Angela Devlin

Background: Folic acid supplementation is recommended for women of childbearing age to prevent birth defects. Women with pregestational obesity (BMI≥30kg/m²) and/or diabetes are recommended to take up to 12x the recommended dose of folic acid. Concerns have been raised that elevated folic acid during pregnancy may have negative impacts on the cardiometabolic health of the mother and child. The objective of my thesis was to determine the effects of folic acid supplementation in gestational obesity/prediabetes on maternal and fetal health. Methods: Female (C57BL/6J) mice were fed from weaning a control diet (10% kcal fat; control dams) or western diet (45% kcal fat; western dams; model of obesity/prediabetes). Diets contained supplemental (10mg/kg diet) or recommended level (2mg/kg diet) folic acid. Dams (n=11-14/diet) were fed for 13 weeks prior to breeding with control males. Tissue from dams and fetal offspring were collected at embryonic day (E)18.5. Results: Prior to breeding, western dams had greater body weight and adiposity accompanied by glucose intolerance and impaired β cell function. Folic acid supplementation reduced insulin sensitivity in control dams and improved insulin sensitivity in western dams. At E18.5, western dams had larger livers and key methyl donor, S-adenosylmethionine (SAM), and it’s demethylated product, S-adenosylhomocysteine (SAH), were lower in liver, compared to control dams. Male offspring from western dams had smaller livers and higher hepatic betaine and choline compared to offspring from control dams. Female offspring from western dams had smaller livers and higher hepatic betaine compared to those from control dams. Maternal folic acid supplementation increased hepatic SAM in female offspring, but not in male offspring. Maternal diet did not affect fetal pancreas size, but male offspring from folic acid supplemented dams had greater β cell mass and β cell density compared to those from non-supplemented dams; no effect of maternal diet on β cell mass was observed in female offspring. Conclusion: Folic acid supplementation does not exacerbate adiposity and glucose tolerance in dams with gestational obesity/prediabetes. However, maternal folic acid supplementation and gestational obesity/prediabetes has sex-specific effects on hepatic one-carbon metabolism and pancreatic β cell mass in fetal offspring.

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Background: Preterm infants (37 weeks of gestation) often require hospitalization in the neonatal intensive care unit and experience painful procedures due to medical care. Oral sucrose treatment for analgesia is the non‑pharmacological standard of care for minor procedural pain relief. The objective of my MSc thesis research was to determine the long‑term effects of repeated neonatal oral sucrose treatment on growth, adiposity, and glucose homeostasis in a mouse model. Methodology: Neonatal female and male mice (C57BL/6J) were randomly assigned to one of four treatments (n=7‑10 mice/group/sex): water, sucrose, fructose, or glucose. Pups were treated 10 times/day for the first six days of life with 0.2g/kg body weight of respective treatments (24% solution; 1‑4 μL/dose) orally to model what is given to preterm infants. Mice were weaned onto a control diet and fed until age 16 weeks (adulthood). Pups were weighed daily from birth to weaning and weekly thereafter. Longitudinal growth and body composition were assessed at adulthood. Physiological assessments of glucose homeostasis (intraperitoneal glucose and insulin tolerance tests; glucose-stimulated insulin secretion test) were performed at weaning and adulthood. Insulin-like growth factor-1 (IGF-1) and liver water-soluble choline metabolites were also assessed. Results: Female and male sucrose-treated mice gained less weight during the suckling period (p0.01 vs other groups) and were smaller at weaning compared to the water- and glucose-treated mice (p0.05). At age 16 weeks, female sucrose-treated mice had smaller tibias (p0.001 vs all) and lower serum IGF-1 concentrations (p0.05 vs water). This was accompanied by lower (p0.05 vs water) liver free choline, phosphocholine, and glycerophosphocholine concentrations, and higher betaine (p0.01 vs water) concentrations in the sucrose-treated compared to the water-treated female mice. No differences in growth or liver choline metabolites were observed in male mice. Neonatal treatments had no effect on adiposity or glucose homeostasis in female or male mice. Conclusion: My findings suggest that repeated neonatal sucrose treatment affects growth in female mice, perhaps through an IGF‑1‑dependent pathway and alters liver choline metabolism. Further research is required to determine the functional consequences of these alterations.

