and rehydrated in a series of graded ethanol baths and water (2x 100% EtOH, 1x 95% EtOH, 1x 70% EtOH; 10 mins each, 1x 50% EtOH, 1x 30% EtOH; 2 mins each, 1x distilled water; 1 min, 1x 1X PBS; 10 mins). Slides were then submerged in 500 mL of antigen retrieval buffer (10 mM sodium citrate dihydrate, 0.05% Tween-20, pH 6.0) and heated to 95-100C in the microwave for 6 mins at high power followed by 20 mins on a timed defrost setting to expose antigenic sites for antibody binding. Once the buffer containing the slides was cooled to room temperature, sections were briefly dried off and placed in a humid chamber. An outline was 28  traced around the perimeter of each section using a hydrophobic pen (Diagnostic BioSystems) followed by the addition of blocking solution (Dako) for 1 hr at room temperature to block non-specific binding. Blocking solution was removed and a primary antibody cocktail of insulin (1:100, rabbit anti-mouse; Cell Signaling) and glucagon (1:500, mouse monoclonal anti-mouse; Sigma-Aldrich) in antibody diluent (Dako) was added for an overnight incubation at 4C. Slides were subsequently washed 3x in 1X PBS with gentle shaking at room temperature. Sections were briefly dried off, and a secondary antibody cocktail (4 g/mL, Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG; Invitrogen) in antibody diluent was added to incubate for 1 hr at room temperature in a dark humid chamber. Slides were then washed 3x in 1X PBS with gentle shaking and briefly dried off. Mounting medium with DAPI (Vectashield) was added to each section, followed by the addition of a coverslip and a clear nail polish seal. As secondary antibodies are light-sensitive, all steps following overnight incubation with primary antibodies were carried out in a dark room. Images of whole pancreas and insulin and glucagon-positive cells were visualized and tiled with a BX61 fluorescence microscope (Olympus; Tokyo, Japan) and quantified by Fiji ImageJ software 104.  cell mass and  cell mass were calculated as the insulin- or glucagon-positive area expressed as a percentage of the whole pancreas area, respectively.  cell density was calculated as the number of insulin-positive particles per 100 mm2 of whole pancreas area.  2.7 Quantification of Fetal Liver Triglyceride Concentrations Whole frozen fetal livers (40-70 mg) were homogenized in 100 L of dH2O by sonication followed by centrifugation at 8000 rpm for 10 mins at 4C. The supernatant was diluted 1:100 in ddH2O and protein concentration was immediately determined by the Bradford 29  Protein Assay using the Quick Start Bradford 1X Dye Reagent (Bio-Rad) 105. Total lipids were extracted from homogenized liver using a modified Folch et al. method 106. A solvent proportion of 2:1:0.75 v/v/v chloroform/methanol/homogenate was thoroughly vortexed followed by centrifugation at 2500 rpm for 5 mins at room temperature. The lower organic layer was then transferred to a new microcentrifuge tube to dry the lipids using a nitrogen evaporator with additional heat for 15 mins. Dried lipids were subsequently resuspended in 75 L 1:1 v/v Triton X-100 (Sigma-Aldrich)/methanol, vortexed thoroughly, and sonicated for 15 mins at 40C. Lipid extracts were stored at -80C.   Triglycerides were quantified using a colorimetric kit (Triglyceride Reagent Set; Pointe Scientific, Canton, MI, USA) and a triglyceride standard (Pointe Scientific) was diluted to create a standard curve (200, 160, 100, 50, and 25 mg/dL). In a 96-well plate, 10 L of sample was mixed with 180 L of triglyceride reagent prewarmed to 37C. Samples were incubated for 5 mins with constant shaking at 700 rpm before reading the absorbance at 500 nm. Triglyceride concentrations were normalized to protein concentration.   2.8 Quantification of Fetal Liver Water Soluble Choline Metabolite Concentrations Hepatic water soluble choline metabolites (free choline, phosphocholine, glycerophosphocholine, and betaine) were quantified by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using stable isotope-labeled internal standards. These analyses were performed by Roger Dyer, Senior Laboratory Technician at the Analytical Core for Metabolomics and Nutrition at BCCHRI.  30  2.9 Quantification of Liver SAM and SAH Concentrations   Maternal and fetal livers were dissected and weighed. To prevent enzymatic conversion of SAM and SAH during the procedure 107, stabilize SAM from degradation using an acidic pH 108, and deproteinize the tissue, 0.4 M perchloric acid (50 L per 0.01 g tissue) was immediately added to excised tissue and homogenized in a Bullet Blender Tissue Homogenizer (Next Advance Inc., Troy, NY, USA). Homogenates were centrifuged at 13,000 rpm for 10 mins at 4C and the supernatant was flash frozen in liquid nitrogen followed by storage at -80C.  Tissue extracts were packaged on dry ice and sent to Dr. Joshua Miller’s laboratory at Rutgers University, New Brunswick, NJ, USA for the quantification of SAM and SAH by high-performance liquid chromatography (HPLC) with UV detection using the methods of Fell et al. 109 with modifications by Miller et al. 110.  2.10 Statistical Analyses Two-way analysis of variance (ANOVA) was used to determine the effect of maternal diet and folic acid supplementation. If a significant (p<0.05) interaction between maternal diet and folic acid supplementation was observed, a subsequent t-test was performed to determine the effect of folic acid supplementation separately in dams fed the control diet (CD vs CDF) and dams fed the western diet (WD vs WDF). Data from one male and one female offspring per litter were used for analyses and were conducted separately. Analyses were performed using SPSS Statistics Software (version 27, IBM) with p<0.05 considered statistically significant. Individual data points were graphed using GraphPad Prism 5 software and bar graphs represent mean  standard deviation (SD).  31  Chapter 3: Effects of Folic Acid Supplementation on Maternal Adiposity and Glucose Homeostasis This chapter presents the findings for Aim 1: To determine if maternal folic acid supplementation before and during pregnancy affect maternal adiposity, glucose and insulin tolerance, and β cell function in dams with or without diet-induced obesity. 3.1 Rationale Folic acid has been identified to reduce the risk of having a NTD-affected pregnancy. As such, women of childbearing age are recommended to consume 0.4 mg of folic acid per day to reduce this risk 35. Furthermore, those with pregestational obesity or diabetes are at a higher risk of having a NTD-affected pregnancy and are recommended to take up to 5.0 mg of folic acid per day which is 12.5X the recommended dose for women at low risk 35. There is evidence in both population-based and rodent studies to suggest that consuming high levels of folic acid can have negative consequences for the offspring 49,50,55,56. Moreover, there may be additional risks for the mother herself. Epidemiological studies have outlined contrasting associations between folic acid supplementation during pregnancy and risk for GDM 111,112. In a prospective cohort study of Chinese women (n=4353 pregnancies), high doses of folic acid supplements ( 0.8 mg/day) during pregnancy were positively associated with increased risk for GDM 112. In contrast, a prospective cohort study from the Nurses’ Health Study II (n=20,199 pregnancies) reported that increasing habitual intakes of supplemental folic acid (up to  0.6 mg/day) before pregnancy were associated with lower risk for GDM 111. Another adverse consequence for the mother is increased adiposity. In female Sprague-Dawley rats supplemented with folic acid during mating, pregnancy, and lactation (40 mg/kg diet, 20X AIN recommendations), dams gained more weight 32  after weaning of their offspring but had smaller gonadal fat pads compared to control dams 59. Persistent exposure to glucose intolerance either as GDM or milder forms of glucose intolerance, as well as increased adiposity can lead to the development of T2D later in life as well as other cardiometabolic diseases 113–115. Women with obesity and/or diabetes are being prescribed very high doses of folic acid prior to pregnancy despite the lack of large-scale epidemiological evidence to suggest that this is necessary. Investigation into the effects on maternal health has been minimal. In particular, the effect of folic acid supplementation in combination with obesity on maternal adiposity and glucose homeostasis has not been studied.  