@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Pathology and Laboratory Medicine, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Aleliunas, Rika"@en ; dcterms:issued "2013-08-02T09:10:17Z"@en, "2013"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Prenatal and early postnatal nutrient status influences metabolic health in adulthood. A study from India reported that children born to women with high folate and low vitamin B12 status during pregnancy had increased adiposity and insulin resistance. Many countries, including Canada, fortify their grain products with folic acid. In Canada, women have adequate folate status but 1 in 20 may be vitamin B12 deficient. Currently, the metabolic consequences of developmental exposure to high folic acid, with or without vitamin B12 deficiency, are unknown. Female C57BL/6J mice were fed diets high in folic acid, with (HFA+B12) and without (HFA-B12) adequate vitamin B12, or a control diet throughout breeding, pregnancy and lactation. Offspring were weaned onto a control or western-type diet (WTD) for 30 weeks. Maternal and post-weaning diets affected visceral (P<0.01) and subcutaneous fat accumulation (P<0.05) at 20 weeks post-weaning. An interaction between maternal and post-weaning diet was observed in the retroperitoneal fat depot; WTD-fed mice with developmental exposure to HFA-B12 and HFA+B12 diets had decreased retroperitoneal fat accumulation relative to those exposed to maternal control diet. Maternal and post-weaning diets interacted at 30 weeks post-weaning to modify fasting insulin concentrations; in control-fed mice, those with developmental exposure to HFA-B12 had increased fasting insulin concentrations (P<0.05) relative to those with exposure to maternal control and HFA+B12 diets. Maternal and post-weaning diets interacted to affect vascular function of the mesenteric artery at 20 weeks post-weaning. Endothelium-mediated smooth muscle relaxation was decreased in WTD-fed mice with developmental exposure to HFA-B12 diet relative maternal control and HFA+B12 diet-exposed groups (P<0.05). Control-fed mice with developmental exposure to HFA-B12 and HFA+B12 diet had decreased mesenteric artery basal nitric oxide production relative to maternal controls (P<0.05). Developmental exposure to high folic acid (with and without vitamin B12) was associated with decreased NADPH oxidase subunit expression in the aorta (P<0.05). These findings demonstrate that developmental exposure to folic acid and vitamin B12 imbalance interacts with post-weaning diet to program the metabolic response in adult mice. Further studies are warranted to determine the mechanisms behind such effects and to determine the need for vitamin B12 supplementation during pregnancy."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/44742?expand=metadata"@en ; skos:note """ PROGRAMMING OF ADULT METABOLIC PHENOTYPE BY MATERNAL DIETARY FOLIC ACID AND VITAMIN B12 IMBALANCE IN MICE by RIKA ALELIUNAS B.Sc., The University of British Columbia, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2013 © Rika Aleliunas, 2013 ii Abstract Background: Prenatal and early postnatal nutrient status influences metabolic health in adulthood. A recent birth cohort study from India reported that children born to women with high folate and low vitamin B12 status during pregnancy had greater adiposity and insulin resistance at age 6. These findings are particularly relevant to countries, including Canada, which fortify their grain products with folic acid, the synthetic form of folate. In Canada, women have adequate folate status but 1 in 20 may be vitamin B12 deficient in the early stages of pregnancy. Currently, the long-term metabolic consequences of developmental exposure to high folic acid, with or without vitamin B12 deficiency, are unknown. Method/Results: Female wild-type C57BL/6J mice were fed diets high in folic acid, with (HFA+B12) and without (HFA-B12) adequate vitamin B12, or a control diet for six weeks prior to breeding, and during pregnancy and lactation. Offspring were weaned onto control or a western-type diet (WTD) consisting of 45% fat and 35% carbohydrate, for 30 weeks. Body composition, glucose homeostasis, and vascular function were assessed in male offspring (n=55). Maternal and post-weaning diets affected both visceral (P<0.01) and subcutaneous fat accumulation (P<0.05) at 20 weeks post-weaning. An interaction between maternal and post-weaning diet was observed in the retroperitoneal fat depot; WTD-fed mice with developmental exposure to HFA-B12 and HFA+B12 diets had decreased retroperitoneal fat accumulation relative to those exposed to maternal control diet. Maternal and post-weaning diets interacted at 30 weeks post-weaning to modify fasting insulin concentrations; in control-fed mice, those with developmental exposure to HFA-B12 had increased fasting insulin concentrations (P<0.05) relative to those with iii exposure to maternal control and HFA+B12 diets at 30 weeks post weaning. Maternal and post-weaning diets interacted to affect vascular function of the mesenteric artery at 20 weeks post weaning. WTD-fed mice with developmental exposure to HFA-B12 diet did not reach the maximum relaxation achieved by maternal control and HFA+B12 diet- exposed groups (P<0.05). Control-fed mice with developmental exposure to HFA-B12 and HFA+B12 diet had decreased mesenteric artery basal nitric oxide production relative to maternal controls (P<0.05). In contrast, WTD-fed mice with developmental exposure to HFA+B12 had increased basal nitric oxide production relative to maternal controls (P<0.05). At 20 weeks post-weaning NADPH oxidase subunit (Nox2) expression in the aorta was affected by maternal diet (P<0.05). Conclusion: These findings demonstrate that developmental exposure to folic acid and vitamin B12 imbalance interacts with post-weaning diet to program the metabolic response in adult mice. The effects include modification of body composition, glucose homeostasis, and vascular function. Further investigation is warranted to elucidate the mechanisms by which this programming occurs and to determine the need for vitamin B12 supplementation during pregnancy. iv Preface This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science in Pathology and Laboratory Medicine and contains works done from January 2011 to July 2013. I have prepared this thesis in its entirety under the direction and supervision of Dr. Angela Devlin. This thesis was revised by Dr. Angela Devlin, Dr. Ismail Laher, and Dr. Tim Green. None of the text is taken from previously published or collaborative articles. All the work presented henceforth was conducted at the Child and Family Research Institute (CFRI) in Vancouver, BC and at Dr. Laher’s laboratory at the University of British Columbia, Vancouver campus. The breeding and feeding portions of the study described in Chapter 4 were conducted by me at the animal unit at CFRI with the help of E. Wang. Experiments in Chapter 5 were planned by me and executed via a collaborative effort by M. Glier, J. Olson, A. Aljaadi, and myself. I conducted the vascular function experiments presented in Chapter 6 at Dr. Laher’s lab with the assistance of S. Golbidi. All experiments and associated methods were approved by the University of British Columbia Animal Care Committee (Protocol: A09-0346). v Table of Contents Abstract .............................................................................................................................. ii   Preface ............................................................................................................................... iv   Table of Contents .............................................................................................................. v   List of Tables ................................................................................................................... vii   List of Figures ................................................................................................................. viii   List of Abbreviations ....................................................................................................... ix   Acknowledgements ........................................................................................................... ii   CHAPTER 1: Introduction .............................................................................................. 1   1.1 A Mouse Model of Maternal Folic Acid and Vitamin B12 Intake ........................... 1   1.2 Obesity and Cardiometabolic Risk ........................................................................... 2   1.2.1 Classification of Obesity ........................................................................................ 2   1.2.2 Regional Fat Distribution and Metabolic Health ................................................... 3   1.2.3 Definitions: Metabolic and Cardiovascular Disease .............................................. 5   1.3 Developmental Programming ................................................................................... 5   1.3.1 Programming of Metabolic Disease ....................................................................... 5   1.3.2 Programming of Vascular Disease ......................................................................... 7   1.4 Epigenetics: Gene and Environment Interaction ...................................................... 9   1.5 Folic Acid Fortification ........................................................................................... 10   1.5.1 Overview of Mandatory Folic Acid Fortification ................................................ 10   1.5.2 Folate and Vitamin B12 Metabolism ................................................................... 12   1.5.3 Folate and Vitamin B12 Imbalance during Pregnancy ........................................ 15   CHAPTER 2: Rationale and Hypothesis ...................................................................... 16   CHAPTER 3: Materials and Methods .......................................................................... 18   3.1 Breeding scheme and Animal Diets ........................................................................ 18   3.2 Body Composition .................................................................................................. 21   3.3 Glucose Tolerance .................................................................................................. 21   3.4 Serum and Plasma Protein Concentrations ............................................................. 21   vi 3.5 Quantification of mRNA ......................................................................................... 22   3.6 Western Blotting ..................................................................................................... 23   3.8 Vascular Endothelial Function ................................................................................ 24   3.9 Statistics .................................................................................................................. 25   CHAPTER 4: Maternal Vitamin B12 Status, Body Weight, and Litter Size ............ 29   4.1 Maternal Body Weight and Litter Size ................................................................... 29   4.2 Maternal Serum Vitamin B12 Concentrations ........................................................ 32   CHAPTER 5: Body Composition and Metabolic Characteristics of Offspring ....... 33   5.1 Male Offspring Growth and Body Weight ............................................................. 33   5.2 Glucose Tolerance at 20 Weeks Post-Weaning ...................................................... 35   5.3 Glucose Tolerance at 30 Weeks Post-Weaning ...................................................... 37   5.4 Body Composition at 20 Weeks Post-Weaning ...................................................... 39   5.6 Circulating Adiponectin Concentrations at 20 Weeks Post-Weaning .................... 43   5.7 Adipose Tissue Gene Expression ............................................................................ 44   CHAPTER 6: Vascular Endothelial Function ............................................................. 46   6.1 Vascular Function of Aortae and Mesenteric Arteries ........................................... 46   6.2 Concentration-Response Curves to PE in the Presence L-NAME ......................... 50   6.3 NADPH Subunit (Nox2) Expression in Aorta at 20 Weeks Post-Weaning ........... 51   CHAPTER 7: General Discussion ................................................................................. 53   7.1 Programming of Offspring Body Fat Distribution .................................................. 55   7.2 Programming of Offspring Glucose Homeostasis .................................................. 57   7.3 Programming of Offspring Vascular Function ....................................................... 59   7.4 Conclusion and Future Directions .......................................................................... 