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INTRODUCTION: In Canada, nearly half of women of child-bearing age are overweight (BMI 25-29.9 kg/m²) or obese (BMI ≥30 kg/m²). Current guidelines recommend moderate exercise for all pregnant women. However, little is known about the effect of exercise during pregnancy on the developing offspring. Rodent studies demonstrate that offspring from dams fed a high-fat diet have vessel-specific impairment in endothelium-dependent vascular function, greater adiposity, and impairments in glucose homeostasis. Further, offspring from exercised dams have improved glucose homeostasis. OBJECTIVE: In this thesis, I determined if maternal exercise during pregnancy mitigates the adverse effects of maternal obesity on offspring adiposity, glucose homeostasis, and vascular health in female offspring.METHODS: Dams (C57BL/6) were fed a control (10% energy from fat) or western diet (45% energy from fat) from weaning for 13 weeks and through breeding, pregnancy, and lactation. Just prior to breeding, dams were put into cages with or without a running wheel for voluntary exercise through breeding, pregnancy, and lactation. Offspring were weaned onto the control or western diet and maintained on the diet for 20 weeks; only female offspring were studied. Lean and fat mass were quantified by ¹H-NMR. Intraperitoneal insulin tolerance (IPITT), glucose tolerance (IPGTT) and insulin secretion tests (IPIST) were performed prior to the end of the feeding period. Vascular endothelial-dependent and independent dilatation were assessed ex vivo by isometric force measurements of aortic rings in response to phenylephrine, acetylcholine, and sodium nitroprusside.RESULTS: Weaning weights were greater in offspring from western-fed dams than control-fed dams. Control-fed offspring from western-fed dams had higher heart and kidney weights than those from control-fed dams. Control-fed offspring from exercised dams had higher blood glucose concentrations at IPGTT₃₀minutes than those from sedentary dams. Western-fed offspring from exercised dams had greater body weights, retroperitoneal fat weights, and Il10 mRNA in retroperitoneal adipose tissue, and lower blood glucose concentrations at IPGTT₉₀minutes than those from sedentary dams. Endothelium-dependent vasorelaxation of thoracic aortae was likely unaffected by maternal western diet or maternal exercise in control-fed and western-fed offspring.CONCLUSION: These findings suggest maternal exercise is beneficial in improving glucose homeostasis and adiposity in western-fed female offspring.

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Developmental programming suggests that perinatal environmental conditions can impact risk for chronic diseases. Population studies have reported greater insulin resistance and adiposity in offspring from mothers with adequate folate but low vitamin B12 (B12) status during pregnancy. Rodent studies have reported that these effects are sex-specific. Folate, a methyl nutrient, is metabolically linked to B12. Low B12 status, even when folate is adequate, can trap folate in a metabolically inactive form. Folate deficiency is rare in Canada due to folic acid fortification of grains, yet one in 20 Canadians are estimated to be B12-deficient. The objective of this thesis is to determine the mechanisms underlying the relationship between maternal B-vitamin status during pregnancy and offspring adiposity and glucose homeostasis.In vitro experiments assessed direct effects of folic acid on adipocyte energy metabolism. In 3T3-L1 adipocytes, cells treated with 1.6μM folic acid had lower (p≤0.05) mitochondrial respiration rates than cells treated with 0.16μM folic acid. Adipocytes treated with 1.6μM 5-methyltetrahydrofolate (5-MTHF), the circulating form of folate, had higher (p≤0.01) mitochondrial respiration rates than cells treated with 0.16μM 5-MTHF.Female mice (C57BL/6J) were fed one of three maternal diets six weeks prior to breeding and through pregnancy/lactation: control (M-CON), supplemental folic acid with adequate B12 (SFA+B12), or SFA without B12 (SFA-B12). One male and one female from each dam were weaned onto either a control or western diet (45% kcal fat). Sex-specific differences by maternal diet were observed in the offspring. Female control-fed SFA-B12 offspring had lower (p≤0.05) serum IGF-1 (insulin-like growth factor-1) concentrations than M-CON and SFA+B12 offspring. This was accompanied by higher (p≤0.05) hepatic Cpt1a mRNA in SFA-B12 and SFA+B12 offspring than M-CON offspring. Female western-fed SFA+B12 offspring had higher (p≤0.05) hepatic FADS2 and ELOVL2 protein than M-CON offspring. Conversely, male control-fed SFA-B12 offspring had higher (p≤0.05) hepatic linoleic acid (C18:2n-6) and lower (p≤0.05) eicosapentaenoic acid (C20:5n-3) concentrations, and lower (p=0.08) hepatic ELOVL2 protein than M-CON offspring.These findings suggest programming of offspring adiposity and glucose homeostasis by maternal B-vitamin status occurs through sex-specific alterations in IGF-1, and adipose tissue and hepatic lipid metabolism.

