Maternal folic acid supplementation and its effects on metabolic and epigenetic regulatory gene networks in offspring

Abstract

Periconceptional folic acid supplementation is a highly prevalent public health intervention and is known to reduce the incidence of neural tube defects (NTDs) and the risk of small for gestational age (SGA) infants. Increased maternal folate status during mid-gestation is associated with increased adiposity and insulin resistance in children. In rodents, maternal folic acid supplementation (MFAS) reduces plasma triacylglyceride (TAG) and cholesterol in adolescent and adult offspring but increases plasma glucose in adult female offspring. Overall, this suggests that MFAS, while beneficial perinatally, may affect metabolic regulation and susceptibility of offspring to chronic metabolic disease long term; however, the potential mechanisms involved remain incompletely understood. One mechanism by which MFAS may alter long-term metabolic regulation in offspring is perturbed DNA methylation. DNA methylation is a heritable form of epigenetic modification, which establishes a signature of inactive gene expression via the addition of a methyl group to the 5’ position of a cytosine residue; folate, a key methyl group donor, may modulate this epigenetic process and state. Consistently, MFAS, before and during gestation, increases DNA methylation in the regulatory region of IGF2 in blood of the newborn infant. In rodents, MFAS, during pregnancy and lactation, decreases hepatic activity of DNA methyltransferases (DNMTs), which are key enzymes for maintaining and establishing DNA methylation, in adult offspring. Maternal folic acid supplementation may act through such pathways to affect epigenetic state and phenotype of offspring long term. Besides affecting protein-coding genes, DNA methylation also regulates the expression of small non-coding microRNAs (miRs). These short single-stranded RNA molecules can repress post-transcriptional expression of their mRNA targets through 3’UTR base-pairing interactions. Many miRs target genes are involved in glucose, lipid and cholesterol metabolism; miRs can act in concert to orchestrate co-ordinated post-transcriptional changes by targeting multiple sites and, in turn, forming regulatory networks. In addition, miRs can target repressors in DNA methylation, enabling for dynamic regulation between these two distinct epigenetic processes. This may be a third pathway whereby MFAS influences phenotype of offspring. The present thesis describes studies, in the rodent, of the effects of MFAS, from preconception to term, on the expression of key metabolic regulatory genes, DNMTs and non-coding miRs in the major glucoregulatory tissues, liver and skeletal muscle, of offspring during prenatal and postnatal stages of development. Maternal folic acid supplementation altered hepatic expression of 13 genes (P < 0.05) in all adult offspring as well as in a sexually dimorphic manner, as shown by microarray analysis. Maternal folic acid supplementation altered the hepatic mRNA transcriptome of adult offspring, with 22 genes in males and 36 genes in females being differentially expressed (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes in liver of offspring following MFAS were closely associated with lipid and cholesterol metabolism. Maternal folic acid supplementation increased hepatic expression of Ppara, an upstream regulator of genes closely related to lipid metabolism, in all offspring in late gestation (1.22 fold, P = 0.024) but reduced it in adulthood (-0.41 fold, P = 0.002). Maternal folic acid supplementation also altered hepatic expression of lipogenic genes in offspring in a sexually dimorphic and age-dependent manner. In male offspring, MFAS decreased hepatic expression of Acaca in late gestation (-0.32 fold, P = 0.018) but increased it in adulthood (1.56 fold, P = 0.011); in female offspring, it was not affected by MFAS in late gestation nor in adulthood. Maternal folic acid supplementation decreased hepatic expression of Scd1 in the female offspring in late gestation (-0.37 fold, P = 0.017) but increased it in adulthood (1.38 fold, P = 0.035); in male offspring, it was not affected by MFAS in late gestation nor in adulthood. Maternal folic acid supplementation altered expression of genes involved in cholesterogenesis in female offspring in an age-dependent manner, with hepatic expression of Sc4mol being increased in late gestation (1.92 fold, P = 0.003), but it was unchanged in adulthood. Maternal folic acid supplementation also decreased hepatic expression of a cholesterogenic gene, Idi1 (-0.64 fold, P < 0.0001), in adult female offspring. In terms of associations, hepatic expression of Ppara was positively correlated with that of Idi1 (r = 0.434, P = 0.036) and Sqle (r = 0.528, P = 0.012) in the adult female offspring. Hepatic expression of Akr1b10 and Acaca (r = 0.846, P < 0.0001) were also positively correlated in the adult female offspring. Collectively, MFAS induces distinct changes in hepatic expression of genes related to lipid and cholesterol metabolism and their regulatory networks in offspring during prenatal and postnatal stages of development, particularly, in a manner that appears to augment the capacity for hepatic lipid and cholesterol homeostasis in later life. Maternal folic acid supplementation reduced hepatic expression of Dnmt1 (-0.36 fold, P = 0.039) and Dnmt3b (-0.21 fold, P < 0.0001) in the female foetus in late gestation. In addition, MFAS altered hepatic miR expression of 36 miRs (P < 0.05) in the female foetus in late gestation, with four miRs being up-regulated (1.18 to 1.22 fold) and 32 miRs being down-regulated (-0.21 to -1.18 fold). Maternal folic acid supplementation did not alter hepatic expression of DNMTs nor that of miRs in the male foetus. This suggests that MFAS could partly mediate its effects on hepatic gene expression in a sexually dimorphic manner through altering the capacity to regulate DNA methylation and miR expression in female foetuses but not in males. Effects of MFAS on hepatic expression of miRs and DNMTs in the adult offspring remain to be further determined. Maternal folic acid supplementation also altered skeletal muscle expression of six genes (P < 0.05) in adult offspring, as shown by microarray analysis. Gene ontology analysis further revealed these differentially expressed genes in offspring following MFAS were closely associated with lipid metabolism, cytoskeletal remodelling and potassium ion homeostasis. Overall, these observations show that MFAS alters hepatic and skeletal muscle expression of genes related to lipid and cholesterol metabolism in the offspring during prenatal and postnatal development. Maternal folic acid supplementation induces sex-specific differences in hepatic expression of DNMTs and non-coding miRs in foetal life, with these changes suggested to contribute towards altered expression of metabolic regulatory genes and, in effect, an altered capacity for lipid and cholesterol homeostasis after birth.