3.2 Results 3.2.1 Western diet-fed dams had increased body weight prior to pregnancy Female mice were weaned on to the experimental diets at age 3 weeks and weighed weekly throughout the study until 13 weeks on diet. From weeks 1-13 on diet, western dams had significantly greater body weight (p<0.05) compared to control dams (Figure 3.1). There was no effect of folic acid supplementation on body weight.   Figure 3.1. Body Weight of Dams Throughout 13 Week Feeding Period. Dams were weaned at age 3 weeks onto the experimental diets and fed for 13 weeks. Body weight was measured weekly. Each time point on the line graph was analyzed by two-way ANOVA. Data are presented as mean  SD (n=11-14/group). *p<0.05, effect of diet. 13 Week Growth CurveWeeks on DietWeight (g)0 1 2 3 4 5 6 7 8 9 10 11 12 13010203040CDCDFWDWDF* ** ** * * ** * * **33  3.2.2 Folic acid supplementation altered insulin sensitivity prior to pregnancy  Prior to breeding, physiological assessments of glucose homeostasis were carried out on the dams. At 11 weeks on diet, ISTs were performed on fasted dams as a physiological indicator of  cell function. Fasting insulin was higher (p=0.032) in western dams compared to control dams (Figure 3.2A). No difference in glucose-stimulated insulin secretion was observed following IP injection of glucose (Figure 3.2B), however IST AUC was higher (p=0.042) for western dams compared to control dams (Figure 3.2C); there was no effect of folic acid supplementation. At 12 weeks on diet, IPITTs were conducted on fasted dams as an indicator of insulin sensitivity. An interaction between diet and supplement (p<0.05) was observed in insulin clearance at the 90 minute and 120 minute time points, and for overall IPITT area over the curve (AOC) (Figure 3.2D, E). Control dams supplemented with folic acid had reduced insulin sensitivity compared to non-supplemented control dams. The opposite effect was observed in dams fed the western diet; those supplemented with folic acid had improved insulin sensitivity compared to western dams with no folic acid supplementation. At 13 weeks on diet, glucose tolerance was assessed by IPGTT. Dams fed the western diet had significantly higher fasting glucose concentrations and were glucose intolerant (p<0.01) compared to dams fed the control diet (Figure 3.2F-H); no effect of folic acid supplementation was observed. 34   Figure 3.2. Physiological Assessments of Glucose Homeostasis Before Breeding. Dams were fasted for 5 hours before assessing their glucose tolerance, insulin sensitivity, and  cell function. (A) Fasting insulin concentrations before IST. (B) Serum insulin concentrations after IP injection of glucose for IST and (C) area under the curve during IST. (D) Blood glucose expressed as a percent from fasting blood glucose after IP injection of insulin for IPITT and (E) area over the curve during IPITT. (F) Fasting glucose concentrations before IPGTT. (G) Glucose excursion after IP injection of glucose for IPGTT and (H) area under the curve during IPGTT. Bar graphs and each time point on line graphs were analyzed by two-way ANOVA. Data are presented as mean  SD (n=11-14/group). *p<0.05, effect of diet; **p<0.05, effect of supplement.  3.2.3 Western diet feeding increased adiposity before and during pregnancy Body composition data were collected just before glucose homeostasis assessments were conducted. Prior to pregnancy, dams fed the western diet had greater fat mass and lean mass Fasting Insulin Before BreedingSerum Insulin (ng/mL)CD WD CDF WDF0.00.51.01.52.0Beta Cell Function Before BreedingTime (minutes)Serum Insulin (ng/mL)0 5 10 15 20 25 300123Beta Cell Function AUCAUC (ng x min/mL)CD WD CDF WDF020406080Insulin Tolerance Before BreedingTime (minutes)% Baseline Glucose0 30 60 90 120050100150Insulin Tolerance AOCAOC (% x min)CD WD CDF WDF0123Fasting Glucose Before BreedingBlood Glucose (mmol/L)CD WD CDF WDF05101520Glucose Tolerance Before BreedingTime (minutes)Blood Glucose (mmol/L)0 30 60 90 1200510152025Glucose Tolerance AUCAUC (mmol x min/L)CD WD CDF WDF0500100015002000A B CD EF G H13 Week Growth CurveWeeks on DietWeight (g)0 1 2 3 4 5 6 7 8 9 10111213010203040CDCDFWDWDFMaternal Diet* ***** * ***** **diet x supplement; p= 0.031*** *35  (p<0.001) compared to control diet-fed dams (Figure 3.3A, B). At E18.5, both subcutaneous and visceral fat depots, including the gonadal and retroperitoneal fat pads, were larger (p0.001) in western dams compared to control dams (Figure 3.3C-E). Folic acid supplementation did not affect adiposity during pregnancy. In addition to fat depots, organ weights differed with diet. Western dams had larger livers (p=0.008) and smaller pancreata (p=0.001) than control dams (Figure 3.4A, B). An interaction (p<0.05) between the diet and supplement was observed for the kidney and heart weight. Control dams supplemented with folic acid had larger kidneys and hearts (p<0.05) compared to non-supplemented control dams (Figure 3.4C, D). This was not observed in the western dams.   Figure 3.3. Body Composition in Dams Before Breeding and at E18.5. Dam’s body composition was measured before breeding to calculate (A) total fat mass and (B) lean mass expressed as percent of body weight. At E18.5, fat pads were collected and weighed, including (C) subcutaneous fat, (D) retroperitoneal fat, and (E) gonadal fat. Bar graphs were analyzed by two-way ANOVA. Data are presented as mean  SD (n=11-14/group). *p<0.05, effect of diet.  Gonadal FatGonadal Fat(% body weight)CD WD CDF WDF0.01.02.03.04.05.06.0Retroperitoneal FatRetroperitoneal Fat(% body weight)CD WD CDF WDF0.00.51.01.52.0Inguinal FatSubcutaneous Fat(% body weight)CD WD CDF WDF0.01.02.03.04.05.0Fat Mass before BreedingFat Mass (%)CD WD CDF WDF01020304050Lean Mass before BreedingLean Mass (%)CD WD CDF WDF081624A BC D E* * * ** * * * **36   Figure 3.4. Organ Weights in Dams at E18.5. At tissue collection, (A) liver, (B) pancreas, (C) kidneys, and (D) heart were collected, weighed, and expressed as percent of body weight. Bar graphs were analyzed by two-way ANOVA. Data are presented as mean  SD (n=11-14/group). *p<0.05, effect of diet; **p<0.05, effect of supplement.   3.2.4 Maternal diet-induced obesity and glucose intolerance affected liver one-carbon metabolism at E18.5 of pregnancy Folate plays an essential role in the remethylation of homocysteine to methionine for the generation of key methyl donor, SAM 15,21. For this reason, a portion of liver was prepared for quantification of SAM and SAH by HPLC. Western dams had lower liver SAM and SAH concentrations (p<0.05) compared to control dams (Figure 3.5A, B). No differences in SAM/SAH ratio were observed (Figure 3.5C). Folic acid supplementation had no effect on hepatic SAM and SAH concentrations at E18.5 of pregnancy.  LiverLiver Weight(% body weight)CD WD CDF WDF0.02.04.06.08.0PancreasPancreas Weight(% body weight)CD WD CDF WDF0.00.51.01.52.0KidneysKidney Weight(% body weight)CD WD CDF WDF0.00.51.01.5HeartHeart Weight(% body weight)CD WD CDF WDF0.00.20.40.60.8A BC D****diet x supplement; p= 0.028 diet x supplement; p= 0.021****37   Figure 3.5. Hepatic SAM and SAH Concentrations in Dams at E18.5. Quantification of liver (A) SAM and (B) SAH of dams during pregnancy. (C) Ratio of SAM to SAH. Bar graphs were analyzed by two-way ANOVA. Data are presented as mean  SD (n=5-6/group). *p<0.05, effect of diet.  3.2.5 Folic acid supplementation did not affect litter development  At E18.5, all viable pups and non-viable resorption sites were counted. There was no difference in total litter size (Figure 3.6A). Similarly, no difference in the number of resorption sites per litter, calculated as embryonic loss, was observed (Figure 3.6B). Fetal sex was determined in all pups and there was no difference in the proportion of male to female offspring (Figure 3.6C).   