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Approximately one in six Canadian children suffer from mental health conditions (MHC), including: anxiety, bipolar disorder, and depression. Second-generation antipsychotics (SGAs) are increasingly used to treat children with MHCs. Recent evidence suggests that SGAs are implicated in rapid weight gain, increased measures of adiposity, and cardiometabolic dysfunction in the pediatric population. The mechanisms for these adverse side effects have yet to be elucidated. One postulation is that SGAs may increase dietary energy intakes; however, little is known about the diets of SGA-treated children. My thesis research explored dietary intake and its potential association with adiposity and cardiometabolic dysfunction, in SGA-treated children.This cross-sectional study recruited SGA-treated (n=25) and SGA-naïve (n=20) children (6-19 yrs) with MHCs, from the Psychiatry Department at BC Children’s Hospital. Demographics, medical history, and anthropometrics were obtained. In a subset, fasting plasma lipids, glucose, and insulin were obtained and the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated. Three 24-hour food records were collected and analyzed for average three-day total energy (kcal), macronutrient, saturated fat, sugar, fibre, and sodium intakes. There were no statistically significant differences between groups for energy intakes (mean ± SD; 2036.5±715.8 vs. 1725.5 ± 545.6, P = 0.352) or macronutrient, saturated fat, sugar, fibre, and sodium intakes; adjusting for sex, height, pubertal status, and psychostimulant medications. SGA-treated children had higher zBMI (p=0.001), waist circumference (p=0.019), and HOMA-IR (p=0.009), compared to SGA-naïve children. There were no associations of energy intake and measures of adiposity. There were positive associations of sodium intake and HOMA-IR (p=0.017; 95% CI: 0.013, 0.109) and saturated fat intake and low-density lipoprotein cholesterol (LDL-c) (p=0.041; 95% CI: 0.004, 0.170), in the SGA-treated group; adjusting for sex, pubertal status, overweight/obesity, height, and psychostimulant treatment. These data suggest that SGA-treated children do not have greater dietary energy intakes compared to SGA-naïve children, and dietary energy intakes may not be responsible for greater measures of adiposity in SGA-treated children. However, the positive associations of sodium intake with HOMA-IR, and saturated fat intake with LDL-c, in SGA-treated children, suggest that dietary intakes in SGA-treated children may contribute to cardiometabolic dysfunction in this high-risk population.

