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Nutrients and Gene Expression in Development
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57 Nutrients and Gene Expression in Development Dolores Busso, Nicola´s Santander, Francisca Salas-Pe´rez, Jose´ Luis Santos Department of Nutrition, Diabetes, and Metabolism, School of Medicine, Pontificia Universidad Cato´lica de Chile, Santiago, Chile
STAGES OF EMBRYONIC AND FETAL DEVELOPMENT
Glossary CpG site DNA sequence where a cytosine sits next to a guanine. CpG islands Clusters of CpG sites. Developmental Origins of Health and Disease Concept linking the rise in the prevalence of noncommunicable diseases to intrauterine programming. DHA Polyunsaturated fatty acid highly concentrated in the brain that might promote neural development. DNA methylation Epigenetic change involving the addition of methyl groups to DNA. Epigenetic Potentially heritable changes regulating gene expression that do not involve changes in DNA sequence. Epigenome Total epigenetic modifications to the genome. Extraembryonic tissues Tissues for protection and nutrition of the developing embryo, such as the placenta, yolk sac, and amnios. Gene promoter DNA sequence that defines the initiation of gene transcription by RNA polymerase. Genomic imprinting Epigenetic mechanism that involves DNA and histone methylation in a parent of originespecific manner. Histones Family of basic proteins that associate with DNA in the nucleus and help it condense into chromatin. Intrauterine programming Group of fetal adaptations to intrauterine environment that lead to diseases in adulthood. microRNA Small noncoding RNA molecules that block RNA transcription. Neural tube Embryonic structure that develops into the brain and spine. ROS (reactive oxygen species) Molecules generated by oxidative metabolism that act in cell signaling in physiological concentrations but can be harmful to cells in high concentrations. Royal jelly Honeybee secretion given exclusively to certain larvae in a beehive to produce a queen. Teratogenic Any substance capable of disrupting normal development and generating malformations. Trophoblast Cells of embryonic origin derived from the blastocyst trophectoderm that differentiate to form extraembryonic tissues (i.e., the yolk sac and placenta). Vitamin B9 (folic acid) Water-soluble vitamin that promotes DNA methylation and protects against neural tube closure defects. Window of susceptibility Time frame during development when the embryo is more likely to be programmed by environmental factors.
Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00057-4
Embryonic and fetal intrauterine development encompasses a series of complex, well-concerted events of cell, tissue and organ differentiation and growth, starting immediately after spermeegg fertilization and ending at birth. Although prenatal development is a continuum, it can be subjectively categorized into three main stages. The first stage, preimplantation development, begins with the unicellular zygote and includes embryonic cleavage and the differentiation of the totipotential blastomeres forming morulae into the two cell types in the blastocyst: trophoblast and inner mass cells. The second stage, embryonic development, starts with attachment of the blastocyst to the uterine wall, in a process known as implantation. This stage is also composed of gastrulation, involving cell migration concomitant to differentiation, resulting in the three germ cell layers, which later combine and continue differentiating to form all of the tissues and organs in the body. The first organs that are formed in the embryo are the neural tube, which later originates the brain and the spine, and the heart. During this stage of development, extraembryonic tissues are also formed. The amniotic membrane, the yolk sac, and the early placenta are essential during early development, because they prevent dehydration and provide a suitable milieu for the embryo, allowing nutrient and gas exchange with the mother. The human yolk sac disappears before the 12th week of pregnancy, whereas in rodents this organ is functional until the end of gestation. Finally, the third stage, known as fetal development, involves mainly organ maturation and growth until parturition. The placenta matures and becomes fully functional during this stage. The timing of the different stages varies among different
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species: in humans, preimplantation development takes around 5 days after conception, embryonic development ends at around week 8, and fetal development lasts until the end of gestation, around 7 months.
EPIGENETICS IN DEVELOPMENT The formation of a complex organism, starting from a unicellular zygote, and the coordinated physiological responses that take place during development require massive changes in gene expression across several cells and tissues. Besides being primarily determined by the DNA sequence, gene expression is regulated by the epigenome through a combination of stable and dynamic epigenetic modifications that make the DNA more or less accessible to transcriptional regulators. One of best-described epigenetic mechanisms is the addition of a methyl group to the 50 position of a cytosine sitting next to a guanine in DNA (CpG site), yielding 5methylcytosine (5 mC) through the process of DNA methylation. This is established in mammals during embryogenesis and maintained by replication of cell division in somatic cells. DNA methylation has important roles in gene expression regulation, genomic imprinting, X-chromosome inactivation, and retrotransposon silencing. In general, it is thought that methylation of CpG sites by DNA methyltransferases (Dnmts) renders the DNA less accessible to transcription factors, resulting in repression of gene expression. Three main Dnmts have been described in mammals: Dnmt3a and Dnmt3b function primarily to establish de novo DNA methylation patterns during early embryogenesis, whereas Dnmt1 is the major maintenance enzyme that reproduces CpG methylation patterns after DNA replication. A relevant percentage of mammalian gene promoters contains clusters of CpG sites, denominated CpG islands, which potentially regulate gene expression. However, many gene promoters remain scarcely methylated, especially those from ubiquitously housekeeping genes. Two waves of global DNA demethylation occur during specific stages of embryonic development. One wave occurs during germ cell differentiation, when nearly complete demethylation and subsequent remethylation take place. The methylation pattern is again reprogrammed shortly after fertilization, in the zygote. After fertilization, paternal 5 mC is oxidized to 5hydroxymethylcytosine (5 hmC) by the Teneleven translocation (Tet3 member) dioxygenase in such a way that the resulting paternal 5 hmC together with maternal 5 mC, gradually declines during subsequent divisions. A new pattern of DNA methylation, known as de novo methylation, is established some time later, during the blastocyst stage. Appropriate reprogramming of
methylation at this stage is crucial for the development, survival, and even postnatal health and behavior of individuals. Once lineage-specific methylation patters are established, aging and different environmental factors such as dietary components, toxins, drugs, or diseases may regulate the DNA methylation processes in different periods of life. In some genes, DNA methylation allows the differential expression of alleles, depending on whether they come from the spermatozoa or the egg. Around 1% of autosomal genes undergo this maternal or paternal imprinting in mammals, which is settled during gametogenesis. If an allele is maternally imprinted, marks are imposed on both the maternally and paternally inherited genes during oogenesis and demethylation takes place in both maternally and paternally inherited during spermatogenesis. Thus, after fertilization, the offspring inherits a silenced (imprinted) allele from the mothers and functional (nonimprinted) allele from the father. The fact that most imprinted genes have roles in the control of embryonic growth and development, including development of the placenta, has led researchers to suggest that they function as an adaptive mechanism to obtain the best nutritional provision to the embryo without affecting the mother’s nutritional status. In fact, several methylated genes code for growth factors. Those paternally expressed genes generally enhance embryonic growth, whereas those that are maternally expressed appear to suppress embryonic growth. Besides being related to embryonic growth, imprinting has been linked to some human diseases. On the one hand, defective imprinting mechanisms and aberrant gene expression during development have been shown to explain congenital syndromes such as Beckwith-Wiedemann, PradereWilli, and Angelman syndromes. On the other hand, the phenotypic consequences of a mutation can remain inactive if the mutated allele is silenced by imprinting, which explains the parent-of-origin effect of certain diseases such as schizophrenia and epilepsy. In the rest of the genes that are not subjected to imprinting, methylation is also important, because some genes need to be expressed specifically during defined windows during embryonic development and then have to be inhibited. Besides DNA, histones can also be subject to modifications that confer genomic plasticity. These proteins pack DNA into chromatin, the structural component of chromosomes. Posttranslational modifications such as methylation and acetylation can change the affinity of histones for DNA, modifying chromatin compaction and modulating the accessibility of transcription factors to interact with genes. Histone deacetylases, acetyltransferases, and methyltransferases have been described in different cell types. Whereas histone acetylation makes chromatin more active and promotes gene expression,