62   References ........................................................................................................................ 64   vii List of Tables TABLE 1. Maternal and Post-Weaning Diet Composition .............................................. 20   TABLE 2: Insulin ELISA Inter-Assay CV ....................................................................... 26   TABLE 3: Insulin ELISA Intra-Assay CV Plate 1 ........................................................... 26   TABLE 4: Insulin ELISA Intra-Assay CV Plate 2 ........................................................... 26   TABLE 5: Adiponectin ELISA Intra-Assay CV .............................................................. 27   TABLE 6: Real-time PCR Inter-Assay CV (AdipoR1) .................................................... 27   TABLE 7: Real-time PCR Intra-Assay CV (AdipoR1) Plate 1 ........................................ 27   TABLE 8: Real-time PCR Intra-Assay CV (AdipoR1) Plate 2 ........................................ 28   TABLE 9: Real-time PCR Intra-Assay CV (AdipoR1) Plate 3 ........................................ 28   TABLE 10: Real-time PCR Intra-Assay CV (AdipoR1) Plate 4 ...................................... 28   TABLE 11: Average Litter Size ....................................................................................... 30   TABLE 12: Comprehensive List of Male Offspring Studied ........................................... 31   viii List of Figures FIGURE 1. Cellular Folate and Methionine Cycle ........................................................... 14   FIGURE 2. Breeding Scheme ........................................................................................... 19   FIGURE 3. Maternal Body Weight During Pregnancy and Lactation. ............................ 29   FIGURE 4. Maternal Serum Vitamin B12 Concentration ................................................ 32   FIGURE 5. Male Offspring Growth and Body Weight .................................................... 34   FIGURE 6. Glucose Homeostasis at 20 Weeks Post-Weaning ........................................ 36   FIGURE 7. Glucose Homeostasis at 30 Weeks Post-Weaning ........................................ 38   FIGURE 8. Body Composition and Fat Pads at 20 Weeks Post-Weaning ....................... 41   FIGURE 9. Absolute Fat Mass and Lean Mass at 20 Weeks Post-Weaning ................... 42   FIGURE 10. Serum Adiponectin Concentration at 20 Weeks Post-Weaning .................. 43   FIGURE 11. Retroperitoneal Fat mRNA Expression at 20 Weeks Post-Weaning .......... 45   FIGURE 12. Endothelium-Dependent Relaxation of Aortae ........................................... 47   FIGURE 13. Endothelium-Dependent Relaxation of Mesenteric Arteries ...................... 49   FIGURE 14. Basal Nitric Oxide Production in Vessels ................................................... 51   FIGURE 15. Nox2 Protein Expression in Aorta ............................................................... 52   ix List of Abbreviations CVD Cardiovascular disease T2D Type 2 diabetes BMI Body mass index MONW Metabolically obese, normal-weight VAT Visceral adipose tissue SAT Subcutaneous adipose tissue MRI Magnetic resonance imaging CT Computed tomography DEXA Dual-energy X-ray absorptiometry NAFLD Non-alcoholic fatty liver disease IDF International Diabetes Federation MetS Metabolic syndrome HDL-C High-density lipoprotein cholesterol LDL-C Low-density lipoprotein cholesterol FPG Fasting plasma glucose BP Blood pressure DOHAD Developmental origins of health and disease eNOS Endothelial nitric oxide synthase NTD Neural tube defect RBC Red blood cell MRC Medical Research Council RCT Randomized controlled trial IOM Institute of Medicine CDC Centers for Disease Control and Prevention MS Methionine synthase THF Tetrahydrofolate DHF Dihydrofolate 5-MTHF 5-methyltetrahydrofolate ii MMA Methylmalonic acid WTD Western-type diet IPGTT Intraperitoneal glucose tolerance test ELISA Enzyme-linked immunosorbent assay PCR Polymerase chain reaction TBS-T Tris-buffered saline-tween KCl Potassium chloride NaCl Sodium chloride Ach Acetylcholine SNP Sodium nitroprusside PE Phenylephrine L-NAME L-NG-nitroarginine methyl ester NO Nitric oxide ANOVA Analysis of variance AUC Area under the curve HMW High molecular weight PSS Physiological saline solution NADPH Nicotinamide adenine dinucleotide phosphate DGAT Diglyceride acyltransferase ACC Acetyl-CoA carboxylase RIPA Radioimmunoprecipitation assay buffer CV Coefficient of variability ii Acknowledgements I wish to take this opportunity to acknowledge a number of individuals who have been instrumental in my progression through my Master’s degree. I would like to thank my supervisor, Dr. Angela Devlin, who took me on a graduate student and, in doing so, granted me exposure to the world of research. Because of her mentorship, I have had the privilege to attend and present at conferences and the opportunity to grow through the challenge of planning and carrying out my research project. I thank her for her support and guidance. I would also like to thank the members of my committee, Dr. Issy Laher, who strictly enforced 10 am tea time and ensured that blood sugar levels were maintained throughout the course of my experiments, and Dr. Tim Green, for his constant support during my project. I thank both for their expertise and insight. Thank-you to Saeid Golbidi for his surgical skills and dexterity –I wouldn’t have been able to do a huge section of my project without his help. In addition, I would like to express my gratitude to Dr. Haydn Pritchard, Advisor for the UBC Pathology and Laboratory Medicine Program, for checking in with me periodically and keeping me on track. I would also like to acknowledge the CFRI animal unit staff for their patience and diligence in accommodating such an involved study. Finally, I would like to extend my heartfelt thanks to past and present members of the Devlin lab at CFRI who have been beyond helpful and supportive during my time here. They are: Melissa Glier, Dian Sulistyoningrum, Jesse Olson, Eugene Wang, Tiffany Ngai, Abeer Aljaadi, Julia Wei, Kaia Hookenson, Kaela Barker, and Dr. Anita Cote. Lastly, a great many thanks to friends and family who served as editors, moral support, and comic relief. 1 CHAPTER 1: Introduction 1.1 A Mouse Model of Maternal Folic Acid and Vitamin B12 Intake Traditionally associated with poor diet and sedentary lifestyle, obesity (BMI ≥ 30 kg/m2) and related metabolic disorders are increasingly prevalent in children and adults throughout the world1-3. Since 1980, worldwide obesity has nearly doubled: in 2008, 35% of adults aged 20 and over were overweight, and 11% were obese 4. A growing body of evidence links cardiovascular and metabolic disease to nutritional adversity in the womb and in early postnatal life5, 6. Countless environmental stressors have the potential to affect offspring metabolic health. The focus of my thesis is on one of these stressors, maternal nutrition. The overall objective of my thesis is to investigate the role of maternal dietary folic acid and vitamin B12 intake on offspring cardiovascular and metabolic health. I used an experimental mouse model of developmental exposure to maternal nutrition to address my objective. Specifically, I determined the effect of maternal dietary folic acid and vitamin B12 intake during pregnancy and lactation on adult offspring: (i) body composition and metabolic health; and (ii) vascular function as an early indicator of cardiovascular health. The following introduction will cover relevant background information pertaining to obesity and cardiometabolic risk in humans. I will discuss developmental programming and its role in chronic diseases such as cardiovascular disease (CVD) and type 2 diabetes (T2D). I will review current folic acid food fortification policies in North America, provide an overview of folic acid and vitamin B12 metabolism, and summarize the current literature on folate and vitamin B12 status of Canadian women. Lastly, I will discuss potential programming mechanisms and provide rationale to support my 2 hypothesis for the role of folic acid and vitamin B12 in developmental programming of metabolic disease. 1.2 Obesity and Cardiometabolic Risk 1.2.1 Classification of Obesity Overweight and obesity are defined by BMI category and are considered as abnormal or excessive fat accumulation that may impair health 4. To date, an adult body mass index (BMI) of 25-30 kg/m2 is considered overweight, and a BMI ≥ 30 kg/m2 is considered obese. This method of defining obesity has come under scrutiny by the medical community as it fails to take into account differences in body composition and body fat distribution due to sex, age, ethnicity, and fitness level. Obesity is a heterogeneous classification and a spectrum of risk for metabolic disease exists among individuals of the same BMI. The assumption that all weight should be treated the same, regardless of which tissue comprises the excess weight, is one of the major pitfalls of using BMI as the measurement standard for obesity. A prime example of this failure can be seen in metabolically obese, normal-weight (MONW) individuals who have what is considered a healthy BMI (< 25 kg/m2) but have metabolic complications usually associated with obesity 7, 8. Similarly, research has documented clinically obese individuals (BMI ≥ 30 kg/m2) who are metabolically healthy and do not manifest complications such as insulin resistance and dyslipidemia 9-11. Body composition and body fat distribution, rather than total body weight, is of primary importance in determining metabolic health. One of the simplest observations for providing insight into determining cardiometabolic risk, is to classify an individual as having either an “apple” (android) or a “pear” (gynoid) body shape 12, 13. Individuals with metabolic health 3 problems typically exhibit more centralized abdominal adiposity and are thus described as “apple-shaped”. “Pear-shaped” individuals, or those that store fat primarily in their buttocks, hips, and thighs, are considered less at risk for cardiovascular disease and diabetes than those with a propensity for abdominal adiposity. As such, methods to quantify abdominal adiposity are more informative in determining metabolic health. At one point, waist circumference was touted as the best anthropometric measure of abdominal adiposity 14. However, it cannot distinguish between visceral and subcutaneous adiposity and is thus a poor predictor of relative metabolic risk among individuals with the same waist circumference. Determining the amount of visceral adiposity is useful as it is well established that visceral adipose tissue (VAT), rather than subcutaneous adipose tissue (SAT), is associated with metabolic risk. Currently, imaging technologies such as magnetic resonance imaging (MRI) and computed tomography (CT) scanning are used to quantify VAT whereas dual energy x-ray absorptiometry (DEXA) is used to quantify lean mass and fat mass 15. 1.2.2 Regional Fat Distribution and Metabolic Health Traditional roles for adipose tissue include cushioning and insulating the body and acting as a depot for energy storage in the form of fat. Normally functioning adipose tissue acts as the body’s storage site for fat in the form of triglycerides and mobilizes fat during times of fasting or high-energy demand. When this feedback system becomes overwhelmed by nutritional excess, adipocyte storage capacity is maxed out and fat begins to accumulate ectopically in other tissues such as skeletal muscle, liver, and heart 16. Fatty liver (liver steatosis) is found in a large proportion of obese individuals and may 4 contribute to the development of nonalcoholic fatty liver diseases (NAFLDs)17. Current views have broadened to define adipose tissue as a metabolically active organ, involved in hormone production and metabolic regulation. Adipocytes produce adipocytokines, such as adiponectin and leptin that contribute to the regulation of insulin sensitivity and appetite regulation, respectively 18, 19. Although there are general similarities between fat depots in terms of vascularization and lipid content, differences in biochemical and metabolic features exist between specific regional depots 20. Adipose tissue distribution, specifically VAT, is more important than overall body fat percentage in determining metabolic health and risk for metabolic diseases 21. Although the definition is simplistic and alternate categorizations exist, body fat can be divided into VAT and SAT. VAT is located within the abdominal cavity and surrounds the abdominal organs such as the liver, stomach, kidneys, and intestines. SAT is located outside of the abdominal cavity, on top of the muscle, and under the skin. VAT represents an independent risk factor for CVD and T2D 22, 23. VAT is associated with development of insulin resistance whereas total fat mass and SAT are not 24-26. Some studies have even suggested a protective role for lower body SAT, specifically in the form of increased thigh fat and hip size, in the development of metabolic disease 27, 28. However, recent research has suggested that the truncal depot may play a role distinct from abdominal and gluteofemoral depots in the development of insulin resistance 29. Thus, despite efforts to identify and assign distinct roles to various depots, adipose tissue should be considered as a heterogeneous, highly variable tissue, with numerous roles in the development of CVD and metabolic disease. 