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Folate is a B-vitamin required for cell growth and division, and its metabolism is linked to vitamin B12 (B12). Food fortification with folic acid (FA) has improved folate status but approximately 5% of Canadian adults, including pregnant women, are B12 deficient. This is concerning because an association between gestational exposure to high maternal folate and low B12 status and greater adiposity and insulin resistance in children has been reported. My thesis examined the effect of developmental exposure to maternal FA/B12 imbalance on programming of liver gene expression in adult offspring using an animal model. Female C57BL/6 mice were fed a high FA/adequate B12 (HFA+B12), high FA/no B12 (HFA-B12), or control diet 6 weeks prior to mating and through pregnancy and lactation. At weaning, offspring mice from each maternal diet group were randomly assigned to receive the control diet or a Western diet (45% fat, 35% carbohydrate) for 20 weeks (n=6 male mice/group) or for 40 weeks (n=6 female mice/group). Serum folate and B12 concentrations were quantified by microbiological assays. Relative mRNA expression of key enzymes in methyl metabolism in liver from adult offspring was quantified by real-time PCR. Male offspring mice from dams fed the HFA-B12 diet had lower Cbs and Mthfr mRNA expression and this was unaffected by post weaning diet. Male offspring mice fed the Western diet had higher Mtr mRNA expression compared to control-fed offspring mice, regardless of maternal diet. Female offspring from dams fed the HFA-B12 diet had lower Mtr mRNA expression and this was not affected by post weaning diet. Moreover, female offspring from dams fed the HFA-B12 diet had higher Mthfr mRNA expression when they were fed the Western diet. No effect of maternal and post weaning diets was observed for serum folate and B12 concentrations.In summary, developmental exposure to maternal FA/B12 imbalance was found to program expression of genes involved in folate and methionine metabolism in the liver of adult offspring mice. The functional consequences of this effect requires further investigation in order to consider B12 screening of pregnant women and to inform the debate on whether B12 fortification should be considered.

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Of particular importance to mental health across the life span is our capacity to regulate neuroendocrine responses to stressful events via the hypothalamic pituitary adrenal (HPA) axis. Prenatal exposure to maternal depression/anxiety may be among the earliest adverse experiences shown to influence the developing HPA system, possibly via changes in fetal serotonergic signaling. In addition, the developmental impact of prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressants is often undistinguishable from prenatal maternal mood. The molecular mechanisms underlying how serotonin (5-HT) influences the development of the HPA stress system remain unclear, but may involve epigenetic mechanisms such as DNA methylation. This thesis explored whether prenatal exposure to SRIs and maternal depressed/anxious mood are associated with altered HPA stress reactivity, characterized by variable basal and stress-induced cortisol concentrations in 5 - 7 year-old children. Furthermore, the methylation status in promoter regions of NR3C1 1F (encodes the glucocorticoid receptor) and SLC6A4 (encodes the serotonin transporter) at birth and at 5 - 7 years of age was evaluated and the relationship to children’s cortisol patterns was assessed. Prenatal exposure to SRIs and higher 3rd trimester maternal depressed/anxious mood were associated with reduced cortisol stress responses at 5 - 7 years. Higher NR3C1 1F methylation at 5 - 7 years was associated with higher diurnal cortisol concentrations and a reduced cortisol stress response. Children exposed to mothers with higher 3rd trimester and concurrent anxious mood exhibited lower SLC6A4 methylation, compared to children exposed to higher 3rd trimester maternal anxious mood alone. Furthermore, children with higher SLC6A4 methylation at birth exhibited a reduced cortisol stress response. These findings suggest that the relationship between early life experiences and altered stress responses in early childhood may be moderated by epigenetic mechanisms involving the serotonergic and HPA regulatory systems. In addition, an interactive relationship between pre- and postnatal maternal mood with cortisol stress responses and methylation status at 5 - 7 years suggests that an early adverse environment may confer sensitivity toward altered HPA activity and regulation, and that the postnatal environment may shift the HPA stress response towards vulnerability or resilience to stress-related disorders across the lifespan.

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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 (P0.01) and subcutaneous fat accumulation (P0.05) 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 (P0.05) 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 (P0.05). 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 (P0.05). Developmental exposure to high folic acid (with and without vitamin B12) was associated with decreased NADPH oxidase subunit expression in the aorta (P0.05). 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.