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NUTRITION AND DEVELOPMENT: DEMANDS, SOURCES, AND STAGES OF SUSCEPTIBILITY
histone methylation can render chromatin more or less active, depending on the modified amino acid. Histone methylation and deacetylation, together with DNA methylation, are involved in inactivating one of the X chromosomes in females, to maintain gene dosage. In addition to DNA methylation and histone modifications, microRNAs (miRNAs), have more recently been shown to be involved in the epigenetic regulation of gene expression. These small noncoding RNA molecules are able to silence gene expression at the translational level, preventing messenger RNA (mRNA) from being transcribed or accelerating mRNA degradation. It has been shown that the expression of several genes coding for miRNAs can be regulated either by DNA methylation or histone modifications, which suggests that different components of the cellular epigenome are interdependent. Making the epigenetics scenario even more complex, several miRNAs directly target DNA methyltransferases and histone-modifying enzymes. Several examples of macronutrients and micronutrients and bioactive food components influencing development through epigenetic marks have been described. Mechanisms explaining the effect of nutrients on epigenetics are diverse and include directly modifying the activity of enzymes that catalyze DNA methylation or histone modifications, altering the availability of substrates necessary for those enzymatic reactions, and regulating the expression of miRNAs.
NUTRITION AND DEVELOPMENT: DEMANDS, SOURCES, AND STAGES OF SUSCEPTIBILITY The epigenetic status of the developing organism is modulated by the intrauterine environment, which in turn is defined both by maternal characteristics (e.g., metabolism) and gestational environmental exposures (e.g., diet). Nutrients not only influence the maternal health status but are transported across maternal fetal barriers to reach the embryo or fetus, directly affecting their development. Both nutrient deficiency and excess have been shown to hinder development. The overall effect for each nutrient on the embryo or fetus depends on two main aspects: the difference between its demand and availability and the timing of exposure. In general terms, macronutrients reaching the extraembryonic tissues and the embryo or fetus serve as sources of energy for cell proliferation and function, to sustain proper development and growth. Micronutrients, in turn, are crucial to regulate diverse biological processes, i.e., DNA repair, gene expression, hormone secretion, vascular tone, and protection against cell oxidative damage. The nutritional demand of a developing organism varies qualitatively and quantitatively as it matures
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from an embryo into a fetus, and eventually into a newborn. Fetal nutrient availability is determined by the type and amount of nutrients consumed by the mother, but also by her body composition, her placental function, and adaptations to cope with her own nutritional requirements and those of the developing embryo or fetus. For example, a teenager mother who has not yet achieved her adult body composition will have a significantly higher nutrient demand than will an adult woman. So will be the case of a woman doing intense physical work or coping with disease. In those cases, there will be competition between maternal needs and those of the fetus. As well, multiple pregnancies reduce the nutrient availability of each fetus, and short intervals between pregnancies hinder the appropriate repletion of maternal nutritional reserves. The source of available nutrients also changes along development. During the preimplantation stage, nutrients are first obtained from the newly fertilized egg cytoplasm and later from the maternal oviductal and uterine fluids, by phagocytosis. After implantation, the early embryo starts taking up nutrients from maternal sources. Even before the establishment of a mature placenta, the blastocyst trophoblasts and yolk sac endoderm cells phagocyte secretions from endometrial and uterine glands. This mechanism of nutrient uptake in the early conceptus is called histiotrophic nutrition. Once a mature, functional placenta is established hemotrophic nutrition takes place. This kind of nutrition corresponds to the exchange of blood-borne materials between the maternal and fetal circulations. Whereas histiotrophic nutrition provides for most of the embryonic stage of development, hemotrophic nutrition becomes predominant during fetal development. However, in certain species, such as in rodents, these two pathways may coexist for much of the gestational period. The placenta is formed by embryonic and maternally derived tissues; it regulates the transport of nutrients, excretions, and gases between the fetal and maternal circulations and modulates the maternal immune system to prevent the rejection of the embryo. Inside the placenta, maternal blood lacunae are separated from fetal capillaries by multinucleated cells called syncytiotrophoblasts. These intriguing cells originated by the fusion of numerous trophoblasts allow the wellcontrolled, bidirectional transport of molecules. Whereas gases such as oxygen and carbon dioxide are permeable and can cross syncytiotrophoblasts by passive diffusion, other substrates require channels and transporters. Glucose, for example, crosses the placenta by facilitated diffusion using specific transmembrane proteins. Other nutrients such as calcium, amino acids, and lactate cross the placenta by active transport using energy from adenosine triphosphate (ATP) or ion gradients. Fatty acids are an example of molecules that can
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either diffuse passively or be actively transported by specialized fatty acid transport proteins. There is significant evidence showing that certain maternal conditions such as diabetes and obesity can alter the expression and/or activity of nutrient receptors and transport molecules in syncytiotrophoblasts, influencing nutrient transport across the placental barrier and in some cases hindering fetal development and growth. In addition, structural abnormalities in the placenta that affect placental blood flow and available surface exchange area can interfere with nutrient transport to the embryo and fetus. Periods when genes are more actively predisposed to epigenetic modifications are considered windows of susceptibility (Fig. 57.1). These are stages when developing individuals are particularly sensitive to different environmental cues. One of these is preimplantation development, when reprogramming of methylation takes place. This stage is one of the most important windows of potential environmental influence over the embryo. Once implantation takes place, a high DNA synthesis rate and elaborate DNA epigenetic patterning are required for appropriate cell differentiation and specification, as well as normal tissue and organ
FIGURE 57.1
development. Thus, environmental exposures affecting gene expression during embryonic development can result in changes in phenotype, such as in color or behavior, or in more severe defects ranging from malformations to embryonic lethality, depending on the organ affected and the severity of the effect. One of the first and best described changes in phenotype is observed in the agouti mouse, whose coat color distribution is shifted from yellow to brown, depending on the maternal consumption of methyl-donor nutrients during pregnancy. In humans, maternal nutritional deficiencies during the first trimester have been linked to developmental syndromes. One clear example is severe maternal iodine deficiency, which results in fetal iodine insufficiency and different degrees of mental retardation. The most extreme manifestation of prenatal iodine deficiency is cretinism, a syndrome characterized by severe mental retardation, deaf-mutism, and strabismus, among other defects. Another well-known example is folic acid deficiency, which has been linked to an increased risk for gestating an infant with neural tube defects (NTD). On the other hand, the excessive ingestion of nutrients such as vitamin A (as retinol/retinyl esters) during pregnancy has also been suggested to result in birth defects.