5 1.2.3 Definitions: Metabolic and Cardiovascular Disease Several definitions exist for describing the comorbidities linked to obesity. Metabolic syndrome (MetS) is the umbrella term typically used to describe the cluster of related factors that increase the risk of CVD and T2D. The International Diabetes Federation (IDF) definition of MetS includes central obesity (defined by ethnic-specific waist circumference) plus any two of the following four factors: triglycerides ≥150mg/dL or 1.7mmol/L, HDL cholesterol < 40 mg/dL or 1.03 mmol/L in males and < 50 mg/dL or 1.29 mmol/L in females, elevated blood pressure (systolic BP ≥ 130 or diastolic BP ≥ 85 mm Hg), and fasting plasma glucose FPG ≥ 100 mg/dL or 5.6 mmol/L 30. In place of MetS, the term “cardiometabolic risk” has recently crept into the medical lexicon as an agglomeration of factors, which promote atherogenic CVD and/or the development of T2D 31. Cardiometabolic risk factors include smoking, elevated blood pressure, elevated LDL, low HDL, elevated blood glucose, abdominal adiposity, insulin resistance, and elevated triglycerides and inflammatory markers 31. The list or factors is expected to expand with new discoveries. The increased use of the term “cardiometabolic risk” mirrors the evolution of our understanding of obesity, CVD, and metabolic disorders as complex, multi-factorial related issues. 1.3 Developmental Programming 1.3.1 Programming of Metabolic Disease Nutrition plays an important role in our overall health and wellbeing and is one of the most modifiable lifestyle factors for the treatment and prevention of obesity, T2D, 6 and CVD. Once considered an affliction of western-world affluence and excess, obesity has reached epidemic proportions in many countries and is now considered a worldwide problem 32. In 2011, 40 million children under the age of 5 were overweight 33. Explanations such as the thrifty gene theory, which suggests that the genes selected for in our ancestors to help us survive periods of starvation and are now making us sick, are not sufficient to explain the huge surge in obesity and development of metabolic disorders. As of late, more emphasis has been placed on the role that nutrition during development plays in the etiology of chronic disease later in life. There is increasing evidence that nutrient status during development (prenatal or early postnatal) can have lasting effects on metabolic health into adulthood. The developmental origins of health and disease (DOHAD) theory proposes that the environmental conditions during prenatal and early postnatal development have the capacity to program metabolic health in adulthood 34, 35. This theory was first pioneered by David Barker who reported that low birth weight and malnutrition during development was associated with greater risk of developing CVD. Research that followed focused largely on the effects of caloric restriction during pregnancy. The Dutch Famine Birth Cohort study consists of individuals conceived during the Dutch famine from 1944 to 1945 and thus subjected to nutritional deprivation during development. The study shows early onset and increased incidence of CVD and risk for T2D in individuals exposed to poor maternal energy intake during the famine 36- 39. Since then, our understanding has been broadened to include maternal obesity and over nutrition as stressors to offspring development. Vitamin deficiency can also occur when the primary types of food consumed are nutrient poor despite being high in energy/calories. As is the case in North America and increasingly throughout the rest of 7 the world, processed food and foods containing high levels of saturated fats and sugar are easily accessible and consumption is up in many demographics. The number of obese or overweight children has increased alarmingly 40 and we are starting to look for explanations, other than sedentarism and caloric excess, to explain the surge in childhood obesity. The number of overweight or obese women of child-bearing age (18-44 years) rose by 50% between 1992 and 2004 in Canada 41. An increasing number of women are overweight or obese when they become pregnant, which places them at risk for pregnancy complications such as gestational diabetes 42. This has led to the suggestion that, if the development of an infant in the womb can be affected by nutrient deprivation, such plasticity could also be affected by caloric excess. Currently, the relative contributions of maternal obesity, high fat intake, and poor nutrient status to metabolic adversity later in life are unknown. The concept of developmental programming has initiated a great amount of research related to fetal exposures during gestation. Already, there are studies to support the role of maternal stress, exposure to chemicals, and diet on offspring physical and mental health 43-46. The focus of my thesis is on the role of maternal diet on offspring cardiometabolic health. 1.3.2 Programming of Vascular Disease Obesity is an established risk factor for insulin resistance and CVD 47. The relationship between visceral obesity and vascular disease is complex and the linking molecular mechanisms unclear. In fact, the relationship between adiposity and vascular disease is so strong that it becomes difficult to study each in isolation. As such, the 8 current obesity epidemic potentially confounds other possible independent causes of CVD. Genetic and lifestyle risk factors have been identified but, as with other aspects of metabolic disease, there is evidence to suggest that adverse prenatal and early postnatal environments can produce vascular disease later in life. Maternal under nutrition often manifests in low birth weight children. In accordance with David Barker’s original discoveries, a systematic review of over 444, 000 male and female individuals across a spectrum of age and ethnicity showed that low birth weight and accelerated postnatal growth are associated with raised blood pressure 48. Independent associations between low birth weight and risk of hypertension and CVD have been observed across a variety of diverse populations 49-53. Low birth weight has also been associated with thickening of the aortic wall, increased arterial stiffness, and increased risk for atherosclerosis 54-56. Such human studies have shown associations between maternal diet, birth weight, and CVD but are not causal or free from potential confounding. Animal models have proven useful in filling in the mechanistic gaps and allowing for controlled studies. Such animal studies have shown that maternal dietary protein restriction or overall reduction in dietary intake during pregnancy can affect offspring cardiovascular function 57-60. Mother to child transmission of obesity and CVD risk has also been studied in rodent models of maternal diet-induced obesity 61, 62. Several rodent studies report that, despite being raised on a standard chow diet, offspring of obese dams have increased adipose tissue later in life 63- 67. Other studies have reported that offspring of diet-induced obese mice go on to develop hypertension as adults 68. As a harbinger of vascular disease, endothelial function is often assessed as an indicator of vascular health and disease progression 69. A functional epithelium continuously produces basal amounts of nitric oxide and responds to factors 9 such as sheer stress induced by exercise to up-regulate nitric oxide production and induce vascular smooth muscle relaxation 70. Processes affecting endothelial nitric oxide production interfere with modulation of vascular tone. For example, research has shown improved endothelial dysfunction, reduced blood pressure, and increased endothelial nitric oxide synthase (eNOS) expression in the offspring of dams supplemented with soy protein during gestation 71. Thus, there is accumulating evidence that the developmental environment may program the development of vascular disease. 1.4 Epigenetics: Gene and Environment Interaction Proponents of classical genetics uphold that the genetic code is, for all intents and purposes, static, and that millions of years are required for natural selection and evolution to occur 72. Until recently, scientists proposed that an individual was dealt a genetic deck of cards at birth and that his risk for disease was fixed. Recent research has revealed a layer of control, referred to as the ‘epi’- genome, which literally sits ‘on top’ of and is more plastic in nature than the genome. Epigenetics is defined as heritable changes in gene expression that occur without changes to the fundamental genetic sequence 73. The epigenome, itself, consists of a set of cellular machinery designed to package DNA to regulate gene expression. Currently studied epigenetic mechanisms include DNA methylation, histone modifications, and siRNA 74. The advancing field of epigenetics has provided evidence to suggest that the epigenome is highly plastic and, although epigenetic patterns can be passed from generation to generation, the epigenome can also be altered during one’s lifetime through interaction with the environment 75. Many 10 chronic diseases, such as cardiovascular disease have well-defined genetic determinants as well as environmental risk factors. In the development of cardiovascular and metabolic disease, many of these environmental factors are related to diet and, as such, nutrient- gene interaction is a promising candidate for studying genomic plasticity and the developmental origins of disease. There is evidence to suggest that the epigenome is susceptible to modification both in adult life and in early development but the DOHAD hypothesis proposes that programming of metabolic disease occurs during fetal and early postnatal development 76. 1.5 Folic Acid Fortification 1.5.1 Overview of Mandatory Folic Acid Fortification Prenatal and early postnatal nutrient status influences metabolic health in adulthood; however, the mechanisms behind these effects are not fully understood. Folate is one of the most routinely recommended prenatal vitamins because of its role in preventing neural tube defects (NTDs) 77. Research suggests that a decrease in serum red blood cell (RBC) folate concentrations is one of the physiological changes found in women not consuming supplements during pregnancy and that adequate fetal folate supply is so crucial that cord blood folate status is maintained at the expense of the mother’s serum folate concentrations 78-81. The increased folate requirement is, in part, due to the production of cells and rapid period of fetal growth during pregnancy. Folate is a one-carbon source for the synthesis of DNA and RNA and metabolism of key amino acids, both of which are crucial for growth and development 82. Other potential causes for decreased maternal circulating folate during pregnancy include increased folate 11 catabolism and clearance in pregnant women relative to nonpregnant women 83-85. The British Medical Research Council (MRC) and Hungarian randomized controlled trials (RCTs) of vitamins were stopped early in 1991 and 1992, respectively, when it became clear that maternal folic acid intake was strongly associated with a decreased incidence of NTDs86, 87. Similarly, a health campaign conducted in China between 1993 and 1995 showed that periconceptual folic acid use reduced the incidence of NTDs by 41 percent in the southern region of China88. Based on the dramatic results shown by these three RCTs , it became widely accepted in the 1990s that consumption of folic acid during pregnancy could prevent most NTDs. It is now recognized that early folic acid use is critical as the development and closure of the neural tube occurs within the first 28 days post- conception, a time when many women are unaware of their pregnancy89. In 1998, the Institute of Medicine (IOM) in the United States recommended that women of child- bearing age consume 400µg of folic acid daily from fortified food or a supplement, in addition to folate consumed from naturally occurring dietary sources90. The importance of periconceptional folate supplementation for normal fetal development led to mandatory addition of folic acid to enriched flour and enriched cereal grains in the United States and to white flour and enriched pasta and cornmeal in Canada, in the 1990s91. Mandatory folic acid fortification efforts have since reduced the incidence of NTDs in the United States, Canada, Costa Rica, and Chile92-95. As assessed by the Centers for Disease Control and Prevention (CDC), as of 2007 there were more than 50 countries, which had implemented mandatory flour fortification programs96. Due to mandatory folic acid fortification policies overall dietary folic acid intake and blood folate concentrations have increased97-99. Although fortification programs have been 12 successful in reducing the rates of NTDs, they have also resulted in increased circulating folic acid concentrations in the entire population, which has been shown to potentially depress immune function100. Folic acid is metabolically linked to other vitamins, such as vitamin B12, and it has been proposed that high serum folate could be detrimental to individuals with marginal vitamin B12 status101. The great success achieved by folic acid fortification programs in reducing the incidence of NTDs has overshadowed some of the potential adverse consequences of increased folic acid intake. Folic acid and vitamin B12 are metabolically linked and the widespread implementation of folic acid fortification programs has led to the situation where many people have very high blood folate concentrations and marginal vitamin B12 status. The majority of Canadians have high folate status but approximately 5% are vitamin B12 deficient99, 102. In Canada as many as 1 in 20 women may be vitamin B12 deficient in the early stages of pregnancy103. Currently, the metabolic consequences of high folate status and vitamin B12 deficiency during pregnancy are unknown. 