Figure 3.6. Litter Size and Distribution at E18.5. All viable pups and resorption sites were counted to give (A) total litter size. (B) Embryonic loss was calculated as the number of resorption sites per litter. Fetal sex was confirmed by PCR of the Sry gene to determine (C) percentage of female pups to male pups in each litter. Bar graphs were analyzed by two-way ANOVA. Data are presented as mean  SD (n=11-14/group).  Dam SAMSAM (nmol/g)CD WD CDF WDF010203040Dam SAM/SAHSAM/SAHCD WD CDF WDF0.00.51.01.52.0Female SAHSAH (nmol/g)CD WD CDF WDF0102030Dam SAHSAM (nmol/g)CD D CDF WDF01020304050* ***A B CSex Distribution% FemaleCD WD CDF WDF020406080100Total Litter SizeImplantation Sitesper LitterCD WD CDF WDF036912Embryonic Loss% Resorptionper LitterCD WD CDF WDF01020304050A B C38  Chapter 4: Effects of Maternal Diet-Induced Obesity/Glucose Intolerance and Folic Acid Supplementation on Fetal Offspring Liver One-Carbon Metabolism This chapter presents the findings for Aim 2: To determine if maternal diet-induced obesity/glucose intolerance and folic acid supplementation affect liver one carbon metabolism in fetal offspring. 4.1 Rationale The liver is a major organ involved in one-carbon metabolism. As discussed in Chapter 1, few studies have investigated the impact of maternal folic acid supplementation on fetal offspring liver methyl metabolism. In C57BL/6 mice, maternal folic acid supplementation (20 mg/kg diet; 10X AIN recommendations) reduced expression of several one-carbon enzymes including MTHFR in the E17.5 fetal liver 61. This study additionally reported reduced concentrations of water soluble choline metabolites including betaine, choline, and phosphocholine in the E17.5 fetal liver 61. In contrast, a study of low maternal folate intake (0.4 mg/kg diet; 0.2X AIN recommendations) reported that E17.5 fetal C57BL/6 mice had altered hepatic gene expression including many genes involved in one-carbon metabolism, adipogenesis, and fatty acid biosynthesis 116.  Maternal obesity and T2D have the potential to affect the development of the offspring and lead to long term negative consequences such as impaired glucose homeostasis and increased adiposity 76,77. These adverse effects may manifest during fetal life. For example, female C57BL/6J mice fed a high fat diet (60% kcal fat as soybean oil + lard) for 6 weeks before breeding and during pregnancy reported that at E17.5, fetal offspring had hepatic triglyceride 39  accumulation and upregulated lipogenic gene expression in the liver 117. Furthermore, fetal offspring from the high fat diet-fed dams had higher choline and lower betaine levels in the liver compared to offspring from control dams 117. It is evident that maternal diet composition, such as folic acid supplementation or high fat intakes during pregnancy, have the potential to program fetal adiposity and one-carbon metabolism. However, little is known about the effects of maternal folic acid supplementation in pregnancies complicated with gestational obesity and prediabetes on offspring health.  4.2 Results 4.2.1 Maternal diet-induced obesity and glucose intolerance reduced fetal liver weight  Fetal offspring were collected and tissues were weighed at E18.5. The placenta and extraembryonic tissues were removed from the fetus to obtain body weight; female offspring from western dams were smaller (p=0.011) than those from control dams; this was not observed in male offspring (Figure 4.1A, B). There were no differences in placental weight of male or female offspring (Figure 4.1 C, D). Interestingly, male fetal offspring from western dams had smaller livers (p=0.033) compared to males from control dams (Figure 4.1E). A similar trend was observed in female offspring, although not significant (p=0.058) (Figure 4.1F). Maternal folic acid supplementation had no effects on fetal body weight, or placenta and liver size.  40   Figure 4.1. Body, Placenta, and Liver Weights of Fetal Offspring at E18.5. Fetal offspring were collected and tissues were weighed. Body weight of (A) male and (B) female fetuses. Placenta weight of (C) male and (D) female fetuses. Liver weight, expressed as a percentage of body weight, of (E) male and (F) female fetuses. Bar graphs were analyzed by two-way ANOVA. Each data point represents one offspring representative from each litter. Data are presented as mean  SD (n=10-12/group). *p<0.05, effect of diet.  4.2.2 Maternal diet did not influence fetal liver triglyceride content  Given the difference in liver size observed in the E18.5 fetal offspring, hepatic triglyceride content was quantified to assess if this difference in weight was due to changes in the accumulation of triglycerides in the liver. However, I observed no differences in liver triglyceride concentrations in male or female offspring (Figure 4.2A, B).  Fetal LiverLiver Weight (% body weight)CD WD CDF WDF0.02.04.06.08.010.0Placenta WeightPlacenta Weight (g)CD WD CDF WDF0.000.050.100.150.20Fetal LiverLiver Weight (% body weight)CD WD CDF WDF0.02.04.06.08.010.0Fetal WeightBody Weight (g)CD WD CDF WDF0.00.51.01.52.0Fetal WeightBody Weight (g)CD WD CDF WDF0.00.51.01.52.0Placenta WeightPlacenta Weight (g)CD WD CDF WDF0.000.050.100.150.20A BC DE F* ** *p=0.058, effect of diet41   Figure 4.2. Fetal Liver Triglyceride Concentrations at E18.5. Hepatic triglyceride content was quantified in (A) male and (B) female fetal offspring. Bar graphs were analyzed by two-way ANOVA. Each data point represents one offspring representative from each litter. Data are presented as mean  SD (n=3-6/group).  4.2.3 Maternal diet-induced obesity and glucose intolerance increased hepatic water soluble choline metabolite concentrations in fetal offspring  Sex-specific differences in hepatic water soluble choline metabolite concentrations were observed. Male offspring from western dams had higher betaine and free choline concentrations (p<0.05), compared to male offspring from control dams (Figure 4.3A, C). Female offspring from western dams had higher liver betaine concentrations (p=0.026), compared to those from control dams (Figure 4.3B). No differences in phosphocholine or glycerophosphocholine (GPC) were observed in offspring (Figure 4.3E-H). Maternal folic acid supplementation had no effect on water soluble choline metabolite concentrations in the fetal liver at E18.5.  Liver TriglyceridesTriglycerides (mg/mg protein)CD WD CDF WDF0.000.020.040.06Liver TriglyceridesTriglycerides (mg/mg protein)CD WD CDF WDF0.000.020.040.06A B42   Figure 4.3. Fetal Liver Water Soluble Choline Metabolite Concentrations at E18.5. Hepatic water soluble choline metabolites were quantified in fetal offspring. Betaine in (A) male and (B) female fetuses. Free choline in (C) male and (D) female fetuses. Phosphocholine in (E) male and (F) female fetuses. Glycerophosphocholine (GPC) in (G) male and (H) female fetuses. Bar graphs were analyzed by two-way ANOVA. Each data point represents one offspring representative from each litter. Data are presented as mean  SD (n=5-7/group). *p<0.05, effect of diet.  