Windows of susceptibility to nutritional exposures during intrauterine development and consequences in the offspring.
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NUTRITION AND DEVELOPMENT: DEMANDS, SOURCES, AND STAGES OF SUSCEPTIBILITY
Although there are only a few cases of retinol excess in humans, experimental models have supported the teratogenic effects of this vitamin. Environmental exposures can also take place during the fetal stage once the vital organs are differentiated. Epigenetic changes in the fetal stage of development are usually not lethal but may affect fetal growth, organ maturation, and/or the postnatal phenotype of the offspring. The process by which insults at critical stages of fetal development lead to permanent changes in the offspring (mostly postnatally) is known as intrauterine programming. Growing evidence shows that both maternal undernutrition and overnutrition have a significant impact on the offspring’s postnatal susceptibility to disease. It has been shown that fetuses, in particular males, can adapt to malnutrition using the limited nutrients available for survival of vital organs, such as the brain, at the expense of growth of other organs, such as the liver or pancreas. It has also been hypothesized that in undernourished fetuses, epigenetic modifications in key organs remodel the metabolism of the fetus to adapt to this nutrient-deprived environment. These fetal adaptive epigenetic responses, however, can render individuals susceptible to metabolic imbalances when exposed postnatally to a nutrientrich environment. Overnutrition, on the other hand, has also been linked by both epidemiological and experimental data to programming of metabolic syndrome, diabetes, and cardiovascular disease (CVD). That the prevalence of chronic diseases has increased substantially in most developing countries, as well as in countries undergoing economic transitions, has led many researchers around the world to reconsider how earlylife environment can affect later health in an approach known as Developmental Origins of Health and Disease. Among a significant number of nutritional exposures on different developmental stages and diverse effects via interdependent epigenetic mechanisms, this chapter focuses on some illustrative cases describing how nutrigenomics can shape development.
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physiology compared with workers. Epigenetic modifications in DNA methylation are induced by the provision of larvae with royal jelly or worker jelly, resulting in a nutrient-derived caste differentiation that leads to different gene and protein expression patterns in queens compared with workers. The second example, extensively studied, on the effect of diet in reversible epigenetic marks and subsequent phenotype determination corresponds to the agouti viable yellow (Avy) mouse. This mouse carries an insertion known as intracisternal A-particle (IAP) retrotransposon within the agouti gene, producing stochastic differences in DNA methylation that lead to variable ectopic expression of this gene, resulting in variability among individuals in traits such as coat color, energy intake, and obesity. Both coat color and body adipose tissue content can be modified by supplementing pregnant mothers’ diets with compounds favoring methylation (e.g., folic acid, vitamin B12, choline, and betaine). Increased DNA methylation upstream the intracisternal A particle element in the agouti gene results in partial reversion of the phenotypic features of Avy mice, so methyl-donor fed dams give birth to pseudoagouti (brown) slim mice instead of yellow obese mice. Dietary supplementation of pregnant Avy mice with the polyphenol genistein, present in soybeans, also increases DNA methylation and shifts the offspring phenotype to pseudoagouti. Apart from this striking example in mice, maternal nutrition and phenotype determination have been studied in women from rural Gambia, because they are exposed to seasonal variations in methyl-donor nutrients. This natural experiment has led to the idea that maternal methyl donor consumption during specific stages of pregnancy may modulate DNA methylation of metastable epialleles in the offspring. However, observational studies measuring methyl-donor intakes in relation to global DNA methylation have not yet yielded conclusive evidence for a causal association among diet, methylation marks in the DNA, and phenotype in this population.