1.5.2 Folate and Vitamin B12 Metabolism One of the current pervasive theories as to how the developmental environment programs metabolic health later in life is through alteration of DNA methylation patterns early in development. The connection between folate and fetal central nervous system development has long since been established but due to the critical role folate plays in supplying methyl groups for cellular reactions, researchers have begun to examine the arguably more subtle relationship between dietary methyl donors, DNA methylation, and 13 metabolically related genes. Mammals lack the ability to synthesize folates de novo and must rely on their diet to supply preformed folates. Naturally occurring folates are synthesized by microorganisms and plants and exist in the reduced form. Folate-rich foods include certain fruits, vegetables, and organ meats – largely liver104. Folic acid is the oxidized form of folate and is generally more stable than naturally occurring folate. As such folic acid is the form added to supplements and used in fortification programs. Vitamin B12 is required as a cofactor by two enzymes in the body: methylmalonyl CoA mutase and methionine synthase (MS). MS is part of the methionine cycle and catalyzes the remethylation step of homocysteine to methionine for subsequent downstream methyl group donation (Figure 1). To become part of the naturally occurring folate pool, folic acid must be reduced to dihydrofolate (DHF) and further to tetrahydrofolate (THF) by DHF reductase. Before methyl groups are delivered to the methionine cycle THF must be further metabolized to 5-methyl-tetrahydrofolate (5- MTHF) in the small intestine or liver. Folate, in the form of 5-MTHF, supplies the methyl group for the reaction and is converted into THF in the process. In the cell, THF can participate in the methionine cycle after reduction to 5-MTHF or be used for the biosynthesis of purines and pyrimidines. Folate compounds are retained in the cell by polyglutamylation by folate polyglutamate synthase. In vitamin B12 deficiency, folate is trapped as 5-MTHF and unable to participate in the production of purines and pyramidines, resulting in megaloblastic anemia. Because vitamin B12 is required by MS for the remethylation step, it is feasible that vitamin B12 deficiency has the potential to affect the methylation capacity of the methionine cycle by decreasing the pool of available one-carbon donors and thus, the subsequent methylation of downstream targets. 14 In fact, recent animal studies have associated changes in DNA methylation (hypomethylation) with vitamin B12 deficiency105, 106. High folic acid intake masks the hematological signs of vitamin B12 deficiency in that folic acid is able to directly enter the cell unmetabolized, be converted to THF and participate in purine and pyramidine synthesis, bypassing the methionine cycle. Vitamin B12 deficiency is distinguishable from folate deficiency by examination of the methymalonyl CoA mutase pathway such that in vitamin B12 deficiency methylmalonic acid (MMA) concentrations are elevated in urine and blood. FIGURE 1. Cellular Folate and Methionine Cycle FA folic acid; 5-MTHF 5-methyltetrahydrofolate; DHF dihydrofolate; THF tetrahydrofolate; Hcy homocysteine; AdoHcy S-adenosylhomocysteine; AdoMet S- adenosylmethionine; MS methionine synthase; MT methyl transferase. 15 1.5.3 Folate and Vitamin B12 Imbalance during Pregnancy Folate metabolism is a fundamental process for all mammals but little is known about the potential adverse effects of high folic acid intakes. The imbalance of high folic acid status and vitamin B12 deficiency created by mandatory folic acid fortification may be of particular concern during pregnancy. Recent studies have suggested such an imbalance my affect the metabolic health of the developing child. A 2008 longitudinal birth cohort study from India reported that children born to women with high erythrocyte folate concentrations at 28 weeks had greater adiposity and insulin resistance at age six107. Low vitamin B12 status also predicted greater insulin resistance at age six, with the greatest insulin resistance observed in offspring from mothers with high folate and low vitamin B12 concentrations107. In 2011, Stewart and colleagues found no association with maternal folate status but reported that maternal B12 deficiency (<148ρmol/L) was associated with an increased risk for insulin resistance108. 16 CHAPTER 2: Rationale and Hypothesis Thus far, the literature suggests a role of the developmental environment in programming offspring health later in life. I hypothesize that maternal folic acid and vitamin B12 intake in mice contributes to cardiometabolic risk in the offspring and programs the metabolic response to adult diet. Folic acid and vitamin B12 are good candidates for programming of metabolism because of the role of DNA methylation in the epigenetic regulation of gene expression and the necessity of folate and vitamin B12 for providing the methyl groups for such cellular reactions. Maternal methyl nutrient intake was reported to affect gene expression patterns in mice109 and insulin resistance and adiposity in sheep and humans, respectively110. Based on the findings of the Pune Maternal Nutrition Study in India and Canadian populations studies which have reported that 1 in 20 women are vitamin B12 deficient despite having adequate folate status 102, work is required to understand the connection between maternal B vitamin intake and offspring metabolism. These early epidemiological studies are purely correlational and, although they can expose important health issues, causality cannot be extrapolated from them; therefore, mechanistic studies are required. The mice used in my studies are the widely used C57BL/6J inbred strain, which is susceptible to diet-induced obesity and glucose intolerance (http://jaxmice.jax.org). Mechanistic studies on the effects of folic acid and vitamin B12 imbalance during pregnancy are lacking and inbred mouse strains provide the means to study such pathways in a controlled setting. Although human metabolism is not identical to that of mouse, many metabolic pathways share similarities between the two species and mouse 17 studies provide the mechanistic foundation upon which to base future research, both clinical and basic science. In this thesis I examined the cardiometabolic phenotype of adult male C57BL/6J mice exposed to maternal dietary folic acid and vitamin B12 imbalance in utero and during early postnatal development. Using the breeding scheme and methods described in Chapter 3, I simulated the high folic acid intake and vitamin B12 deficiency often observed in Canadian women and assessed the metabolic phenotype of the offspring. I further challenged a group of offspring with a WTD to induce excess adiposity. Data on weight gain, body composition, glucose homeostasis, and adipose tissue gene expression were collected and are presented in Chapter 5. I further analyzed vascular function and protein expression in the aorta from the same offspring mice; these results are presented in Chapter 6. 18 CHAPTER 3: Materials and Methods 3.1 Breeding scheme and Animal Diets Male and female C57BL/6J mice [purchased from the UBC Centre for Disease Modeling and Charles River (Montreal), respectively] were acclimatized to the CFRI animal facility for one week and mated to generate dams for our study. Virgin dams on standard laboratory chow (9% fat, PicoLab Mouse Diet 20 (5058), LabDiet®, PMI Nutrition International, St. Louis, MO) were bred at 6 weeks of age to age-matched males. After the first litter had been weaned, the same dams were fed one of the following diets: high folic acid/ no vitamin B12 (HFA-B12), high folic acid/adequate vitamin B12 (HFA+B12), or adequate folic acid/adequate vitamin B12 (control) for 6 weeks prior to breeding, during pregnancy and lactation. The control diet conformed to the nutrient requirements set by the National Research Council (1995). The HFA-B12 and HFA+B12 diets contained five times more folic acid than the accepted nutritional requirements for mice 111, in the absence and presence of adequate vitamin B12 (Table 1). At three weeks offspring mice were weaned onto the control or WTD for the duration of the study. Mice were housed in groups of 3 to 5 animals per cage under a standard 12-hr light, 12-hr dark cycle and had ad libitum access to food and water. At the end of the feeding period, animals were anesthetized with isofluorane and blood was collected via cardiac puncture. After cervical dislocation, tissues were harvested and snap-frozen in liquid nitrogen for storage at -80°C. All procedures were performed according to the guidelines and with the approval of the University of British Columbia Animal Care Committee. 19 FIGURE 2. Breeding Scheme 20 TABLE 1. Maternal and Post-Weaning Diet Composition HFA+B12 HFA-B12 Control Western Vitamin B12 (µg/kg) 50.0 0 50.0 50.0 Folic acid (mg/kg) 10.0 10.0 2.0 2.0 Fat (% energy) 16% 16% 16% 45% Protein (% energy) 20% 20% 20% 20% Carbohydrate (% energy) 64% 64% 64% 35% Total energy (kcal/kg) 3948 3948 3948 4700 Soybean oil and cornstarch were the primary sources of fat and carbohydrate in the control, HFA-B12, and HFA+B12 diets. Fat in the western diet was comprised of a mixed source including soybean oil, butter, lard, and vegetable shortening; carbohydrate content was 70% sucrose and 30% cornstarch. All other dietary components were consistent between diets. 21 3.2 Body Composition Body composition was quantified using quantitative magnetic resonance technology, which distinguishes differential proton states between lipids, lean tissues, and free water (EchoMRI-100 Echo Medical Systems, Houston, TX) 112. The qMRI machine is located in the animal unit of the Child and Family Research Institute and its use kindly donated by Dr. William Gibson. Lean mass and fat mass were calculated by averaging the values from three separate scans per animal. The scans were done in retrospect of the findings at 20 weeks on post-weaning diet and thus coincided with the second time point (30 weeks on post-weaning diet). 3.3 Glucose Tolerance Glucose tolerance tests were performed on weeks 19 and 29 (one week before the end of the post-weaning feeding period) on 5 hour fasted mice using a 25% dextrose solution administered intraperitoneally at a dosage of 0.75g/kg body weight. Blood was collected at baseline from the saphenous vein in EDTA-treated tubes for insulin measurement. Blood glucose was quantified at baseline, and at 15 min, 30 min, 60 min, and 90 min post-injection using a glucometer (Breeze®2 Glucometer, Bayer). 3.4 Serum and Plasma Protein Concentrations At time of sacrifice blood was collected via cardiac puncture and allowed to clot at room temperature for 15 minutes. The blood samples were spun at 8000 rpm at 4°C for 15 minutes and the serum recovered and stored at -80°C. Total and high molecular weight adiponectin levels were quantified in the serum samples using the mouse ultrasensitive adiponectin ELISA (ALPCO Diagnostics). Fasting insulin concentrations 22 were quantified in the plasma samples collected in EDTA-treated tubes at IPGTT baseline using the mouse ultrasensitive insulin ELISA (ALPCO Diagnostics). Maternal serum vitamin B12 concentrations were quantified via microbiological assay (ID-Vit® Vitamin B12, Immundiagnostik AG). 