GlycerophosphocholineGPC (nmol/g liver)CD WD CDF WDF0500100015002000PhosphocholinePhosphocholine (nmol/g liver)CD WD CDF WDF05001000150020002500Free CholineFree Choline (nmol/g liver)CD WD CDF WDF0500100015002000Liver BetaineBetaine (nmol/g liver)CD WD CDF WDF050010001500200025003000PhosphocholinePhosphocholine (nmol/g liver)CD WD CDF WDF05001000150020002500GlycerophosphocholineGPC (nmol/g liver)CD WD CDF WDF0500100015002000Liver BetaineBetaine (nmol/g liver)CD WD CDF WDF050010001500200025003000Free CholineFree Choline (nmol/g liver)CD WD CDF WDF0500100015002000A BC DE FG H*** ***43  4.2.4 Maternal folic acid supplementation increased hepatic SAM in female offspring  Female offspring from control and western dams supplemented with folic acid had higher liver SAM concentrations (p=0.04; Figure 4.4B) and SAM/SAH ratios (p=0.046; Figure 4.4F) compared to female offspring from dams not supplemented with folic acid. No effect of maternal folic acid supplementation on fetal liver SAH concentrations were observed (Figure 4.4D). Maternal diet had no effect on hepatic SAM and SAH concentrations in E18.5 male offspring (Figure 4.4A, C, E).     44   Figure 4.4. Hepatic SAM and SAH Concentrations in E18.5 Fetal Offspring. At E18.5, fetal offspring liver was prepared to quantify SAM in (A) male and (B) female offspring, and SAH in (C) male and (D) female offspring. The SAM/SAH ratio was calculated for (E) male and (F) female offspring. Bar graphs were analyzed by two-way ANOVA. Each data point represents one offspring representative from each litter. Data are presented as mean  SD (n=4-6/group). **p<0.05, effect of supplement. Female SAM/SAHSAM/SAHCD WD CDF WDF0.01.02.03.04.0Female SAHSAH (nmol/g)CD WD CDF WDF020406080Female SAMSAM (nmol/g)CD WD CDF WDF020406080100Male SAMSAM (nmol/g)CD WD CDF WDF020406080100Male SAHSAH (nmol/g)CD WD CDF WDF020406080Male SAM/SAHSAM/SAHCD WD CDF WDF0.01.02.03.04.0A BC DE F****** **45  Chapter 5: Effects of Maternal Diet-Induced Obesity/Glucose Intolerance and Folic Acid Supplementation on Fetal Offspring Islet Morphology This chapter presents the findings for Aim 3: To determine if maternal diet-induced obesity/ glucose intolerance and folic acid supplementation affect β and  cell mass in fetal offspring. 5.1 Rationale Proper development of the pancreas is critical for the regulation of glucose homeostasis. As discussed in Chapter 1, population-based studies and rodent studies have provided evidence to suggest that exposure to a perturbed maternal environment, such as obesity or diabetes during pregnancy can result in impaired glucose homeostasis and increased adiposity in the offspring later in life 77,94,95. This manifestation may be due to improper development of  cells in the offspring that begins during fetal development. In rodent models of maternal diet-induced obesity (high fat diet feeding), both neonatal and adult offspring display alterations in  and  cell mass, as well as altered glucose tolerance 79,118,119. Elevated blood glucose, reduced  cell mass, and increased  cell mass were reported in 1 day old neonatal Wistar rat offspring from dams fed a high fat diet (40% kcal fat; fat source not specified) 118. Furthermore, impaired glucose tolerance and elevated  and  cell mass were reported in adult male C57BL/6 mouse offspring from dams fed a high fat diet (49% kcal fat from soybean oil + lard) 79.  Parallel to maternal obesity, in utero exposure to maternal hyperglycemia, as in uncontrolled GDM, results in negative consequences for the offspring 92,93. Interestingly, a recent study in China reported that folic acid supplementation ( 0.8 mg/day) before and during pregnancy is associated with an increased risk of developing GDM 112. However, the effect of 46  folic acid supplementation in pregnancies complicated by obesity and GDM, on fetal offspring  and  cell mass is unknown.  5.2 Results 5.2.1 Maternal folic acid supplementation increased fetal  cell mass in male offspring  To assess if maternal folic acid supplementation in combination with maternal obesity and glucose intolerance conferred any changes in fetal islet morphology, fetal  and  cell mass were quantified. No effects of maternal diet or folic acid supplementation on pancreas weight were observed in male and female offspring (Figure 5.1A, B). A trend towards greater  cell mass (p=0.058) was observed in male offspring from control and western dams supplemented with folic acid (Figure 5.1C). No differences in  cell mass were observed in female offspring (Figure 5.1D). Additionally, no difference in  cell mass was observed in male or female offspring (Figure 5.1E, F). The increase in  cell mass in male fetal offspring from control and western dams supplemented with folic acid was accompanied by greater  cell density (p=0.039) (Figure 5.2A). No differences in  cell density were observed in female offspring (Figure 5.2B). Further, no differences in the size distribution of islets were observed in male or female offspring (Figure 5.2C-H).  47    Figure 5.1. Fetal  and  cell mass at E18.5. Fetal pancreas expressed as a percentage of body weight for (A) male and (B) female pancreata. Beta cell area for (C) male and (D) female offspring represents the insulin-positive area expressed as a percentage of the whole pancreas area. Alpha cell area for (E) male and (F) female offspring represents the glucagon-positive area expressed as a percentage of the whole pancreas area. (G) Representative immunofluorescence images of islets stained with insulin, glucagon, and Dapi; scale bar= 50 m. Bar graphs were analyzed by two-way ANOVA. Each data point represents an average of three sections per offspring representative from each litter. Data are presented as mean  SD (n=5-8/group). **p<0.05, effect of supplement. Female Alpha Cell AreaAlpha Cell Area(% Pancreas Area)CD WD CDF WDF0.00.20.40.60.81.0Male Beta Cell AreaBeta Cell Area(% Pancreas Area)CD WD CDF WDF0.01.02.03.04.0Fetal PancreasPancreas Weight(% body weight)CD WD CDF WDF0.00.20.40.60.81.01.2MaleInsulin Glucagon DapiGFemaleCD CDF WD WDFFetal PancreasPancreas Weight(% body weight)CD WD CDF WDF0.00.20.40.60.81.01.2Female Beta Cell AreaBeta Cell Area(% Pancreas Area)CD WD CDF WDF0.01.02.03.04.0Male Alpha Cell AreaAlpha Cell Area(% Pancreas Area)CD WD CDF WDF0.00.20.40.60.81.0A BC DE Fp=0.058, effect of supplement48   Figure 5.2. Fetal Islet Density and Distribution at E18.5. Beta cell density for (A) male and (B) female offspring expressed as number of particles per 100 mm2 of whole pancreas area. Small islets for (C) male and (D) female offspring are particles <100 m2. Medium islets for (E) male and (F) female offspring are 100-200 m2. Large islets for (G) male and (H) female offspring are >200 m2. All islet sizes are expressed as percent of islet particles. Bar graphs were analyzed by two-way ANOVA. Each data point represents an average of three sections per offspring representative from each litter. Data are presented as mean  SD (n=5-8/group). **p<0.05, effect of supplement. Male Islet DensityParticles/ 100 mm2CD WD CDF WDF0246810Female Islet DensityParticles/ 100 mm2CD WD CDF WDF0246810A B****Beta Cell DensityBeta Cell DensityMale Small Islets% Small Islets(<100 um^2)CD WD CDF WDF020406080100Male Medium Islets% Medium Islets(100-200 um^2)CD WD CDF WDF020406080100Male Large Islets% Large Islets(>200 um^2)CD WD CDF WDF020406080100Female Small Islets% Small Islets(<100 um^2)CD WD CDF WDF020406080100Female Medium Islets% Medium Islets(100-200 um^2)CD WD CDF WDF020406080100Female Large Islets% Large Islets(>200 um^2)CD WD CDF WDF020406080100C DE FG H49  Chapter 6: Discussion and Conclusions 6.1 Discussion For my Master’s thesis research, I utilized a mouse model to evaluate the effects of maternal folic acid supplementation and diet-induced obesity/ glucose intolerance on the health of the mother and fetal offspring. I assessed adiposity and physiological indicators of glucose tolerance, insulin sensitivity, and  cell function in dams before pregnancy, quantified one-carbon metabolites in maternal and fetal liver, and assessed pancreatic  and  cell mass in fetal islets. My research provides insight into the health of mothers with pregnancies complicated by gestational obesity and prediabetes who are recommended to supplement with high doses of folic acid, and how this affects the development of the fetal offspring. 