Nutrients and Gene Expression in the Brain: Preventing Neural Tube Defects and Promoting Brain Cognitive Function
To Be or Not to Be: Phenotype Can Be Determined by Nutrients That Modulate Methylation There are two remarkable examples in the animal kingdom of the interaction between diet and epigenome affecting phenotype. In the honeybee (Apis mellifera), queens and workers are genetically identical in such a way that larvae may develop into a queen or a worker, depending on the environmental conditions. In a striking case of phenotypic plasticity, only queens are reproductively competent and have an increased life span and other important differences in morphology and
NTDs constitute the second most common malformation in humans. NTD are determined by maternal and embryonic genes and have a great influence on the environment. Significant prevention of NTD in humans and in animal models is achieved by supplementation with vitamin B9 (also known as folic acid). The success of this intervention to decrease the risk for NTD in the general population led several countries, particularly in the Americas, to implement the mandatory folic acid fortification of flour. Maintaining this policy resulted in the
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reduction of the NTD prevalence in 50%e70%, despite controversial evidence linking folic acid fortification with increased risk for colorectal cancer, epilepsy, or twin birth. Work with animal models has been invaluable in trying to unravel mechanisms linking folic acid consumption and NTD. Several murine models with natural or artificial mutations recapitulate different aspects of human NTD. Interestingly, some of the mouse mutants are completely sensitive to folic acid supplementation whereas others are totally resistant to this treatment. Folic acid, as well as other molecules involved in one-carbon metabolism such as vitamin B12 and choline, serves as donors of methyl groups necessary for DNA synthesis and methylation. In agreement with this role for folic acid, maternal folate status has been associated with the methylation level and expression level of certain genes in the embryo, including those involved in neural tube closure (e.g., Alx3, Pax3). Folic acid has also been shown to act as an antioxidant by regulating the activity of the nicotinamide adenine dinucleotide phosphate oxidase, an enzyme that produces large quantities of reactive oxygen species (ROS). In rodent models of NTD, overt ROS production has been extensively documented. Besides folate, other antioxidants have been successfully used to prevent NTD in experimental models. In neural cells, oxidative imbalance has been shown not only to be toxic but also to regulate the expression of genes involved in migration and differentiation. For example, exposing mouse embryos to oxidative stress in vitro or in vivo leads to a reduction in Pax3 expression, whereas mutations in this gene are associated with NTD in mice and humans. After neural tube closure, the ventricles expand and the different areas of the brain form by the concerted migration and differentiation of neural progenitors. Nutrients such as long-chain polyunsaturated fatty acids (PUFA) have been suggested to modulate these processes. Maternal supplementation with the PUFA docosahexanoic acid (DHA) during pregnancy has been associated with improvements in specific cognitive functions in children aged 9 years or younger. Observational studies comparing high versus low DHA status have also supported the beneficial effect of DHA on cognitive tasks in infants. However, the long-term effects of DHA and the epigenetic mechanisms explaining its effects are still unknown.
Ying and Yang: Maternal Undernutrition and Overnutrition and the Epigenetic Programming of Chronic Diseases The idea that abnormal fetal growth resulting from undernutrition caused the 20th century epidemic of coronary heart disease in Western countries was pioneered by the English epidemiologist David Barker in his
studies on newborns in England around 1940. His hypothesis was strongly supported by posterior data provided by the Dutch Famine in 1944, when a military-occupied region of The Netherlands was subjected to limited rations of food for nearly 6 months. In those pregnant women, undernourishment from mid to late gestation was associated with reduced birth weight, whereas exposure to famine during early gestation did not affect birth weight but increased susceptibility to developing CVD, obesity, renal dysfunction, and type 2 diabetes. Studies on the methylation pattern of genes from individuals conceived during the Dutch famine demonstrated that embryonic undernutrition caused several epigenetic changes that persisted throughout life, including reduced DNA methylation of the insulin-like growth factor (IGF)2 (IGF2) gene and increased DNA methylation in interleukin-10, leptin, and ATP-binding cassette subfamily A member 1 genes. Similarly, individuals exposed to the Chinese Great Famine (1959e61) during the first trimester of pregnancy have higher risk for type 2 diabetes and hypertension in adulthood, although the epigenetic mechanisms explaining this phenomenon remain largely underexplored. Another important human study showing the influence of maternal nutrient consumption on perinatal outcomes examined pregnancies in Gambia, where seasonal food availability determines seasonal increase in the incidence of intrauterine growth restriction and prematurity. Studying methylation patterns in tissues derived from the three germ lines demonstrated that the embryonic epigenome is altered before gastrulation by periconceptional undernutrition in these women. Animal models, primarily rodents and sheep, revealed some information about the complex mechanisms of the programming of CVD by intrauterine nutrient deficiency. Gestational caloric restriction in different animal models has linked nutrient deficiency with altered expression of IGFs and abnormal fetal growth. In sheep, maternal global nutrient restriction during early pregnancy affected the offspring’s hypothalamusepituitarye adrenal axis response through downregulation of the adrenal glucocorticoid receptor (GR) expression. In pregnant rats, consumption of reduced protein diets leads to promoter hypomethylation in fetal liver genes such as peroxisome proliferator-activated receptor a and GR, key regulators of carbohydrate and lipid metabolism. Folic acid supplementation in that model can prevent some of the metabolic abnormalities in the offspring, which supports the idea that interventions with nutrients can reduce the long-term risk for diseases associated with undernutrition. Besides affecting gene methylation, prenatal undernutrition can induce epigenetic modifications in histones and in genes coding for miRNAs regulating metabolism.
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CONCLUDING REMARKS: UNDERSTANDING NUTRIGENOMICS TO IMPROVE PREGNANCY OUTCOME
The population in most developing countries faces the opposite of malnutrition, caused by consumption of diets with low-quality nutrients and excessive calories. Maternal overweight and obesity are independent risk factors for maternal and fetal pregnancy complications, including gestational diabetes mellitus, embryonic malformations, and fetal macrosomia. Women who are obese before conception or gain excess weight during pregnancy are more likely to have babies who develop obesity, insulin resistance and early markers of CVD during their infancy or adult life, including higher blood pressure and inflammation. Patients with obesity and metabolic syndrome exhibit different global DNA methylation, histone acetylation, and miRNA expression marks than do healthy patients, which suggests that epigenetic modifications on key genes for metabolism could be a mechanism explaining metabolic abnormalities in the offspring of obese mothers. A major target of epigenetic influence is peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a), a transcriptional coactivator that regulates mitochondrial biogenesis and integrates different metabolic signals. Hypermethylation of the PGC-1a promoter has been linked to a reduction in PGC-1a expression in skeletal muscle and pancreatic islets from patients with type 2 diabetes. A variety of animal models have been established to study developmental programming by overnutrition. Pregnant rodents fed highly palatable, hypercaloric diets are being used as experimental models of gestational obesity. Their offspring exhibit hyperphagia, insulin resistance, hypertension, and glucose intolerance during adulthood. Several different, yet mutually interacting epigenetic mechanisms have been proposed to explain intrauterine programming by maternal obesity. Different metabolically relevant fetal tissues are epigenetically modified in response to obesity. In the hypothalamus, specific neuropeptides involved in energy homeostasis (e.g., Neuropeptide Y and proopiomelanocortin) and receptors for metabolites such as insulin and estrogen are differentially expressed in offspring from obese mice compared with controls. In livers of mice exposed to maternal obesity, high triglyceride content is associated with changes in histone marks at the liver X receptor, an important regulator of cholesterol, fatty acid, and glucose metabolism.