3.5 Quantification of mRNA Total RNA was extracted from frozen whole retroperitoneal fat pads using the RNeasy Lipid Tissue Mini Kit (QIAGEN) as per manufacture’s instructions. On-column DNase digestion was performed for complete removal of DNA. RNA concentration and purity (A260/A280 ratio of between 1.9 and 2.1) were determined using a nanodrop spectrophotometer. RNA integrity was confirmed via gel electrophoresis and visual observation of intact 18s and 28s ribosomal RNA bands and 500ng of RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Adipor1, Acaca, and Dgat1 mRNA levels were quantified by real-time PCR with primers and TaqMan MGB probes (Applied Biosystems) specific for murine Adipor1 (Mm01291334_mH), Acaca (Mm01304257_m1), and Dgat1 (Mm00515643_m1). The internal control was 18s rRNA (VIC-MGB) and relative gene expression was calculated using the ΔΔCt method of relative quantification113. Each sample was run in duplicate and the experiment repeated twice. All results were normalized to findings in maternal control diet offspring fed the post-weaning control diet. 23 3.6 Western Blotting Frozen aortae were homogenized by sonication of tissue (~15mg) on ice for two 15-second pulses in 200 µL of a lysis buffer [1x RIPA buffer (Cell Signaling: 20mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin -) and EDTA-free protease inhibitor tablet (Roche Diagnostics) dissolved in dH2O]. This was followed by centrifugation at 10,000 rpm at 4°C. The protein concentration of the resulting supernatant was determined by the colorimetric Bradford Assay114. For Western blot analyses, lysed tissue homogenates (30 µg protein) were boiled for 15 minutes in loading sample buffer consisting of 950 µL 2x Laemlli buffer (BioRad: 65.8 mM Tris-HCl, pH 6.8, 23.6% glycerol, 2.1% SDS, 0.01% Bromophenol Blue) and 50 µL β-mercaptoethanol and were resolved on 10% SDS-PAGE denaturing gel for 90 minutes at 110V. Protein was transferred in ice-cold transfer buffer (25mM Tris base, 190 mM glycine) onto polyvinylidine difluoride (PVDF) membrane for 90 minutes at 110V. Membranes were stained with Ponceau S staining solution (Sigma) to visualize protein bands and ensure complete transfer, and then rinsed with dH2O. Membranes were blocked overnight with 5% milk powder in Tris-Buffered Saline (20 mM Tris base, 250 mM NaCl), 0.05% Tween-20 (TBS-T) at 4°C. Membranes were incubated with monoclonal mouse anti-human gp91-phox (54.1) primary antibody (sc- 130543, Santa Cruz) at 1:200 dilution in 5% bovine serum albumin (BSA) for two hours at room temperature. After 30-minute wash with TBS-T the membrane was incubated with alkaline phosphatase conjugated goat anti-mouse IgG (sc-2008, Santa Cruz) at 1:2000 in 5% milk in TBS-T for 2 hours. Expression of actin was used as a loading 24 control. For actin detection, membranes were incubated with goat anti-human actin polyclonal primary antibody (I-19:sc-1616, Santa Cruz) at 1:500 in 5% BSA for 2 hours followed by incubation with goat anti-rabbit (IgG-AP: sc-2007, Santa Cruz) at 1:2000 in 5% milk in TBS-T for 2 hours. After incubation with secondary antibody the membranes were washed again in TBS-T and then incubated for 2 minutes with Immun-Star™ AP Substrate (Bio-Rad) before exposure and visualization of protein bands via chemiluminescence. Membranes were exposed for a total of 25 minutes with images captured every 5 minutes and relative protein fluorescence quantified by densitometry using GeneTools software (Perkin Elmer). 3.8 Vascular Endothelial Function Dissected thoracic aortae were cleaned of fat and connective tissue in ice-cold physiological saline solution (PSS: 119 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4, 24.9 NaHCO3, 0.023 mM EDTA, 1.6 mM CaCl2 and 11.1 mM dextrose), cut into 2mm long cylinders, and mounted on a wire myograph system for isometric force measurement of relaxation (JP Trading, Aarhus, Denmark). Each ring was suspended in 5 mL of oxygenated (95% O2 + 5% CO2) PSS solution maintained at 37°C and gradually stretched to an optimal resting tension of 5 mN as determined in previous experiments to allow development of a stable basal tone115. Vessels were allowed to equilibrate for 30 minutes and PSS solution was replaced between tension adjustment and equilibration steps. Each vessel was maximally activated with a KCl solution (80 mM, prepared by equimolar substitution of NaCl in PSS solution) to confirm tissue viability. Following a 30-minute washout period with fresh PSS solution and return to basal tension, aortic rings 25 were pre-constricted to a stable tension with phenylephrine (10-5 M). After stable contraction was achieved, acetylcholine (10-9 to 10-5 M) was added in an incremental manner to observe endothelium-dependent relaxation. After an hour-long washout period, aortic rings were pre-constricted with phenylephrine and sodium nitroprusside (SNP) was added (10-9 to 10-5 M) to observe endothelium-independent relaxation. Responses to vasorelaxants were calculated as a percentage decrease from original force induced by addition of phenylephrine. In a separate set of experiments after challenge with 80 mM KCl solution, phenylephrine was added incrementally (10-9 to 10-5 M) followed by a washout period. Vessels were then incubated with 100µM of nitric oxide synthase (NOS) inhibitor NG –nitro-L-arginine methyl ester (L-NAME) and constricted again with phenylephrine to further assess the role of nitric oxide in the relation of the vessels. 3.9 Statistics Statistical significance of differences between groups was tested by a two- way analysis of variance (ANOVA) with maternal diet and post-weaning offspring diet as the independent variables. If significant interactions were found, the effect of maternal diet was assessed separately from post-weaning offspring diet by one-way ANOVA. Results were normalized to findings in maternal control diet offspring fed the post- weaning control diet. All statistical analyses were performed using SPSS statistical software (version 12). P≤0.05 was considered significant. Experimental results are expressed as means ± SEM. 26 3.9.1 Inter- and intra- Assay Variability The inter- and intra- assay coefficient of variability (CV) was calculated for the insulin and adiponectin assays and real-time PCR runs using the following equation: % CV = [Standard deviation between duplicates (SD)/ Mean of duplicates (AVG)] x 100 Insulin ELISA Coefficient of Variability TABLE 2: Insulin ELISA Inter-Assay CV Standard Plate 1 (ng/mL) Plate 2 (ng/mL) SD AVG (ng/mL) CV (%) AVG CV (%) 1 0.15 0.17 0.01 0.16 8.84 1.93 2 0.53 0.52 0.01 0.53 1.35 3 1.24 1.24 0.00 1.24 0.00 4 3.75 3.75 0.00 3.75 0.00 5 6.9 6.90 0.00 6.90 0.00 6 1.24 1.27 0.02 1.26 1.69 7 5.50 5.63 0.09 5.57 1.65 TABLE 3: Insulin ELISA Intra-Assay CV Plate 1 Standard Measure 1 (ng/mL) Replicate (ng/mL) SD AVG (ng/mL) CV (%) AVG CV (%) 1 0.17 0.14 0.02 0.15 16.14 7.59 2 0.50 0.55 0.03 0.53 6.09 3 1.11 1.36 0.17 1.24 14.02 4 3.69 3.81 0.08 3.75 2.24 5 6.53 7.27 0.53 6.90 7.62 6 1.27 1.22 0.03 1.24 2.44 7 5.68 5.32 0.25 5.50 4.61 TABLE 4: Insulin ELISA Intra-Assay CV Plate 2 Standard Measure 1 (ng/mL) Replicate (ng/mL) SD AVG (ng/mL) CV (%) AVG CV (%) 1 0.16 0.18 0.01 0.17 6.49 7.15 2 0.46 0.57 0.07 0.52 14.32 3 1.13 1.36 0.16 1.24 12.90 4 3.65 3.85 0.14 3.75 3.72 5 6.63 7.17 0.38 6.90 5.49 6 1.22 1.32 0.07 1.27 5.30 7 5.70 5.55 0.10 5.63 1.82 Insulin ELISA inter-assay CV (%) = 1.93 Insulin ELISA intra-assay CV (%) = (7.59 + 7.15)/2 =7.37 27 Adiponectin ELISA Coefficient of Variability (%) TABLE 5: Adiponectin ELISA Intra-Assay CV Standard Measure 1 (ng/mL) Replicate (ng/mL) SD AVG (ng/mL) CV (%) AVG CV (%) 1 7.19 7.25 0.05 7.22 0.63 1.11 2 4.49 4.54 0.04 4.52 0.87 3 2.53 2.55 0.02 2.54 0.72 4 1.38 1.40 0.02 1.39 1.15 5 0.76 0.76 0.00 0.76 0.38 6 0.48 0.49 0.01 0.49 1.35 7 0.33 0.32 0.01 0.32 2.69 Adiponectin ELISA intra-assay CV (%) = 1.11 Traditionally accepted inter- assay CV values are less than 15% and less than 10% and for intra- assay CV. The inter- and intra- assay CV calculated for the insulin ELISA were 1.93% and 7.37%, respectively, and 1.11% for the adiponectin ELISA intra- assay CV (only one plate was used). Real-Time PCR Coefficient of Variability (%) TABLE 6: Real-time PCR Inter-Assay CV (AdipoR1) Control AVG Ct 1 AVG Ct 2 AVG Ct 3 SD AVG Ct CV (%) AVG CV (%) 1 23.45 27.43 27.52 2.32 26.13 8.88 8.35 2 23.19 27.44 27.37 2.43 26.00 9.34 3 23.72 27.66 27.50 2.23 26.29 8.47 4 23.52 27.58 - 2.87 25.55 11.24 5 23.70 27.97 - 3.02 25.83 11.67 6 27.69 27.87 - 0.13 27.78 0.47 TABLE 7: Real-time PCR Intra-Assay CV (AdipoR1) Plate 1 Control Ct 1 Ct 2 SD AVG Ct CV (%) AVG CV 1 23.43 23.47 0.03 23.45 0.11 0.65 2 23.43 22.96 0.34 23.19 1.45 3 23.77 23.68 0.07 23.72 0.28 4 23.50 23.55 0.04 23.52 0.16 5 23.91 23.49 0.30 23.70 1.25 28 TABLE 8: Real-time PCR Intra-Assay CV (AdipoR1) Plate 2 Control Ct 1 Ct 2 SD AVG Ct CV (%) AVG CV 1 27.49 27.36 0.09 27.43 0.32 0.33 2 27.53 27.35 0.13 27.44 0.47 3 27.69 27.63 0.04 27.66 0.15 6 27.61 27.76 0.11 27.69 0.38 TABLE 9: Real-time PCR Intra-Assay CV (AdipoR1) Plate 3 Control Ct 1 Ct 2 SD AVG Ct CV (%) AVG CV 4 27.63 27.54 0.06 27.58 0.23 0.30 5 28.01 27.93 0.06 27.97 0.20 6 27.97 27.78 0.13 27.87 0.47 TABLE 10: Real-time PCR Intra-Assay CV (AdipoR1) Plate 4 Control Ct 1 Ct 2 SD AVG Ct CV (%) AVG CV 1 27.57 27.47 0.07 27.52 0.25 0.15 2 27.38 27.36 0.01 27.37 0.05 3 27.52 27.47 0.04 27.50 0.14 Real-time PCR inter-assay CV (%) = 1.93 Real-time PCR intra-assay CV (%) = (0.65 + 0.33 + 0.30 + 0.15)/4 = 0.36 Calculated real-time PCR inter- and intra- assay CV for the AdipoR1 gene target were 1.93% and 0.36%, respectively, which is acceptable. 29 CHAPTER 4: Maternal Vitamin B12 Status, Body Weight, and Litter Size 4.1 Maternal Body Weight and Litter Size The breeding scheme was designed around the idea that each distinct developmental environment represents one experimental unit (i.e. each dam is an n of 1). As such, the goal was to achieve at least 6 litters per diet group. At the completion of the breeding portion of the experiment, the control, HFA-B12, and HFA+B12 groups had 8, 6, and 7 dams, respectively, that had produced and nursed litters until weaning. Maternal growth was plotted from time on diet, throughout breeding and pregnancy, until weaning of the litters. I observed no effect of maternal diet on maternal body weight during breeding, pregnancy, and lactation (Figure 3). FIGURE 3. Maternal Body Weight During Pregnancy and Lactation. Data are presented as mean ± SEM. 0 5 10 15 0 20 40 60 Week M as s (g ) Control HFA-B12 HFA+B12 Maternal Diet Birth Breed Wean 30 At weaning, litters were separated into male and female, and half of the pups from each sex randomly assigned to the control diet or the WTD. The folic acid and vitamin B12 imbalance was only present during gestation and the early postnatal period; once weaned onto adult diet, mice received the same about of folic acid and vitamin B12. At time of weaning, analysis revealed no differences in overall litter size or in the number of male and female pups per litter (Table 2). TABLE 11: Average Litter Size Control (n=8) HFA-B12 (n=6) HFA+B12 (n=7) Litter size 6.0 ± 0.60 9.0 ± 0.70 7.6 ± 0.90 Male pups per litter 4.8 ± 0.65 4.7 ± 0.67 3.3 ± 0.60 Female pups per litter 3.6 ± 0.56 4.3 ± 0.43 4.9 ± 0.74 n = number of litters. Values are presented as mean ± SEM. 31 Although both sexes were weaned onto adult diets, my thesis work focused on characterizing the male offspring only. Thus, the offspring data presented in following chapters examines the effect of and interaction between maternal and post-weaning diets on dependent variables in male mice. Although sex effects are typically evident when studying metabolism, the discussion of sex-specific programming is beyond the scope of my thesis. Allocation of male pups to post-weaning diet groups resulted in 6 groups for study (3 maternal diets x 2 post-weaning diets). I will refer to the six groups as follows: C/C, HFA-B12/C, HFA+B12/C, C/W, HFA-B12/W, and HFA+B12/W (maternal diet/post weaning diet) where C=control diet and W=western diet. Table 3 shows the pool of male pups drawn from for proceeding experiments in Chapters 4 and 5. TABLE 12: Comprehensive List of Male Offspring Studied Maternal diet Control HFA-B12 HFA+B12 Post Weaning diet Control 20 13 10 Western 17 15 11 32 4.2 Maternal Serum Vitamin B12 Concentrations Dams were sacrificed at 1 week post-weaning (13 weeks on diet) and tissues collected. Serum vitamin B12 concentrations were quantified to confirm vitamin B12 depletion in the HFA-B12 group. Serum B12 concentrations in the HFA-B12 dams were significantly lower compared to those of dams fed the control and HFA+B12 diets (Figure 4). FIGURE 4. Maternal Serum Vitamin B12 Concentration Values are presented as mean ± SEM. Control HFA-B12 HFA+B12 0 5000 10000 15000 20000 25000 C on ce nt ra tio n (p m ol /L ) Control HFA-B12 HFA+B12 Maternal Diet P<0.001 33 CHAPTER 5: Body Composition and Metabolic Characteristics of Offspring 5.1 Male Offspring Growth and Body Weight Mice were weighed at weaning to observe the effect of maternal diet on pup size before introduction to the post-weaning offspring diet. Analysis revealed no differences in body weight between groups at time of weaning (Figure 5C). Control and WTD-fed mice showed rapid rates of growth from weaning for the first 5 weeks. Between weeks 5 and 10 rate both weaning groups exhibited a constant growth rate with the western group slightly larger than the control. The western group showed an increase in growth rate at week 10, breaking off from the control group and proceeding to gain substantially more weight whereas the control group continued to gain weight at a steady rate (Figure 5A & 5B). The early increased rate of growth exhibited by male offspring was not maintained and decreased steadily over time. At 20 weeks post-weaning, WTD- fed mice were larger than control diet-fed mice and an effect of maternal diet on body weight was observed; regardless of post-weaning diet, mice with developmental exposure to HFA-B12 and HFA+B12 diets weighed less than those exposed to maternal control diet (Figure 5D). At 30 weeks post weaning, WTD-fed mice were still larger than control diet-fed mice but the effect of maternal diet on body weight was no longer observed (Figure 5E). 