6.1.1 Folic acid supplementation before and during pregnancy does not affect adiposity, glucose tolerance, or hepatic one-carbon metabolism in dams with or without diet-induced obesity/glucose intolerance In Chapter 3, I studied the effect of folic acid supplementation on adiposity and glucose homeostasis in the dams before and during pregnancy. Folic acid supplementation altered insulin sensitivity, but this was dependent on the dam’s diet. When supplemented with folic acid, western dams had improved insulin sensitivity and control dams had reduced insulin sensitivity. The greater adiposity and glucose intolerance associated with western-diet feeding resulted in dams with larger livers accompanied by reduced hepatic SAM and SAH during pregnancy, indicating that gestational obesity and prediabetes may have consequences for liver one-carbon metabolism in the dams. The dams were fed the western diet as a model of diet-induced obesity and glucose intolerance. I confirmed the model, and showed that western dams had greater body weight and 50  adiposity accompanied by glucose intolerance and impaired  cell function. My findings are consistent with other studies that have used a comparable model of high fat feeding to induce excess adiposity and glucose intolerance 120–122. For example, female C57BL/6 dams fed obesogenic diets ranging in fat content (30- 60% kcal fat from soybean oil + lard) prior to pregnancy are more adipose 120,121 and glucose intolerant before 120 and during breeding 121,122.  Interestingly, I found that folic acid supplementation before and during pregnancy increased the size of the heart and kidney in the dams at E18.5. During pregnancy, the maternal cardiovascular system adapts to the physiological changes of pregnancy and this includes increased maternal blood pressure, blood volume, and cardiac output to support the development of the fetus 123. Furthermore, erythropoietin, a hormone produced primarily by the kidneys for the production of red blood cells, is stimulated by placental lactogen during pregnancy for increased red blood cell production 124,125. In order to accommodate these changes during a healthy pregnancy, the heart and kidney undergo structural changes, including enlargement 126. The reason why folic acid supplementation would augment increases in heart and kidney size during pregnancy is not understood but may involve direct effects of folic acid or circulating folate on heart and kidney metabolism and its structure and function. Contrary to my hypothesis, I did not find that folic acid supplementation exacerbated the effects of maternal obesity on glucose tolerance or  cell function. However, I did observe improved insulin sensitivity in western dams supplemented with folic acid. The effect of supplemental folic acid on insulin sensitivity in the context of diet-induced obesity has been previously explored, but only in adult male rats; little is known about this during pregnancy although physiological changes associated with pregnancy contribute greatly to alterations in insulin sensitivity in females. Buettner et al. reported that adult male Wistar rats fed a high fat 51  diet with supplemental folic acid (25% kcal fat from lard; 40 mg/kg diet, 20X AIN recommendations) had improved insulin sensitivity 127. The findings from my study and that of Buettner et al. suggest a beneficial effect of folic acid supplementation on insulin sensitivity in the context of diet-induced obesity in male and female rodents.  Interestingly, the difference I observed in dams was only after 90 minutes following an insulin bolus. As insulin has a short half-life of approximately 10 minutes 128, all exogenous insulin should be cleared following the first 30 minutes of an IPITT 129. Therefore, later differences following insulin administration, particularly after the initial fall at 30 minutes post-insulin injection, may not reflect a direct effect on insulin action but may be due to secondary effects such as counterregulatory hormone responses like changes in endogenous insulin production 129,130. This suggests that the effect of folic acid supplementation with or without gestational obesity that I observed may not have a big impact on whole body insulin action despite an overall difference in IPITT AOC. Further investigation into the role of folic acid supplementation on insulin regulation is warranted.  I found that dams fed the western diet had larger livers accompanied by lower hepatic SAM and SAH compared to control dams. These changes in hepatic SAM and SAH during pregnancy complicated by diet-induced obesity and glucose intolerance are novel and have not been previously reported. Diet-induced obesity is associated with the development of nonalcoholic fatty liver disease (NAFLD) and is characterized by hepatic accumulation of fat from increased fatty acid uptake, de novo fatty acid synthesis, impaired fatty acid oxidation, or reduced triglyceride export by very-low-density-lipoprotein (VLDL) particles into circulation. Assembly of VLDL particles is dependent on the presence of hepatic PC, which is derived from PEMT-mediated methylation of PE using SAM as a methyl donor 23. Further, SAM can be 52  synthesized through betaine-dependent remethylathion of homocysteine to methionine via BHMT. The lower SAM that I observed could potentially be related to impaired BHMT activity. Lower availability of SAM may affect PEMT enzymatic activity, and thus, lower the production of PC from PE for VLDL assembly and export of triglycerides. It would be intriguing to quantify the hepatic triglyceride content in the dams of my study, as well as histologically assess the liver for signs of NAFLD. Furthermore, investigating choline metabolism in the liver would provide additional insight into the regulation of one-carbon metabolism in dams with diet-induced obesity and glucose intolerance. A link between glucose and lipid metabolism is well known and involves insulin signalling and regulatory factors such as, sterol regulatory element-binding protein 1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP) 131; these transcription factors are stimulated by insulin and glucose, respectively. The first steps of hepatic de novo fatty acid synthesis from acetyl-CoA are catalyzed by acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) 131. Insulin stimulates FAS expression via the PI3K pathway, and both ACC and FAS are transcriptionally activated by SREBP-1c and ChREBP 131. The western dams in my study had hyperglycemia and hyperinsulinemia, suggesting that higher levels of glucose and insulin may have promoted SREBP-1c and ChREBP-mediated hepatic lipid accumulation and subsequently resulted in larger livers.  Interestingly, I found no effect of folic acid supplementation on liver SAM and SAH which contradicts what I predicted based on reports by others. Christensen et al. reported that female BALB/cAnN mice supplemented with folic acid (10 mg/kg diet; 5X AIN recommendations) before and during pregnancy had lower hepatic SAM, higher SAH, and reduced SAM/SAH ratio compared to control dams at E10.5 132. The reason for the lack of 53  change in liver SAM and SAH with folic acid supplementation in my study is not understood but may be due to differences in rodent species, or the gestational time point at which these metabolites were quantified.  6.1.2 Maternal diet-induced obesity/glucose intolerance and folic acid supplementation affect liver one-carbon metabolism in fetal offspring In Chapter 4, I investigated the effects of maternal folic acid supplementation and diet-induced obesity/glucose intolerance on fetal offspring liver one-carbon metabolism and observed disturbances in body weight, as well as methionine and choline metabolism. Female fetuses from western dams were smaller in body weight but this was not observed in male fetuses. Fetal offspring from western dams also had higher liver choline metabolites and smaller livers without changes in liver triglyceride concentrations. Maternal folic acid supplementation increased liver SAM in female, but not in male fetal offspring.  