THE ROLE OF THE FATHER Evidence described previously showed how different nutrients consumed by females can affect embryonic and fetal development and modulate the offspring’s health. Besides the intrauterine influence on embryos and fetuses, studies in both humans and
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animal models support the idea that maternal prepregnancy and even paternal nutrition can also contribute to reprogramming of the offspring. Germ cells resulting in sperm and eggs start differentiation during fetal life, so DNA damage, mutations, and epigenetic marks on those embryonic germ cells can also affect the following generation. Existing evidence demonstrates that feeding male mice a suboptimal diet (e.g., lowprotein, high-fat, or folate deficient) can alter the global methylation and miRNA content in germ cells, which persist in sperm and may influence the offspring phenotype. Thus, epigenetic changes resulting from malnutrition in both mothers and fathers might have important roles in the transgenerational persistence of metabolic diseases. Different studies show that exposure to a high-fat diet (HFD) in utero causes metabolic disturbances in the offspring and that epigenetic modifications in certain genes, i.e., those coding for adiponectin and leptin, may persist for several generations. Paternal body mass index (BMI) also affects the offspring’s BMI via epigenetic marks, and these effects are additive to the influence of maternal BMI. Interestingly, the transgenerational reprogramming effects are reversible: a study showed that consumption of a normal diet by the offspring during subsequent generations after maternal or paternal HFD exposure first diminished and then completely abolished the effect of HFD exposure on the metabolic traits and the epigenetic changes in the offspring.
CONCLUDING REMARKS: UNDERSTANDING NUTRIGENOMICS TO IMPROVE PREGNANCY OUTCOME Nutrients encompass a large, diverse, and complex family of molecules with many known roles, and probably more yet to be discovered. These molecules can serve as energy sources, substrates, or cofactors of other physiologically relevant molecules, antioxidants, methyl donors, and enzymes, among many functions. In this chapter, we focused on nutrients affecting epigenetics and gene expression during intrauterine development. Pregnancy is a critical period when maternal and paternal nutrition choices can influence the embryonic, fetal, and postnatal health of the offspring. Inadequate levels of nutrients during critical stages of intrauterine development can lead to birth defects, suboptimal fetal development, and reprogramming of fetal tissues, predisposing the individual to chronic conditions later in life. In adolescent and young women, consuming a healthy, balanced diet and keeping a BMI within the recommended range for the gestational stage are required to achieve a healthy pregnancy. Consumption of insufficient,
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excessive, or poor-quality diets, or increased body requirements (e.g., caused by maternal infections, intense physical activity, or growth in adolescents) may promote malnutrition and negatively affect the offspring’s health. Appropriate and timely nutrient supplementation policies, nutritional counseling, and education can aid in preventing some consequences of malnutrition. Our knowledge of nutrigenomics during pregnancy will help us understand how the environment tailors diversity and susceptibility to disease in human beings and provide us with the necessary information to improve the health of future generations globally.
TAKE-HOME MESSAGES • Embryonic development is a complex and dynamic process requiring the well-controlled regulation of gene expression to sustain cell division and differentiation. • Embryonic nutrition can be histotrophic, hemotrophic, or both. In humans, histotrophic nutrition takes place before the establishment of a mature placenta, the specialized organ that allows the maternal fetal exchange of blood-borne nutrients. • Altered nutrient availability (by defect or excess) can induce embryonic lethality, teratogenesis, or organ malformations. In some cases, maternal nutrient consumption determines the phenotype of the offspring via epigenetic marks. These can influence fetal growth, organ maturation, and the postnatal susceptibility to disease. • Epigenetic marks in the embryo (DNA methylation, histone methylation, or acetylation) provide mechanisms to ensure the spatial and temporal organization of gene expression and confer adaptability to the intrauterine environment. • Maternal nutrient intake during pregnancy can modify (enhance or reduce) epigenetic marks in the embryo, changing the expression of critical genes associated with development.
• Both maternal preconceptional and paternal nutrient inadequacies may affect the epigenetic status of germ cells and their derived gametes, explaining the transgenerational effect of nutrition.
Suggested Reading Fleming, T.P., Watkins, A.J., Velazquez, M.A., Mathers, J.C., Prentice, A.M., Stephenson, J., Barker, M., Saffery, R., Yajnik, C.S., Eckert, J.J., Hanson, M.A., Forrester, T., et al., 2018. Origins of lifetime health around the time of conception: causes and consequences. Lancet 391, 1842e1852. Gilbert, S.F., 2006. Developmental Biology, eighth ed. Sinauer Associates, Sunderland, MA. Greene, N.D., Copp, A.J., 2014. Neural tube defects. Annu Rev Neurosci 37, 221e242. Hanson, M.A., Bardsley, A., De-Regil, L.M., Moore, S.E., Oken, E., Poston, L., et al., 2015. The international federation of gynecology and obstetrics (FIGO) recommendations on adolescent, preconception, and maternal nutrition: "think nutrition first". Int J Gynaecol Obstet 131 (Suppl. 4), S213eS253. Jirtle, R.L., Skinner, M.K., 2007. Environmental epigenomics and disease susceptibility. Nat Rev Genet 8 (4), 253e262. Masuyama, H., Mitsui, T., Eguchi, T., Tamada, S., Hiramatsu, Y., 2016. The effects of paternal high-fat diet exposure on offspring metabolism with epigenetic changes in the mouse adiponectin and leptin gene promoters. Am J Physiol Endocrinol Metab 311, E236eE245. Masuyama, H., Mitsui, T., Nobumoto, E., Hiramatsu, Y., 2015. The effects of high-fat diet exposure in utero on the obesogenic and diabetogenic traits through epigenetic changes in adiponectin and leptin gene expression for multiple generations in female mice. Endocrinology 156, 2482e2491. Nilsson, E.E., Sadler-Riggleman, I., Skinner, M.K., 2018. Environmentally induced epigenetic transgenerational inheritance of disease. Environ Epigenet 4, dvy016. Rasmussen, E.M., Amdam, G.V., 2015. Cytosine modifications in the honey bee (Apis mellifera) worker genome. Front Genet 6, 8. Vanhees, K., Vonhogen, I.G., van Schooten, F.J., Godschalk, R.W., 2014. You are what you eat, and so are your children: the impact of micronutrients on the epigenetic programming of offspring. Cell Mol Life Sci 71 (2), 271e285. Warner, M.J., Ozanne, S.E., 2010. Mechanisms involved in the developmental programming of adulthood disease. Biochem J 427 (3), 333e347.