34 FIGURE 5. Male Offspring Growth and Body Weight A. Weight of control-fed mice over time. B. Weight of WTD-fed mice over time. C. Body weight at weaning (3 weeks old). D. Body weight at 20 weeks post-weaning. E. Body weight at 30 weeks post-weaning. Values are presented as mean ± SEM. 0 10 20 30 0 20 40 60 Week M as s (g ) Control Western 0 5 10 15 M as s (g ) Weaning Diet (at wean) Control Western 0 20 40 60 M as s (g ) Weaning Diet (at 20wks) P<0.001, weaning diet P<0.05, maternal diet Maternal Diet Control HFA-B12 HFA+B12 Control Western 0 20 40 60 M as s (g ) P<0.001, weaning diet Weaning Diet (at 30wks) 0 10 20 30 0 20 40 60 Week M as s (g ) Control HFA-B12 HFA+B12 Maternal Diet A B C D E 35 5.2 Glucose Tolerance at 20 Weeks Post-Weaning Intraperitoneal glucose tolerance tests (IPGTTs) were conducted and fasting concentrations of glucose and insulin quantified in mice at 20 weeks post-weaning. Fasting blood glucose (P<0.05) and plasma insulin concentrations (P<0.001) were higher in WTD-fed mice than in control diet-fed mice (Figure 6A & B). Blood glucose concentrations remained higher (P<0.01) in WTD-fed mice at all time points post glucose injection compared to mice fed the control diet (Figure 6C & D). This was accompanied by greater AUC (P<0.001) in WTD-fed mice relative to control diet-fed mice (Figure 6E), suggesting greater glucose intolerance in mice fed the WTD. No effect of maternal diet was observed in the mice at this age. 36 FIGURE 6. Glucose Homeostasis at 20 Weeks Post-Weaning A. Fasting blood glucose concentration. B. Fasting plasma insulin concentration. C. IPGTT in control-fed mice. D. IPGTT in WTD-fed mice. E. IPGTT AUC for control-fed and WTD-fed mice. Values are presented as mean ± SEM. Control Western 0 5 10 15 B lo od g lu co se (m m oL /L ) Weaning Diet P<0.05, weaning diet 0 30 60 90 120 0 5 10 15 20 25 B lo od g lu co se (m m oL /L ) Time (minutes) HFA+B12 HFA-B12 Control Maternal Diet A U C (m m ol ! m in /L ) Control Western 0 1000 2000 3000 Weaning Diet P<0.001, weaning diet Control Western 0 2 4 6 8 P la sm a in su lin n g/ m L Weaning Diet P<0.001, weaning diet Control HFA-B12 HFA+B12 Maternal Diet 0 30 60 90 120 0 5 10 15 20 25 B lo od g lu co se (m m oL /L ) Time (minutes) HFA+B12 HFA-B12 Control Maternal Diet P<0.01, weaning diet A B C D E 37 5.3 Glucose Tolerance at 30 Weeks Post-Weaning I conducted IPGTTs and quantified fasting glucose and insulin concentrations in the same mice at 30 weeks post-weaning to determine if glucose tolerance changed over time. At 30 weeks post-weaning there were no differences in fasting glucose concentrations and IPGTT AUC between control and WTD diet groups (Figure 7A&E). I observed an interaction between maternal and post-weaning diets on fasting insulin concentrations; offspring mice fed the post-weaning control diet that were from dams fed the HFA-B12 diet had higher fasting plasma insulin concentrations (P<0.05) than offspring mice from females fed the control and HFA+B12 diets (Figure 7B). This effect was not observed in offspring mice fed the WTD. 38 FIGURE 7. Glucose Homeostasis at 30 Weeks Post-Weaning A. Fasting blood glucose concentration. B. Fasting plasma insulin concentration. C. IPGTT in control-fed mice. D. IPGTT in WTD-fed mice. E. IPGTT AUC for control-fed and WTD-fed mice. Values are presented as mean ± SEM. B lo od g lu co se (m m oL /L ) Contol Western 0 5 10 15 Weaning Diet Time (minutes) B lo od g lu co se (m m oL /L ) 0 30 60 90 120 0 5 10 15 20 25 Control HFA-B12 HFA+B12 Maternal Diet A U C (m m ol ! m in /L ) Control Western 0 1000 2000 3000 Weaning Diet P la sm a in su lin n g/ m L Control Western 0 2 4 6 8 Weaning Diet * Control HFA-B12 HFA+B12 Maternal Diet * *P<0.05, maternal diet (interaction) Time (minutes) B lo od g lu co se (m m oL /L ) 0 30 60 90 120 0 5 10 15 20 25 Control HFA-B12 HFA+B12 Maternal Diet A B C D E 39 5.4 Body Composition at 20 Weeks Post-Weaning Individual fat pads were dissected and weighed at harvest and lean mass and fat mass were quantified by qMRI technology to determine the relative contributions of maternal diet and post-weaning diet to body composition. Body composition analysis showed that males fed the WTD had a greater proportion of fat mass (P<0.05) and a lower proportion of lean mass (P<0.05) relative to mice fed the control diet (Figure 8A). Epididymal, retroperitoneal, and inguinal fat pads were excised and weighed. Mice fed the WTD had greater adiposity (total fat pad weight) than mice fed the control diet (Figure 8B). The proportion of body weight made up of fat from the visceral compartment (epididymal + retroperitoneal) was greater in WTD-fed mice compared to mice fed the post weaning control diet (P<0.01). There was also an affect of maternal diet on visceral fat percentage (P<0.01); regardless of post-weaning diet, visceral fat made up a smaller proportion of total fat in mice with developmental exposure to the HFA-B12 diet compared to mice developmentally exposed to control and HFA+B12 diets (Figure 8E). Similarly, the subcutaneous (inguinal) fat depot in WTD-fed mice made up a larger percentage of total body weight relative to control-fed males (P<0.0001) and an effect of maternal diet was observed on inguinal fat pad size; in both control-fed and WTD-fed groups, mice with developmental exposure to HFA-B12 and HFA+B12 diets had less subcutaneous fat (P<0.05) than those exposed to maternal control diet (Figure 8F). The proportion of total body weight comprised of epididymal fat was affected by maternal diet but not weaning diet (P<0.05); mice developmentally exposed to HFA+B12 diet had an increased proportion of epididymal fat relative to those exposed to HFA-B12 diet (Figure 8C). The proportion of retroperitoneal fat was increased in WTD-fed mice 40 relative to controls (P<0.0001) and there was an interaction observed between maternal and weaning diets; in the WTD-fed group, the fraction of total body weight made up of retroperitoneal fat was decreased (P<0.05) male mice exposed to HFA-B12 and HFA+B12 maternal diets (Figure 8D). The following data is body composition data from Figure 8 presented in absolute values as opposed to fraction of total body weight to emphasize the differences in body composition between groups. Body composition analysis by qMRI revealed that WTD- fed males had both increased absolute lean mass (P<0.05) and increased absolute fat mass (P<0.01) relative to control diet-fed males (Figure 9A). Total epididymal (P<0.05), retroperitoneal (P<0.0001), and inguinal (P<0.0001) fat pad weight was higher in WTD- fed mice relative to control-fed mice and also affected by maternal diet; independent of post-weaning diet, mice with developmental exposure to HFA-B12 diet had smaller epididymal (P<0.05), retroperitoneal (P<0.001), and inguinal (P<0.05) fat pads than those with developmental exposure to control and HFA+B12 diets (Figure 9C, D&F). Figures 8 and 9 show that although all fat pads increased in size when mice were on WTD diet, the epididymal fat pat was the only one which increased proportionally with weight gain. 41 FIGURE 8. Body Composition and Fat Pads at 20 Weeks Post-Weaning A. Fat mass and lean mass. B. Individual fat depot size. C. Epididymal fat depot. D. Retroperitoneal fat depot. E. Visceral fat (epididymal + retroperitoneal). F. Subcutaneous fat (inguinal fat). Values are presented as mean ± SEM. Control HFA-B12 HFA+B12 Control HFA-B12 HFA+B12 0 5 10 15 P er ce nt to ta l b od y M as s (% ) Control Western Epididymal Depot Retroperitoneal Depot Inguinal Depot Control Western 0 2 4 6 E pi di dy m al fa t ( % to ta l b od y m as s) Weaning Diet P<0.05, maternal diet Control Western 0 2 4 6 8 10 V is ce ra l f at (% to ta l b od y m as s) Weaning Diet P<0.01, weaning diet P<0.01, maternal diet P er ce nt to ta l b od y m as s (% ) Control HFA-B12 HFA+B12 Control HFA-B12 HFA+B12 0 50 100 % Fat mass % Lean mass Control Western Control Western 0 2 4 6 R P fa t ( % to ta l b od y m as s) Weaning Diet * * P<0.0001, weaning diet *P<0.05, maternal diet Control HFA-B12 HFA+B12 Maternal Diet Control Western 0 2 4 6 8 10 In gu in al fa t ( % to ta l b od y m as s) Weaning Diet P<0.0001, weaning diet P<0.05, maternal diet A B C D E F 42 FIGURE 9. Absolute Fat Mass and Lean Mass at 20 Weeks Post-Weaning A. Fat mass and lean mass. B. Individual fat depot size. C. Epididymal fat depot. D. Retroperitoneal fat depot. E. Visceral fat (epididymal + retroperitoneal). F. Subcutaneous fat (inguinal fat). Values are presented as mean ± SEM. Control HFA-B12 HFA+B12 Control HFA-B12 HFA+B12 0 20 40 60 M as s (g ) Fat Mass Lean Mass Control Western Control Western 0.0 0.5 1.0 1.5 2.0 2.5 E pi di dy m al fa t m as s (g ) P<0.05, weaning diet P<0.05, maternal diet Control Western 0 1 2 3 4 V is ce ra l f at m as s (g ) P<0.0001, weaning diet P<0.01, maternal diet Control HFA-B12 HFA+B12 Control HFA-B12 HFA+B12 0 2 4 6 8 M as s (g ) Epididymal Fat Retroperitoneal Fat Inguinal Fat Control Western Control Western 0.0 0.5 1.0 1.5 2.0 2.5 R et ro pe rit on ea l f at m as s (g ) Control HFA-B12 HFA+B12 Maternal Diet * P<0.0001, weaning diet *P<0.001, maternal diet Control Western 0 1 2 3 4 In gu in al fa t m as s (g ) P<0.0001, weaning diet P<0.05, maternal diet A B C D E F 43 5.6 Circulating Adiponectin Concentrations at 20 Weeks Post-Weaning To determine whether the changes in body composition and glucose tolerance are accompanied by changes in circulating adiponectin concentrations, I quantified total and HWM adiponectin concentrations. Unlike other adipose-derived hormones, circulating adiponectin and mRNA are decreased in obesity and inversely correlated with VAT116. Adiponectin circulates in multiple isoforms including globular, low molecular weight (LMW), medium molecular weight (MMW), and HMW117. The various forms circulate at different concentrations and are thought to have different roles. It has been reported that a reduction in the HMW form of adiponectin is associated with CVD, while the other forms remain unchanged or increased118, 119. However, despite differences in adiposity and glucose tolerance, I found no statistically significant differences in serum total and HMW adiponectin concentrations (Figure 10). FIGURE 10. Serum Adiponectin Concentration at 20 Weeks Post-Weaning A. Total adiponectin concentration. B. HMW adiponectin concentration. Values are presented as mean ± SEM. To ta l S er um A di po ne ct in C on c (µ g/ m l) Control Western 0 10 20 30 Weaning Diet H M W S er um A di po ne ct in C on c (µ g/ m l) Control Western 0 2 4 6 8 Weaning Diet Control HFA-B12 HFA+B12 Maternal Diet A B 44 5.7 Adipose Tissue Gene Expression Based on differences I observed in adipose tissue distribution I went on to investigate gene expression in the retroperitoneal adipose depot to explore potential metabolic alternations in the expression profile. DGAT1 catalyzes the terminal step in triglyceride synthesis120. Dgat1-/- knockout mice are resistant to diet-induced obesity and exhibit increased thermogenesis and energy expenditure 121. These mice are also more sensitive to leptin and insulin 122. Similarly, mice which overexpress adiponectin exhibit increased energy expenditure and fatty acid oxidation123. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which is used for the biosynthesis of fatty acids in lipogenic tissue, and hormones and diet regulate its expression124. Relative mRNA expression of metabolically related genes AdipoR1, Dgat1, and Acaca was quantified in the retroperitoneal adipose depot in male mice at 20 weeks post-weaning. AdipoR1 mRNA was unaffected by diet whereas Dgat1 (P<0.01) and Acaca (P<0.05) mRNA was lower in WTD-fed mice compared to control diet-fed mice; no effect of maternal diet was observed (Figure 11). 