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Background: Body fat distribution, in particular visceral adipose tissue (VAT), contributes to risk of cardiovascular disease (CVD). The Multicultural-Community Health Assessment Trial (M-CHAT) reported that South Asians have greater VAT than Europeans despite similar BMIs, putting them at greater risk of CVD. However, the molecular mechanisms underlying ethnic-specific differences in body fat distribution are unclear. Low circulating 25-hydroxyvitamin D (25OHD) and adiponectin concentrations are prevalent in individuals with obesity (BMI ≥ 30kg/m²), and are associated with increased risk of CVD. Furthermore, 25OHD is inversely associated with blood pressure. Adiponectin is an adipokine that has insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties. Adiponectin circulates as isoforms low (LMW), medium (MMW), and the reported most biologically active isoform, high (HMW) molecular weight. This thesis aims to investigate ethnic-specific differences in the relationship between plasma 25OHD and adiponectin concentrations with body fat distribution and CVD risk factors.Methods/Results: Europeans (n=171) and South Asians (n=176) from the M-CHAT cohort were assessed for demographics, plasma 25OHD, total and HMW adiponectin concentrations, and CVD risk factors. A computed tomography (CT) scan was used to quantify VAT and subcutaneous adipose tissue (SAT) deposition. South Asians had lower (p 0.001) 25OHD in comparison to the Europeans (63.0 nmol/L vs. 39 nmol/L, respectively). VAT was inversely associated (p 0.05) with 25OHD even after adjustment for age, sex, BMI, season of blood collection, SAT and total body fat (%). This suggests that VAT mediates the relationship between plasma 25OHD and CVD risk. Furthermore, the inverse association between systolic and diastolic blood pressure and 25OHD was no longer significant after adjustment for VAT. However, circulating plasma adiponectin concentrations remained strongly associated with fasting plasma HDL-cholesterol, triglycerides, insulin, and HOMA-insulin resistance (HOMA-IR), even after adjustment for VAT. Interestingly, South Asians also had lower (p0.001) total and HMW adiponectin concentrations compared to Europeans. Conclusion: These findings demonstrate that plasma 25OHD and total and HMW adiponectin concentrations are associated with VAT deposition in Europeans and South Asians. Furthermore, plasma 25OHD and total and HMW adiponectin concentrations are lower in South Asians than the Europeans, which may contribute to elevated CVD risk in South Asians.

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Depression occurs in 15% of pregnant women and 1/3 are taking selective serotonin reuptake inhibitors (SSRIs) as antidepressants. The neurotransmitter, serotonin, plays a critical role in modulating stress responses and early brain development. Serotonin transporter (SLC6A4) regulates extracellular serotonin levels, and an insertion/deletion variant in the promoter (5HTTLPR) is associated with depression. Maternal mood and SSRIs may program newborns’ behaviour later in life. The underlying molecular mechanism for developmental programming may involve DNA methylation, which requires methyl nutrients as enzymatic cofactors. While low methyl nutrient status (folate and vitamin B₁₂) and a genetic variant in methylenetetrahydrofolate reductase (MTHFR C677T) have been associated with depression in adults, the role of methyl nutrient metabolism in depression during pregnancy remains unclear. Furthermore, little is known about the combined roles of methyl nutrient status and depression in the epigenetic regulation of SLC6A4. The experiments in this thesis explored whether disturbances in methyl nutrient metabolism and depressed mood during the 3rd trimester of pregnancy affect SLC6A4 methylation and expression in mothers and their newborns. Maternal folate status was positively associated with maternal SLC6A4 methylation at CpGs 1, 4, and 8 (P0.05). Maternal 3rd trimester mood was inversely associated with SLC6A4 CpG 10 methylation in both mothers and newborns (P0.05). Methylation at SLC6A4 CpG 8 was lower in newborns with mothers with the MTHFR 677TT genotype, and methylation at CpGs 6 and 10 were lower in newborns with the MTHFR 677TT genotype (P0.05). Maternal SLC6A4 mRNA level was positively associated with mean maternal methylation and methylation at CpGs 5, 7, 8, and 10 (P0.05). Yet, newborn SLC6A4 mRNA level was negatively associated with newborn methylation at CpGs 4 and 7 (P0.05). Homozygosity for the minor allele for MTHFR C677T and 5-HTTLPR insertion/deletion variants in mothers were not associated with maternal mood (P>0.05). These results provide evidence to suggest that maternal mood during pregnancy and methyl nutrient metabolism may program SLC6A4 gene expression through DNA methylation in both mothers and their newborns. Mood during pregnancy and disturbances in methyl nutrient metabolism could set up life-long health consequences in the development of the offspring.

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