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57 Nutrients and Gene Expression in Development Dolores Busso, Nicola´s Santander, Francisca Salas-Pe´rez, Jose´ Luis Santos Department of Nutrition, Diabetes, and Metabolism, School of Medicine, Pontificia Universidad Cato´lica de Chile, Santiago, Chile
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Glossary CpG site DNA sequence where a cytosine sits next to a guanine. CpG islands Clusters of CpG sites. Developmental Origins of Health and Disease Concept linking the rise in the prevalence of noncommunicable diseases to intrauterine programming. DHA Polyunsaturated fatty acid highly concentrated in the brain that might promote neural development. DNA methylation Epigenetic change involving the addition of methyl groups to DNA. Epigenetic Potentially heritable changes regulating gene expression that do not involve changes in DNA sequence. Epigenome Total epigenetic modifications to the genome. Extraembryonic tissues Tissues for protection and nutrition of the developing embryo, such as the placenta, yolk sac, and amnios. Gene promoter DNA sequence that defines the initiation of gene transcription by RNA polymerase. Genomic imprinting Epigenetic mechanism that involves DNA and histone methylation in a parent of originespecific manner. Histones Family of basic proteins that associate with DNA in the nucleus and help it condense into chromatin. Intrauterine programming Group of fetal adaptations to intrauterine environment that lead to diseases in adulthood. microRNA Small noncoding RNA molecules that block RNA transcription. Neural tube Embryonic structure that develops into the brain and spine. ROS (reactive oxygen species) Molecules generated by oxidative metabolism that act in cell signaling in physiological concentrations but can be harmful to cells in high concentrations. Royal jelly Honeybee secretion given exclusively to certain larvae in a beehive to produce a queen. Teratogenic Any substance capable of disrupting normal development and generating malformations. Trophoblast Cells of embryonic origin derived from the blastocyst trophectoderm that differentiate to form extraembryonic tissues (i.e., the yolk sac and placenta). Vitamin B9 (folic acid) Water-soluble vitamin that promotes DNA methylation and protects against neural tube closure defects. Window of susceptibility Time frame during development when the embryo is more likely to be programmed by environmental factors.
Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00057-4
Embryonic and fetal intrauterine development encompasses a series of complex, well-concerted events of cell, tissue and organ differentiation and growth, starting immediately after spermeegg fertilization and ending at birth. Although prenatal development is a continuum, it can be subjectively categorized into three main stages. The first stage, preimplantation development, begins with the unicellular zygote and includes embryonic cleavage and the differentiation of the totipotential blastomeres forming morulae into the two cell types in the blastocyst: trophoblast and inner mass cells. The second stage, embryonic development, starts with attachment of the blastocyst to the uterine wall, in a process known as implantation. This stage is also composed of gastrulation, involving cell migration concomitant to differentiation, resulting in the three germ cell layers, which later combine and continue differentiating to form all of the tissues and organs in the body. The first organs that are formed in the embryo are the neural tube, which later originates the brain and the spine, and the heart. During this stage of development, extraembryonic tissues are also formed. The amniotic membrane, the yolk sac, and the early placenta are essential during early development, because they prevent dehydration and provide a suitable milieu for the embryo, allowing nutrient and gas exchange with the mother. The human yolk sac disappears before the 12th week of pregnancy, whereas in rodents this organ is functional until the end of gestation. Finally, the third stage, known as fetal development, involves mainly organ maturation and growth until parturition. The placenta matures and becomes fully functional during this stage. The timing of the different stages varies among different
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species: in humans, preimplantation development takes around 5 days after conception, embryonic development ends at around week 8, and fetal development lasts until the end of gestation, around 7 months.
EPIGENETICS IN DEVELOPMENT The formation of a complex organism, starting from a unicellular zygote, and the coordinated physiological responses that take place during development require massive changes in gene expression across several cells and tissues. Besides being primarily determined by the DNA sequence, gene expression is regulated by the epigenome through a combination of stable and dynamic epigenetic modifications that make the DNA more or less accessible to transcriptional regulators. One of best-described epigenetic mechanisms is the addition of a methyl group to the 50 position of a cytosine sitting next to a guanine in DNA (CpG site), yielding 5methylcytosine (5 mC) through the process of DNA methylation. This is established in mammals during embryogenesis and maintained by replication of cell division in somatic cells. DNA methylation has important roles in gene expression regulation, genomic imprinting, X-chromosome inactivation, and retrotransposon silencing. In general, it is thought that methylation of CpG sites by DNA methyltransferases (Dnmts) renders the DNA less accessible to transcription factors, resulting in repression of gene expression. Three main Dnmts have been described in mammals: Dnmt3a and Dnmt3b function primarily to establish de novo DNA methylation patterns during early embryogenesis, whereas Dnmt1 is the major maintenance enzyme that reproduces CpG methylation patterns after DNA replication. A relevant percentage of mammalian gene promoters contains clusters of CpG sites, denominated CpG islands, which potentially regulate gene expression. However, many gene promoters remain scarcely methylated, especially those from ubiquitously housekeeping genes. Two waves of global DNA demethylation occur during specific stages of embryonic development. One wave occurs during germ cell differentiation, when nearly complete demethylation and subsequent remethylation take place. The methylation pattern is again reprogrammed shortly after fertilization, in the zygote. After fertilization, paternal 5 mC is oxidized to 5hydroxymethylcytosine (5 hmC) by the Teneleven translocation (Tet3 member) dioxygenase in such a way that the resulting paternal 5 hmC together with maternal 5 mC, gradually declines during subsequent divisions. A new pattern of DNA methylation, known as de novo methylation, is established some time later, during the blastocyst stage. Appropriate reprogramming of
methylation at this stage is crucial for the development, survival, and even postnatal health and behavior of individuals. Once lineage-specific methylation patters are established, aging and different environmental factors such as dietary components, toxins, drugs, or diseases may regulate the DNA methylation processes in different periods of life. In some genes, DNA methylation allows the differential expression of alleles, depending on whether they come from the spermatozoa or the egg. Around 1% of autosomal genes undergo this maternal or paternal imprinting in mammals, which is settled during gametogenesis. If an allele is maternally imprinted, marks are imposed on both the maternally and paternally inherited genes during oogenesis and demethylation takes place in both maternally and paternally inherited during spermatogenesis. Thus, after fertilization, the offspring inherits a silenced (imprinted) allele from the mothers and functional (nonimprinted) allele from the father. The fact that most imprinted genes have roles in the control of embryonic growth and development, including development of the placenta, has led researchers to suggest that they function as an adaptive mechanism to obtain the best nutritional provision to the embryo without affecting the mother’s nutritional status. In fact, several methylated genes code for growth factors. Those paternally expressed genes generally enhance embryonic growth, whereas those that are maternally expressed appear to suppress embryonic growth. Besides being related to embryonic growth, imprinting has been linked to some human diseases. On the one hand, defective imprinting mechanisms and aberrant gene expression during development have been shown to explain congenital syndromes such as Beckwith-Wiedemann, PradereWilli, and Angelman syndromes. On the other hand, the phenotypic consequences of a mutation can remain inactive if the mutated allele is silenced by imprinting, which explains the parent-of-origin effect of certain diseases such as schizophrenia and epilepsy. In the rest of the genes that are not subjected to imprinting, methylation is also important, because some genes need to be expressed specifically during defined windows during embryonic development and then have to be inhibited. Besides DNA, histones can also be subject to modifications that confer genomic plasticity. These proteins pack DNA into chromatin, the structural component of chromosomes. Posttranslational modifications such as methylation and acetylation can change the affinity of histones for DNA, modifying chromatin compaction and modulating the accessibility of transcription factors to interact with genes. Histone deacetylases, acetyltransferases, and methyltransferases have been described in different cell types. Whereas histone acetylation makes chromatin more active and promotes gene expression,