45 FIGURE 11. Retroperitoneal Fat mRNA Expression at 20 Weeks Post-Weaning A. Relative Acaca mRNA expression. B. Relative Dgat1 mRNA expression. C. Relative Adipor1 mRNA expression. Values are presented as mean ± SEM. Control Western 0.0 0.5 1.0 1.5 2.0 R el at iv e E xp re ss io n P<0.01, weaning diet Control Western 0.0 0.5 1.0 1.5 2.0 Control HFA-B12 HFA+B12 Maternal diet R el at iv e E xp re ss io n P<0.05, weaning diet Control Western 0.0 0.5 1.0 1.5 2.0 R el at iv e E xp re ss io n A B C 46 CHAPTER 6: Vascular Endothelial Function 6.1 Vascular Function of Aortae and Mesenteric Arteries I examined vascular function in the offspring as an indicator of endothelial function. Endothelial dysfunction is the precursor to vascular disease and atherosclerosis 69, which develop chronically over time, and ideal to assess in a relatively short-term rodent feeding study. To examine vascular function in the male offspring, mice were injected intraperitoneally with a lethal dose of tribromoethanol and aorta and mesenteric arteries dissected immediately. Analysis of aorta relaxation curves revealed no differences between diet groups in acetylcholine-mediated relaxation (Figure 12). Direct smooth muscle relaxation by sodium nitroprusside (SNP) was predictably unaffected in aorta (data not shown). I observed no effect of diet on endothelium-dependent or - independent relaxation in aorta from mice at 20 weeks post-weaning. 47 FIGURE 12. Endothelium-Dependent Relaxation of Aortae A. Ach-dependent relaxation in control-fed mice. B. Ach-dependent relaxation in WTD- fed mice. C. Maximum vessel relaxation from pre-constricted state (Emax). D. Ach concentration to achieve 50% relaxation (EC50). Values are presented as mean ± SEM. -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 Log Ach Concentration (M) % R el ax at io n Log Ach Concentration (M) % R el ax at io n -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 Control HFA-B12 HFA+B12 Maternal DietA B Control Western 0 50 100 150 E m ax Control Western -8 -6 -4 -2 0 Lo g E C 50 Control HFA-B12 HFA+B12 Maternal DietC D 48 Due to their proximity to and thus likelihood to be exposed to the inflammatory processes of visceral fat, I examined vascular function in the mesenteric arteries. An interaction between maternal and weaning diets was observed in the ability of the mesenteric artery to achieve endothelium-mediated relaxation. There were no differences evident in the control-fed group; however, WTD-fed mice with developmental exposure to HFA-B12 (P<0.05) did not reach the maximum relaxation achieved by the maternal control and HFA+B12 groups (Figure 13C). There were, however, no significant differences between groups in the concentration of acetylcholine required to reach 50% relaxation (Figure 13D). 49 FIGURE 13. Endothelium-Dependent Relaxation of Mesenteric Arteries A. Ach-dependent relaxation in control-fed mice. B. Ach-dependent relaxation in control- fed mice. C. Maximum vessel relaxation from pre-constricted state (Emax). D. Acetylcholine concentration to achieve 50% relaxation (EC50). Values are presented as mean ± SEM. Log Ach Concentration (M) % R el ax at io n -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 Log Ach Concentration (M) % R el ax at io n -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 Maternal Diet Control HFA-B12 HFA+B12 A B Control Western 0 50 100 150 E m ax * *P<0.01, maternal diet (interaction) P = 0.056 Control Western -8 -6 -4 -2 0 Lo g E C 50 Control HFA-B12 HFA+B12 Maternal Diet C D 50 6.2 Concentration-Response Curves to PE in the Presence L-NAME To further examine basal nitric oxide production in aortae and mesenteric arteries, vessel contraction in response to phenylephrine (PE) was observed first in the absence and then in the presence of nitric oxide synthesis inhibitor L-NAME. Phenylephrine was added to the chamber to a final concentration of 10-5 M. Maximum contraction was recorded and the vessels were washed with PSS until basal tension had been re- established. At this point L-NAME was added to the vessel chambers for an incubation period before a second addition of PE to a final concentration of 10-5. Differences in basal nitric oxide production were expressed as the percentage increase in contraction when vessels were contracted with PE in the presence of L-NAME (PEmax with L-NAME / PEmax). No significant effect of either maternal or post-weaning diet was seen in basal nitric oxide production in the aorta. Observation of mesenteric artery contraction curves revealed an interaction between maternal and weaning diets. Mice weaned onto control diet and exposed developmentally to HFA-B12 and HFA+B12 diets showed lower maximum contraction in the presence of L-NAME than maternal controls (P<0.05). In contrast, WTD-fed mice with developmental exposure to maternal HFA+B12 diet showed greater contraction (P<0.05) in the presence of L-NAME relative to those exposed to maternal control diet (Figure 14B). 51 FIGURE 14. Basal Nitric Oxide Production in Vessels A. Percent increase in PE contraction in the presence of L-NAME (Aorta). B. Percent increase in PE contraction in the presence of L-NAME (Mesenteric artery). Values are presented as mean ± SEM. 6.3 NADPH Subunit (Nox2) Expression in Aorta at 20 Weeks Post-Weaning Although physiological changes in vascular function were not detectable in the aorta, western blot analysis was undertaken to look for changes in protein expression that might be indicative of a pre-atherosclerotic state and contribute to vascular dysfunction. The NADPH oxidases are reactive oxygen species-generating enzymes and have been implicated in atherosclerosis 125, 126. High fat diet has been shown to increase oxidative stress through activation of NADPH oxidase and this has been associated with increased mRNA levels of NADPH oxidase subunit, Nox2 127. As such, I examined NADPH subunit protein expression in the aorta. Total protein was extracted from frozen aorta tissue and expression of Nox2 assessed by western blotting as described in Chapter 2. WTD diet did not affect protein expression but an effect of maternal diet on Nox2 expression was observed. In control-fed mice, Nox2 expression was decreased (P<0.05) in mice with developmental exposure to maternal high folic acid supplementation (Figure 15). Control Western 0 100 200 300 400 500 C on tra ct io n (% P E M ax ) Control Western 0 50 100 150 200 Control HFA-B12 HFA+B12 C on tra ct io n (% P E M ax ) Maternal Diet * *P<0.053 * *P<0.05, maternal diet (interaction) A B 52 FIGURE 15. Nox2 Protein Expression in Aorta A. Relative protein expression normalized to actin. B. Sample blot showing relative fluorescence and bands for Nox2 and actin (a, b, c: C/C, HFA-B12/C, HFA+B12/C; A, B, C: C/W, HFA-B12/W, HFA+B12/W). Values are presented as mean ± SEM. 53 CHAPTER 7: General Discussion Prenatal and early postnatal nutrient status influences metabolic health in adulthood. There is increasing evidence that manipulation of maternal diet is accompanied by sex-specific changes in offspring metabolic phenotype. Here I report that maternal folic acid and vitamin B12 intake interacts with postnatal diet to affect body composition and vascular function in male offspring mice. Overall, this study provides support for the general hypothesis that nutrient imbalance during development, programs metabolism later in life. The independent effects of WTD on murine vascular and metabolic health have been well studied and my findings were expected. The novel effects were those induced by maternal diets and the interactions between maternal and post-weaning diets. My novel findings include modified response to post-weaning diet by developmental exposure to maternal folic acid and vitamin B12 intake observed in 1) body fat distribution 2) glucose homeostasis and 3) vascular characteristics. The observation that developmental exposure to folic acid and vitamin B12 imbalance is capable of programming body fat distribution is arguably my most interesting finding in that the effects on glucose homeostasis and vascular function may be derived from changes in adiposity. However, this is purely speculation, as more work is required to distinguish the direct effects of such developmental exposures from related downstream events. Previous studies have reported that varying methyl nutrient content of the diet fed to female mice during pregnancy can affect the phenotype of the offspring. The biological and molecular mechanisms behind such developmental programming are largely unknown but significant evidence points towards epigenetic mechanisms. 54 Waterland et al. showed that altering maternal methyl nutrient intake alters gene expression via DNA methylation, thus altering coat colour in the offspring mice with the agouti gene109. Similarly, Devlin et al. has shown altered methylation status of Fads2, a gene involved in fatty acid metabolism, accompanied by changes in lipid profile in adult mice due to manipulation of methyl nutrient content in the diet128. The resultant phenotype is due to a combination of both developmental exposures and adult diet. Findings from the Dutch Famine Birth Cohort study remain some of the most pivotal evidence for the influence of the developmental environment on future metabolic health. Findings from this cohort have found early onset and increased incidence of CVD and risk for T2D in adults who were exposed prenatally to poor maternal energy intakes because of ration shortage during WWII36-38. As such, I aimed to explore the hypothesis that developing fetus is highly responsive to diet and that the prenatal environment makes use of such plasticity to program metabolism and risk for disease later in life. In North America, current food fortification policies mandate the addition of folic acid to the food supply, specifically to grain products, for the prevention of NTDs. In Canada, as a result of folic acid fortification, unmetabolized levels of circulating folic acid are higher in the population and the consequences, metabolic or otherwise, are unknown99. While the majority of Canadians have high folate status, approximately 5% are vitamin B12 deficient102. As many as 1 in 20 women may be vitamin B12 deficient in the early stages of pregnancy103. A 2008 study from India implicated concern for this potential imbalance during pregnancy by reporting that children born to women with high folate and low vitamin B12 status had increased adiposity and insulin resistance at 6 years of age107. Controlled, mechanistic studies on the effects of folic acid and vitamin 55 B12 imbalance during pregnancy are currently lacking and, as such, I aimed to conduct an experiment, which would shed light on this issue. I focused on the effects of developmental exposure on male offspring metabolic phenotype. 7.1 Programming of Offspring Body Fat Distribution At weaning, there were no differences in body weight between groups and growth followed a smooth trajectory. Males responded quickly to the WTD, breaking off from the control growth curve between weeks 5 and 10. I conducted statistics on all groups but graphed growth separately by post-weaning diet for the sake of visual purposes. Research has revealed an association between increased rate of growth in the early period of life and metabolic and cardiovascular dysfunction as an adult 129, 130. The early increased rate of growth exhibited by males was not maintained and decreased steadily over time. At the end of the post-weaning feeding period (20 weeks and 30 weeks) WTD-fed mice were larger than controls but the effect of maternal diet was observed only at 20 weeks post-weaning. At 20 weeks post-weaning, mice that had been exposed to high folic acid (with and without vitamin B12) during development weighed less than mice exposed to the control diet; this was true for both post-weaning diets. I believe that the lack of maternal influence on male weight at 30 weeks is due to a combination of age and the heterogeneous metabolic adaptation to high-fat diet, which has been reported to exist in C57BL/6J mice131. I analyzed body composition by qMRI and found that WTD increased both absolute lean mass and absolute fat mass in male mice relative to controls. However, in WTD-fed mice, the total percentage of body mass made up by fat was increased whereas 56 the proportion of lean mass was decreased. I found visceral (retroperitoneal and epididymal) and subcutaneous (inguinal) depots to be affected by both maternal and weaning diet in males. As has been shown before, WTD feeding increased both visceral and subcutaneous fat pad weight. An effect of maternal diet was also observed on subcutaneous fat pad size. Some research has reported increased epididymal fat pad weight with maternal vitamin restriction132. I found an effect of maternal diet but not of post-weaning diet on epididymal fat pad size. Overall, retroperitoneal fat pads were larger in WTD-fed males, but I observed an interaction between maternal and post weaning diets on the size of this fat pad. In control-fed mice, maternal diet had no effect on retroperitoneal fat pad size. However, in WTD-fed mice retroperitoneal fad pads were smaller in the HFA-B12 and HFA+B12 relative to the maternal control group. In WTD- fed mice, maternal folic acid (and less so vitamin B12) supplementation appeared to drive retroperitoneal fat pad size. Infiltration of ectopic tissue such as skeletal muscle with fat has been associated with impaired glucose tolerance and insulin sensitivity. Functioning adipose tissue should be able to absorb caloric excess and protect the body from insulin resistance. It is possible that the decrease in fat mass in the visceral compartment in males is accompanied by an increase in ectopic tissue storage but this hypothesis remains to be explored. My main finding was that developmental exposure to maternal folic acid and vitamin B12 intake affected fat distribution in both the subcutaneous and visceral depots and overall body weight in young adult mice. High folic acid intake was associated with reduced adiposity in mice challenged with the WTD. I did not measure food intake or energy expenditure during the course of my study. Recent research has shown that early postnatal nutrition determines adult physical 57 activity and energy expenditure in mice 133. Factors such as food palatability could modify food intake and interpretation of results. Such studies would determine if caloric intake and energy expenditure were similar between diet groups and paint a clearer picture of the metabolic states of these animals. 