Interestingly, female fetuses from dams with diet-induced obesity/glucose intolerance had lower body weight. However, the difference in body weight compared to fetal offspring from control dams is very subtle and the biological significance of such a small difference seems minimal. Nevertheless, maternal gestational obesity is associated with fetal growth restriction in both humans and rodents 133–135. Furthermore, maternal gestational obesity is associated with impaired structure and vascular function in the placenta, therefore impairing blood flow to the fetus and contributing to the development of fetal growth restriction 136,137. In a study of C57BL/6J mice fed an obesogenic diet and sweetened condensed milk (20% kcal fat from lard + 8% kcal fat from milk fat), E13 and E19 fetal male and female offspring had lower body weight and smaller placentae with impaired structural and vascular development 133. Placenta weight was not affected by maternal diet in my study; vascular function was not assessed. Alterations in 54  placental structure and function due to maternal diet-induced obesity/glucose intolerance may be the reason for lower body weight in the female offspring from western dams.  Additionally, I observed that male and female fetal offspring from dams with diet-induced obesity/glucose intolerance had smaller livers accompanied by higher levels of water-soluble choline metabolites but no differences in liver triglyceride concentrations. Male offspring from western dams had higher liver free choline and betaine, whereas female offspring from western dams had higher liver betaine. As discussed in the previous section, a western diet in adult mice is associated with hepatic triglyceride accumulation and characteristic of NAFLD 138,139. Furthermore, fetal offspring from dams with diet-induced obesity have hepatic triglyceride accumulation 117,140. Supplementation with either choline (25mM choline chloride) or betaine (1% betaine anhydrous) in the drinking water of dams fed a high fat-diet (60% kcal fat as soybean oil + lard) resulted in lower liver triglycerides in the liver of fetal offspring at E17.5 117,140. These studies and others who have supplemented rodents with betaine and reported lower hepatic triglycerides, 138,141 suggest that choline and betaine lower hepatic triglyceride accumulation by potentially upregulating BHMT-mediated production of SAM, which can be used for the methylation of PE to PC for VLDL assembly and export of triglycerides. As such, the higher levels of liver choline metabolites I observed in offspring from western dams may protect them from accumulating triglycerides in the liver.  Interestingly, I found no effect of maternal folic acid supplementation on fetal liver choline metabolites, which is contrary to reports by others 61. Lower liver choline and betaine were reported in E17.5 fetal C57BL/6 offspring from folic acid supplemented dams (20 mg/kg diet; 10X AIN recommendations) 61. Folate and choline metabolism are interrelated and both contribute to the synthesis of SAM. Methionine is the precursor of SAM and can be synthesized 55  by the MTR-catalyzed remethylation of homocysteine using 5-MTHF as a methyl donor, or by BMHT using betaine as a methyl donor. In the rat liver, both MTR and BHMT pathways contribute equally under control conditions 142. When disturbances in one of these pathways occur, compensatory changes in the other pathway may follow. For example, MTHFR deficiency is associated with greater demand for betaine-dependent remethylation of homocysteine via BHMT 141. In contrast, BALB/cAnN dams supplemented with folic acid (10 mg/kg diet; 5X AIN recommendations) have reduced MTHFR protein at E10.5, but no changes in hepatic choline or betaine 132. This suggests that folic acid supplementation can lead to changes in folate metabolism without compensation by, or alterations in choline metabolism. Additional experiments on fetal liver are necessary to assess disturbances in folate metabolism, such as changes in MTHFR expression and activity. This would further our understanding of the effect of maternal folic acid supplementation on alterations in fetal choline metabolism and the interrelationship between choline, betaine, and folate in fetal one-carbon metabolism.   I reported that female E18.5 offspring from control and western dams supplemented with folic acid had higher hepatic SAM and SAM/SAH ratio compared to those from non-supplemented dams. My findings are contrary to reports by others 61,132. In the study by Christensen et al., E10.5 BALB/cAnN fetuses from folic acid supplemented dams (10 mg/kg diet; 5X AIN recommendations) had no differences in liver SAM or SAH compared to control fetuses 132. Similarly, Bahous et al. reported that E17.5 fetuses from dams supplemented with folic acid (20 mg/kg diet; 10X AIN recommendations) had no differences in hepatic SAM and SAH concentrations compared to control offspring 61. SAM is a key methyl donor and is central for many biological processes. The SAM/SAH ratio is frequently used as an indicator of cellular methylation potential, such that a lower ratio is indicative of lower methyl group donor 56  availability 143. However, studies have reported that changes in SAH are more strongly associated with methylation potential than changes in SAM alone because SAH is an inhibitor of methyltransferases 20. Further, several studies have investigated relationships between tissue SAM and SAH concentrations and global and gene-specific DNA methylation 143–145. For example, C57BL/6J male mice with higher liver SAH concentrations and lower SAM/SAH had global DNA hypomethylation in the liver as well as other tissues including the kidney, brain, and testes 143. Similarly, Glier et al. reported that male mice with diet-induced hyperhomocysteinemia had higher liver SAH and lower SAM/SAH and this was accompanied by lower gene-specific DNA methylation in the liver 145. This study also found higher gene-specific DNA methylation in the brain with no differences in SAM and SAH, suggesting that the relationship between SAM and SAH to gene-specific DNA methylation is tissue specific and that changes in DNA methylation can occur without changes in these methyl nutrients. I did not observe changes in SAH in the fetal female offspring, however, the Devlin Lab has previously reported increased hepatic SAH in adult female offspring from folic acid supplemented dams 56. This suggests that hepatic SAM and SAH levels are dynamic and can change throughout life. It is not understood why SAM is higher in female livers from folic acid-supplemented dams during fetal life, or if higher SAM-mediated SAM/SAH ratio can improve methylation potential in the fetus. Further investigation of global and gene-specific DNA methylation in the fetal liver would provide insight on the effects of elevated SAM in the fetus. 6.1.3 Maternal folic acid supplementation affects islet morphology in male fetal offspring In Chapter 5, I identified greater  cell mass and density in male fetal offspring from control and western dams supplemented with folic acid; this effect was not observed in female offspring. Alterations in  cell mass are an indicator of impairments in insulin secretion and 57  glucose tolerance. Increased  cell mass has been observed in E18.5 fetal and 1 day old postnatal pups from C57BL/6J dams fed a high fat diet before pregnancy (60% kcal fat from soybean oil + lard) 119. Increased  cell mass was associated with fetal and neonatal hyperinsulinemia and consequently lead to hypoglycemia in the fetus and neonate. Untreated and recurrent severe hypoglycemia in early life is associated with several negative consequences, including neurological impairments 146. This study is one of many examples of how the maternal environment can influence fetal  cell mass and lead to adverse consequences. It further implies that other maternal environments, such as one with folic acid supplementation, may have consequences for offspring health. Typically, a reduction in  cell mass in combination with impaired glucose-stimulated insulin secretion is indicative of increased T2D susceptibility 77,79,147. This suggests that maternal folic acid supplementation may protect against T2D susceptibility by increasing  cell mass in male fetal offspring. This notion compliments previous findings in the Devlin Lab in which maternal folic acid supplementation had no effect on glucose tolerance in adult male offspring, but worsened glucose tolerance in female offspring 55,56. Furthermore, adult female offspring from this previous study did not exhibit any changes in  cell mass 56, which is consistent with my findings in fetal female  cell mass.  