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histone methylation can render chromatin more or less active, depending on the modified amino acid. Histone methylation and deacetylation, together with DNA methylation, are involved in inactivating one of the X chromosomes in females, to maintain gene dosage. In addition to DNA methylation and histone modifications, microRNAs (miRNAs), have more recently been shown to be involved in the epigenetic regulation of gene expression. These small noncoding RNA molecules are able to silence gene expression at the translational level, preventing messenger RNA (mRNA) from being transcribed or accelerating mRNA degradation. It has been shown that the expression of several genes coding for miRNAs can be regulated either by DNA methylation or histone modifications, which suggests that different components of the cellular epigenome are interdependent. Making the epigenetics scenario even more complex, several miRNAs directly target DNA methyltransferases and histone-modifying enzymes. Several examples of macronutrients and micronutrients and bioactive food components influencing development through epigenetic marks have been described. Mechanisms explaining the effect of nutrients on epigenetics are diverse and include directly modifying the activity of enzymes that catalyze DNA methylation or histone modifications, altering the availability of substrates necessary for those enzymatic reactions, and regulating the expression of miRNAs.
NUTRITION AND DEVELOPMENT: DEMANDS, SOURCES, AND STAGES OF SUSCEPTIBILITY The epigenetic status of the developing organism is modulated by the intrauterine environment, which in turn is defined both by maternal characteristics (e.g., metabolism) and gestational environmental exposures (e.g., diet). Nutrients not only influence the maternal health status but are transported across maternal fetal barriers to reach the embryo or fetus, directly affecting their development. Both nutrient deficiency and excess have been shown to hinder development. The overall effect for each nutrient on the embryo or fetus depends on two main aspects: the difference between its demand and availability and the timing of exposure. In general terms, macronutrients reaching the extraembryonic tissues and the embryo or fetus serve as sources of energy for cell proliferation and function, to sustain proper development and growth. Micronutrients, in turn, are crucial to regulate diverse biological processes, i.e., DNA repair, gene expression, hormone secretion, vascular tone, and protection against cell oxidative damage. The nutritional demand of a developing organism varies qualitatively and quantitatively as it matures
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from an embryo into a fetus, and eventually into a newborn. Fetal nutrient availability is determined by the type and amount of nutrients consumed by the mother, but also by her body composition, her placental function, and adaptations to cope with her own nutritional requirements and those of the developing embryo or fetus. For example, a teenager mother who has not yet achieved her adult body composition will have a significantly higher nutrient demand than will an adult woman. So will be the case of a woman doing intense physical work or coping with disease. In those cases, there will be competition between maternal needs and those of the fetus. As well, multiple pregnancies reduce the nutrient availability of each fetus, and short intervals between pregnancies hinder the appropriate repletion of maternal nutritional reserves. The source of available nutrients also changes along development. During the preimplantation stage, nutrients are first obtained from the newly fertilized egg cytoplasm and later from the maternal oviductal and uterine fluids, by phagocytosis. After implantation, the early embryo starts taking up nutrients from maternal sources. Even before the establishment of a mature placenta, the blastocyst trophoblasts and yolk sac endoderm cells phagocyte secretions from endometrial and uterine glands. This mechanism of nutrient uptake in the early conceptus is called histiotrophic nutrition. Once a mature, functional placenta is established hemotrophic nutrition takes place. This kind of nutrition corresponds to the exchange of blood-borne materials between the maternal and fetal circulations. Whereas histiotrophic nutrition provides for most of the embryonic stage of development, hemotrophic nutrition becomes predominant during fetal development. However, in certain species, such as in rodents, these two pathways may coexist for much of the gestational period. The placenta is formed by embryonic and maternally derived tissues; it regulates the transport of nutrients, excretions, and gases between the fetal and maternal circulations and modulates the maternal immune system to prevent the rejection of the embryo. Inside the placenta, maternal blood lacunae are separated from fetal capillaries by multinucleated cells called syncytiotrophoblasts. These intriguing cells originated by the fusion of numerous trophoblasts allow the wellcontrolled, bidirectional transport of molecules. Whereas gases such as oxygen and carbon dioxide are permeable and can cross syncytiotrophoblasts by passive diffusion, other substrates require channels and transporters. Glucose, for example, crosses the placenta by facilitated diffusion using specific transmembrane proteins. Other nutrients such as calcium, amino acids, and lactate cross the placenta by active transport using energy from adenosine triphosphate (ATP) or ion gradients. Fatty acids are an example of molecules that can
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either diffuse passively or be actively transported by specialized fatty acid transport proteins. There is significant evidence showing that certain maternal conditions such as diabetes and obesity can alter the expression and/or activity of nutrient receptors and transport molecules in syncytiotrophoblasts, influencing nutrient transport across the placental barrier and in some cases hindering fetal development and growth. In addition, structural abnormalities in the placenta that affect placental blood flow and available surface exchange area can interfere with nutrient transport to the embryo and fetus. Periods when genes are more actively predisposed to epigenetic modifications are considered windows of susceptibility (Fig. 57.1). These are stages when developing individuals are particularly sensitive to different environmental cues. One of these is preimplantation development, when reprogramming of methylation takes place. This stage is one of the most important windows of potential environmental influence over the embryo. Once implantation takes place, a high DNA synthesis rate and elaborate DNA epigenetic patterning are required for appropriate cell differentiation and specification, as well as normal tissue and organ
FIGURE 57.1
development. Thus, environmental exposures affecting gene expression during embryonic development can result in changes in phenotype, such as in color or behavior, or in more severe defects ranging from malformations to embryonic lethality, depending on the organ affected and the severity of the effect. One of the first and best described changes in phenotype is observed in the agouti mouse, whose coat color distribution is shifted from yellow to brown, depending on the maternal consumption of methyl-donor nutrients during pregnancy. In humans, maternal nutritional deficiencies during the first trimester have been linked to developmental syndromes. One clear example is severe maternal iodine deficiency, which results in fetal iodine insufficiency and different degrees of mental retardation. The most extreme manifestation of prenatal iodine deficiency is cretinism, a syndrome characterized by severe mental retardation, deaf-mutism, and strabismus, among other defects. Another well-known example is folic acid deficiency, which has been linked to an increased risk for gestating an infant with neural tube defects (NTD). On the other hand, the excessive ingestion of nutrients such as vitamin A (as retinol/retinyl esters) during pregnancy has also been suggested to result in birth defects.