7.2 Programming of Offspring Glucose Homeostasis In accordance with the early weight gain in males in response to WTD diet, after 20 weeks of exposure to WTD, mice exhibited decreased glucose tolerance and higher fasting glucose and insulin concentrations than control diet-fed males. This is consistent with their overall increased adiposity and body weight. At this point, I believe that any deviation in glucose tolerance from control was due to WTD exposure and not due to maternal diet. Although I observed differences in body weight and body composition at 20 weeks post weaning, it is possible that developmental exposure to folic acid and vitamin B12 imbalance do not manifest as changes in glucose homeostasis until a later time point. I assessed glucose tolerance and fasting glucose and insulin concentrations again at 30 weeks to observe the change in response to diet over time. At 30 weeks there were no differences in glucose tolerance, and fasting glucose concentrations between control-fed and WTD-fed males. This may be attributed to the predisposition of C57BL/6J mice to become glucose intolerant over time and to a compensatory mechanism on the part of the WTD-fed males. Although there were no significant differences in glucose tolerance, WTD-fed males had higher fasting insulin concentrations and I observed an interaction between maternal and post weaning diets. In 58 the 30-week control-fed males, those with developmental exposure to high folic acid and low vitamin B12 had fasting insulin concentrations more than double those observed in the maternal control and HFA+B12 groups. Nutrient imbalance during development, alone, produced the increase in fasting insulin concentrations typically seen in animals on WTD. Evidence suggests that visceral adipose tissue is a metabolic and inflammatory tissue and represents a risk factor for CVD and T2D. I observed changes in adipose tissue deposition due to maternal diet, post-weaning diet, and an interaction between the two diets. Adiponectin is an adipose-specific adipokine involved in regulating metabolism and inflammation and is inversely correlated with body fat in humans. Adiponectin is insulin sensitizing, increases fatty acid oxidation, and regulates blood glucose. Reduction of adiponectin in mice leads to impaired glucose tolerance and elevated hepatic glucose production134. However, despite changes in fat mass, serum total and HMW adiponectin concentrations remained unchanged between diet groups. I next analyzed mRNA expression of three metabolically related genes in retroperitoneal adipose tissue. Adiponectin enables communication between visceral adipose tissue and peripheral tissues through receptors AdipoR1 and AdipoR2. AdipoR1 is expressed ubiquitously with increased expression observed in skeletal muscle, whereas AdipoR2 is localized to the liver. Analysis of AdipoR1 mRNA expression in male retroperitoneal adipose tissue showed uniform expression levels between groups. I have yet to examine the expression of adiponectin receptors in peripheral tissues and in liver. Dgat1 is expressed ubiquitously and encodes an acyl CoA:diacylglycerol acyltransferase which catalyzes the final step in triglyceride synthesis135. Dgat1-/- mice have a favourable metabolic 59 phenotype in that they are lean and resistant to diet-induced obesity 121. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA which is used for the biosynthesis of fatty acids in lipogenic tissue. The genes coding for ACC1 and ACC2 are controlled by multiple promoters, which are regulated by hormones and diet124. The ACC1 isoform is predominantly expressed in lipogenic tissue whereas the ACC2 isoform is highly expressed in skeletal muscle and heart, but also in the liver136. Analysis of Dgat1 and Acaca (encodes ACC1) mRNA in male retroperitoneal fat pads showed decreases in relative expression of both genes in WTD-fed mice relative to control-fed mice. I observed no effect of maternal diet on relative expression of these three target genes and no effect of post-weaning diet on AdipoR1 relative gene expression. Therefore, changes in mRNA expression of Dgat1 and Acaca do not account for the differences observed in retroperitoneal fat pads between mice with different developmental exposure to folic acid and vitamin B12. 7.3 Programming of Offspring Vascular Function The link between maternal under-nutrition and incidence of offspring CVD later in life is well known. Early-onset obesity (often linked to maternal under-nutrition) is also associated with risk for developing CVD as an adult 35, 137, 138 as is exposure to maternal high fat diet during pregnancy68, 139, 140. There is little to no research on the effects of maternal methyl nutrient imbalance on adult offspring vascular function. Endothelial dysfunction is the central feature of obesity-related CVD141-143 and has been documented in rodents as an indicator of vascular health144. Based on the human findings of increased adiposity in children exposed to folic acid and vitamin B12 balance during 60 development and on the association of maternal under-nutrition, and obesity itself, with adult CVD, I predicted vascular function in offspring mice to be affected by maternal diet and modified by post-weaning diet. The first vessel I examined was the aorta. Several segments from the same vessel were mounted in separate chambers as replicates to account for potential damage to endothelial layer caused by the mounting process. Despite differences in glucose homeostasis and adiposity between groups I did not observe any effect of maternal diet or post-weaning diet on aortic vascular function in the offspring mice. Aortae from all groups achieved maximum endothelial-dependent relaxation when stimulated with acetylcholine. There were also no differences in the acetylcholine concentration required to reach 50% aortic relaxation. Based on cursory observation that aortae from WTD-fed mice did not always respond (contract) to stimulation by phenylephrine to the same extent as aortae from control diet-fed mice, I carried out another set of experiments to examine basal nitric oxide production. I measured maximum aortic contraction achievable by addition of phenylephrine to 10-5 M. I then washed the vessels, and after return to a baseline state I added the nitric oxide synthase inhibitor, L-NAME, and measured maximum aortic contraction. I expressed this measure as contraction in the presence of L-NAME over contraction in the absence of L- NAME. Thus, an increased value is indicative of increased basal nitric oxide production. Observation of contractile responses in aorta revealed no significant differences in vascular function; aortic basal nitric oxide production was unaffected in all groups based on the 2 to 3-fold increase in constriction observed when nitric oxide synthase was inhibited. I did however, observe a trend of the effect of maternal diet on basal nitric oxide production in the aorta but cannot make any conclusions due to the small sample 61 size of the experiment. The aorta is a large and durable conduit vessel, as it must withstand the substantial pressure and sheer forces it is exposed to as a result of its position in circulation immediately after the heart. As such, it is possible that minute nutritional changes (folic acid and vitamin B12) during development are not enough to affect the endothelial lining. It is much more likely that smaller, more delicate vessels (i.e. femoral, cerebral, and mesenteric) are more susceptible to subtle nutritional changes during development. In support of this, a prior study reported endothelial dysfunction in cerebral arterioles but no effect in aorta from Mthfr +/- mice with diet-induced hyperhomocysteinemia145. Following this line of reasoning, I examined vascular function in the mesenteric arteries of the same offspring mice. Based on the relatively small size of the mesenteric arteries and their proximity to potentially inflammatory visceral adipose tissue and portal circulation, I hypothesized that changes in glucose homeostasis and adipose tissue distribution would be reflected in mesenteric artery vascular function. I observed an interaction between maternal and post-weaning diets on maximum relaxation (Emax) reached by mesenteric arteries from different diet groups. There were no differences in Emax observed in mesenteric arteries of mice fed the control post-weaning diet. However, in mice fed WTD post-weaning, mesenteric arteries of mice exposed developmentally to HFA-B12 did not reach the same degree of relaxation as those exposed to control and HFA+B12 diets. Despite this, there were no significant differences in the concentration of acetylcholine required to achieve 50% (EC50) relaxation of mesenteric artery between groups. As in aorta, I next examined basal nitric oxide production in the mesenteric artery and observed an interaction between maternal 62 and post-weaning diets. In mice fed the control post-weaning diet, basal nitric oxide production was decreased in mice with developmental exposure to HFA-B12 diet. In WTD-fed mice, basal nitric oxide production was increased in those developmentally exposed to maternal HFA+B12 diet relative to control. It is interesting that the response to maternal diet was modified by post-weaning diet. Therefore, developmental exposure to changes in folic acid and vitamin B12 status has the potential to program vascular function later in life, and this may be modified by adult diet. 7.4 Conclusion and Future Directions My work demonstrates that maternal folic acid and vitamin B12 imbalance independently affects but also interacts with adult diet to affect vascular function and adiposity in adult male mice. The roles of specific tissues and genes involved in such developmental programming remain to be determined. Because folic acid and vitamin B12 are so closely tied to the methionine cycle and generation of methyl groups, it is plausible that exposure to such disturbances in methyl nutrient balance during development create aberrant gene expression profiles which, in the right environment, manifest later in life as metabolic dysfunction. There are now both human and animal studies to suggest that developmental exposure to folic acid and vitamin B12 imbalance can alter adult metabolism. My work showed that developmental exposure to folic acid and vitamin B12 imbalance in mice produced largely beneficial results in terms of offspring adiposity and vascular function. High folic acid and low vitamin B12 status during development was associated with less visceral and subcutaneous adipose tissue in the offspring. Developmental exposure to high folic acid (regardless of vitamin B12 63 status) was associated with decreased retroperitoneal fat and decreased NADPH oxidase subunit expression in the aorta. Further studies are warranted to determine the mechanisms behind such effects and to further our understanding of the developmental origins of obesity and metabolic disease. 64 References 1. Eckel RH, York DA, Rossner S, et al. 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Blood. 2004;103:2624-2629."""@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2013-11"@en ; edm:isShownAt "10.14288/1.0074019"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Pathology and Laboratory Medicine"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "Attribution-NonCommercial-NoDerivs 3.0 Unported"@en ; ns0:rightsURI "http://creativecommons.org/licenses/by-nc-nd/3.0/"@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Programming of adult metabolic phenotype by maternal dietary folic acid and vitamin B12 imbalance in mice"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/44742"@en .