Others have investigated the effects of maternal folic acid supplementation on  cell mass  in older offspring. Seven week old male and female C57BL/6J offspring from folic acid supplemented dams (40 mg/kg diet; 20X AIN recommendations) had lower  cell mass (assessed as insulin-positive cell diameter) compared to offspring from control dams 148. Additionally, both offspring had lower fasting insulin levels and females were glucose intolerant following an oral GTT. Several reports on adult male and female offspring from folic acid supplemented dams 58  have observed glucose intolerance 56,59,60. Changes during fetal life may program glucose tolerance in adulthood. However, the murine pancreas continues to develop in the early postnatal period, undergoing a wave of  cell apoptosis and neogenesis over the first 20 days of postnatal life until weaning 149,150. This  cell turnover is required for the functional maturation of immature “fetal type”  cells, to fully glucose sensitive adult  cells 150,151. It is therefore possible that the alterations in fetal  cell mass that I observed may differ from the health of the  cells and glucose regulation of these offspring later in life; further studies to follow these offspring into adulthood are necessary. It is reasonable to consider that the effect of maternal folic acid supplementation on fetal  cell mass may not be direct and may occur as a consequence of supplementation in the dams. There have been conflicting reports on the association between folic acid supplementation and risk for GDM 111,112. Diabetes during pregnancy has several negative consequences for the mother and the offspring, including obesity and glucose intolerance later in life 92,93. In my study, folic acid supplementation had no effect on glucose tolerance or  cell function in the dams, suggesting that the effect of maternal folic acid supplementation on male fetal  cell mass is not because of effects of folic acid supplementation on glucose homeostasis in the dams. This implies that either maternal folic acid supplementation has direct effects on male fetal offspring  cell mass, or that there are other unidentified effects in the dams supplemented with folic acid which in turn influence male offspring  cell mass. Further studies are required to elucidate the mechanisms underlying my observations.  Surprisingly, maternal obesity did not influence fetal  and  cell mass, contrary to several other reports of alterations in  and  cell mass from fetal and adult offspring of dams 59  fed high fat diets 79,118,119. Elevated blood glucose, reduced  cell mass, and increased  cell mass has been reported in 1 day old neonatal Wistar rat offspring from dams fed a high fat diet (40% kcal fat; fat source not specified) 118. Likewise in adult offspring, impaired glucose tolerance and elevated  and  cell mass has been reported in male C57BL/6 offspring from dams fed a high fat diet (49% kcal fat from soybean oil + lard) 79. However, similar to my findings, a study of C57BL/6J dams fed a high fat diet and sweetened condensed milk (20% kcal fat from lard + 16% kcal fat from milk fat) prior to pregnancy, reported no effects on  and  cell mass in adult male and female offspring 152. Additionally, Cerf et al., reported reduced  cell mass in one day old neonates but no effect in adult offspring from the same female Wistar rat dams fed a high fat diet (40% kcal fat from lard) before and during pregnancy 153. These findings provide evidence for the dynamic nature of  cell mass that change with age. Considering the  cell turnover that occurs during the neonatal period, it is likely that the  cell mass observed in fetal life will be different from the  cell mass present in adulthood.  Finally, the greater  cell mass that I observed in male fetal offspring from folic acid supplemented dams may be due to the increased  cell density, which I also found. However, the greater  cell density was not accompanied by differences in islet size distribution. The number and size of islets contribute to  cell mass 154. In a study of non-diabetic human islets (n=72 samples), authors reported that islet density, rather than islet size, was more predictive of  cell mass 154. This suggests that although the increase in  cell mass in male offspring from folic acid supplemented dams in my study was not statistically significant (p=0.058), the  cell density was significantly greater and provides some confidence that the change in  cell mass is biologically relevant.  60  6.2 Summary In summary, my thesis research demonstrates that maternal folic acid supplementation induced changes in fetal offspring one-carbon metabolism and pancreatic islet morphology. Dams were supplemented with folic acid before and during pregnancy to investigate if this exacerbates the adverse effects associated with gestational obesity and prediabetes on the mother and fetal offspring. Contrary to my hypothesis, folic acid supplementation did not affect maternal adiposity, glucose tolerance,  cell function, or hepatic one-carbon metabolism before or during pregnancy. Folic acid supplementation did however, mildly alter insulin sensitivity in dams, dependent on the dietary fat content before pregnancy. In the fetal offspring, maternal folic acid supplementation was associated with sex-specific alterations in hepatic one-carbon metabolism and pancreatic  cell mass. Although supplementation did not affect maternal one-carbon metabolism, female offspring had higher liver SAM and SAM/SAH ratio. Also, despite no effect of folic acid supplementation on  cell function and glucose tolerance in dams, male offspring from folic acid supplemented dams had elevated  cell mass likely due to an increase in  cell density.  Interestingly, gestational obesity and prediabetes altered liver size in dams and their offspring, possibly due to disturbances in liver one-carbon metabolism. Dams with diet-induced obesity and glucose intolerance had larger livers with reduced hepatic SAM and SAH levels. In the offspring, maternal diet-induced obesity and glucose intolerance altered hepatic choline metabolism. Male and female offspring from western dams also had smaller livers which I infer may be due to the protective effects of higher choline and betaine concentrations in the liver that lead to the prevention of triglyceride accumulation. Taken together, gestational obesity/ 61  prediabetes and folic acid supplementation before and during pregnancy affect maternal and fetal health. An overarching summary of my findings is depicted in Figure 6.1.  Figure 6.1. Overarching Summary of Findings. Effects of maternal western diet are highlighted in blue and effects of maternal folic acid supplementation are highlighted in gold. “Mouse” icons by Darrin Higgins and zidney, from thenounproject.com. Licensed under Creative Commons Attribution 3.0.   6.3 Strengths and Limitations My thesis research has several strengths. Analyses in fetal offspring were able to be done separately in male and female fetuses. It is well known that offspring exhibit sexually dimorphic phenotypes in regards to maternal obesity and folic acid supplementation 55,56,59,77,78. Many studies conducted in fetal offspring often do not genotype the fetuses for analysis, grouping both together when making conclusions. For example, studies by Christensen et al. and Bahous et al. provide compelling data with regards to fetal one-carbon metabolism 61,132. However, the offspring in these studies were not genotyped for sex, making it difficult to conclude if alterations from maternal folic acid supplementation were related to male and/or female fetal mice. By analyzing the offspring separately in my study, I was able to tease out sex-specific 62  effects of one-carbon metabolism and islet morphometrics in the fetal offspring that may not have been possible, had the fetal offspring been analyzed as one group. This furthers our understanding of how males and females differentially adapt to the maternal environment, allowing for better identification of potential risk factors for male and female offspring health. Another strength of my study is the assessment of  cell density and islet size distribution to compliment assessment of  cell mass. Mass may be altered through changes in the size or number of  cells as well as changes in vascularization or sympathetic innervation 155–157. Addition of these assessments enhanced the findings and provided further insight into the changes seen in  cell mass.  My study is not without limitations. There is no doubt that studies of maternal folic acid supplementation in the literature have been variable in terms of dose and duration of supplementation and the outcomes on offspring health. The dose of folic acid supplementation in the control diet I used in my study met the AIN-93 recommendations of 2 mg/kg diet of folic acid for mice and rats 101,102. This amount provided 0.5 g of folic acid/kcal (the control diet is 3800 kcal/kg diet, see Table 2.1), which is approximately equivalent to a woman consuming 1 mg of folic acid in a 2000 kcal/day diet. The folic acid supplemented diets of my study contained 10 mg/kg diet of folic acid which is 2.5 g of folic acid/kcal, and is equivalent to 5 mg/day of folic acid in humans. This amount is recommended for women at high risk of having a NTD pregnancy 35. However, it should be noted that there are differences between mice and humans in relation to the metabolism of folic acid by DHFR whereby enzyme activity is higher in the liver of rodents compared to humans 158,159. This suggests that mice and humans may have different capacities for handling large doses of folic acid and the supplemental dose that we provided the 63  animals may not be considered “high” intake for a mouse. In addition, the timing and duration of folic acid supplementation may have differential effects on offspring 160, which is similar to findings from the Dutch Famine Birth Cohort where exposure to famine at different points of gestation lead to varying adverse outcomes for offspring 6–11. These differences in study design make it challenging to compare data across studies and formulate solid overarching conclusions. I found that diet-induced obesity and folic acid supplementation did not impact  cell function, as assessed by IST. However, this physiological assessment is only one indicator of  cell function and does not provide a direct functional measure. Further studies should include direct measures of  cell function including islet secretory function and insulin content in the  cell 161. To obtain a full understanding and confirm that  cell function is in fact not impaired would require functional experiments using isolated islets for ex-vivo glucose-stimulated insulin secretion to assess the influence of folic acid supplementation and diet-induced obesity/glucose intolerance on the secretory function of  cells.   6.4 Future Directions The data presented in this thesis provide a starting point for future research to further elucidate the phenotypes observed and understand the underlying mechanisms of maternal folic acid supplementation on maternal and offspring health. The Devlin lab has previously reported that adult offspring from folic acid supplemented dams display alterations in glucose homeostasis 55,56. I found that at E18.5, fetal  cell mass was altered in male offspring, but not in female offspring. The pancreas continues to develop in the early postnatal period until weaning to become mature adult  cells 149,150. Investigating the effect of maternal folic acid supplementation on neonatal  cell maturation and function, including detection of apoptosis, 64  expression of transcription factors crucial for maintaining  cell identity and functional maturation such as Pdx1 and Mafa 162, and glucose-stimulated insulin secretion of isolated islets, would provide insight on how the  cells develop for future adult physiologic handling of glucose. Given that folic acid is an oxidized synthetic form of folate, it may have different metabolic effects compared to other folate forms. Differential effects may be due to accumulation of unmetabolized folic acid, which has been implicated in adverse health consequences associated with folic acid supplementation in both humans and animals 163–166. In a study of folic acid supplemented male BALB/c mice (20 mg/kg diet; 10X AIN recommendations), unmetabolized folic acid was detected in control mice fed the recommended 2 mg folic acid/kg diet, and was increased by 60% in mice fed the folic acid supplemented diet 166. Folic acid supplementation in these males lead to reduced methylation potential, indicated by reduced SAM/SAH ratio. The metabolically useful form of folic acid, 5-MTHF, is as effective as folic acid in raising red blood cell folate levels in humans without the accumulation of unmetabolized folic acid 167–169. It is therefore of interest to test the different forms of folate to investigate if the phenotypes observed in fetal offspring of dams supplemented with folic acid are different with alternate folate forms. Additionally, exploring if differences observed due to exposure to folic acid are elicited by elevated maternal folate status or accumulating unmetabolized folic acid would be of interest.  The placenta plays a critical role in the developing fetus. When pregnancy is accompanied by obesity, placental function may be impaired through structural and functional changes 170,171. Vascular remodeling of the placenta involves the invasion into, and the remodeling of spiral arteries for proper hemochorial blood circulation between mother and fetus 65  172. This process can be impaired by obesity 120,136,137,173. Maternal folic acid supplementation may also impair placental development. For example, in the study by Christensen et al., BALB/cAnN female mice supplemented with folic acid (10 mg/kg diet; 5X AIN recommendations) had abnormal placental development, including the presence/ absence of, and alterations in thickness of the labyrinth layer 132. This study suggested that changes in SAM/SAH ratio observed in the fetal offspring at E10.5 may have been affected by an abnormal placenta. This insinuates that changes observed in my study may have also been affected by alterations in the placenta at E18.5. Given the high doses of folic acid being recommended to women with obesity during pregnancy, it is of interest to understand how nutrient transport across the placenta is impacted by folic acid supplementation, particularly in combination with maternal obesity and glucose intolerance.  In conclusion, maternal environmental exposures during fetal and early postnatal life are influential on offspring development and on the programming of long-term offspring health. The maternal environment may evoke an influence on offspring health through direct changes to fetal development, or through changes in placental development that subsequently affect the growth and development of the fetus. It is well accepted that maternal folic acid supplementation prevents NTDs during pregnancy. 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