Windows of susceptibility to nutritional exposures during intrauterine development and consequences in the offspring.
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Although there are only a few cases of retinol excess in humans, experimental models have supported the teratogenic effects of this vitamin. Environmental exposures can also take place during the fetal stage once the vital organs are differentiated. Epigenetic changes in the fetal stage of development are usually not lethal but may affect fetal growth, organ maturation, and/or the postnatal phenotype of the offspring. The process by which insults at critical stages of fetal development lead to permanent changes in the offspring (mostly postnatally) is known as intrauterine programming. Growing evidence shows that both maternal undernutrition and overnutrition have a significant impact on the offspring’s postnatal susceptibility to disease. It has been shown that fetuses, in particular males, can adapt to malnutrition using the limited nutrients available for survival of vital organs, such as the brain, at the expense of growth of other organs, such as the liver or pancreas. It has also been hypothesized that in undernourished fetuses, epigenetic modifications in key organs remodel the metabolism of the fetus to adapt to this nutrient-deprived environment. These fetal adaptive epigenetic responses, however, can render individuals susceptible to metabolic imbalances when exposed postnatally to a nutrientrich environment. Overnutrition, on the other hand, has also been linked by both epidemiological and experimental data to programming of metabolic syndrome, diabetes, and cardiovascular disease (CVD). That the prevalence of chronic diseases has increased substantially in most developing countries, as well as in countries undergoing economic transitions, has led many researchers around the world to reconsider how earlylife environment can affect later health in an approach known as Developmental Origins of Health and Disease. Among a significant number of nutritional exposures on different developmental stages and diverse effects via interdependent epigenetic mechanisms, this chapter focuses on some illustrative cases describing how nutrigenomics can shape development.
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physiology compared with workers. Epigenetic modifications in DNA methylation are induced by the provision of larvae with royal jelly or worker jelly, resulting in a nutrient-derived caste differentiation that leads to different gene and protein expression patterns in queens compared with workers. The second example, extensively studied, on the effect of diet in reversible epigenetic marks and subsequent phenotype determination corresponds to the agouti viable yellow (Avy) mouse. This mouse carries an insertion known as intracisternal A-particle (IAP) retrotransposon within the agouti gene, producing stochastic differences in DNA methylation that lead to variable ectopic expression of this gene, resulting in variability among individuals in traits such as coat color, energy intake, and obesity. Both coat color and body adipose tissue content can be modified by supplementing pregnant mothers’ diets with compounds favoring methylation (e.g., folic acid, vitamin B12, choline, and betaine). Increased DNA methylation upstream the intracisternal A particle element in the agouti gene results in partial reversion of the phenotypic features of Avy mice, so methyl-donor fed dams give birth to pseudoagouti (brown) slim mice instead of yellow obese mice. Dietary supplementation of pregnant Avy mice with the polyphenol genistein, present in soybeans, also increases DNA methylation and shifts the offspring phenotype to pseudoagouti. Apart from this striking example in mice, maternal nutrition and phenotype determination have been studied in women from rural Gambia, because they are exposed to seasonal variations in methyl-donor nutrients. This natural experiment has led to the idea that maternal methyl donor consumption during specific stages of pregnancy may modulate DNA methylation of metastable epialleles in the offspring. However, observational studies measuring methyl-donor intakes in relation to global DNA methylation have not yet yielded conclusive evidence for a causal association among diet, methylation marks in the DNA, and phenotype in this population.
Nutrients and Gene Expression in the Brain: Preventing Neural Tube Defects and Promoting Brain Cognitive Function
To Be or Not to Be: Phenotype Can Be Determined by Nutrients That Modulate Methylation There are two remarkable examples in the animal kingdom of the interaction between diet and epigenome affecting phenotype. In the honeybee (Apis mellifera), queens and workers are genetically identical in such a way that larvae may develop into a queen or a worker, depending on the environmental conditions. In a striking case of phenotypic plasticity, only queens are reproductively competent and have an increased life span and other important differences in morphology and
NTDs constitute the second most common malformation in humans. NTD are determined by maternal and embryonic genes and have a great influence on the environment. Significant prevention of NTD in humans and in animal models is achieved by supplementation with vitamin B9 (also known as folic acid). The success of this intervention to decrease the risk for NTD in the general population led several countries, particularly in the Americas, to implement the mandatory folic acid fortification of flour. Maintaining this policy resulted in the
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reduction of the NTD prevalence in 50%e70%, despite controversial evidence linking folic acid fortification with increased risk for colorectal cancer, epilepsy, or twin birth. Work with animal models has been invaluable in trying to unravel mechanisms linking folic acid consumption and NTD. Several murine models with natural or artificial mutations recapitulate different aspects of human NTD. Interestingly, some of the mouse mutants are completely sensitive to folic acid supplementation whereas others are totally resistant to this treatment. Folic acid, as well as other molecules involved in one-carbon metabolism such as vitamin B12 and choline, serves as donors of methyl groups necessary for DNA synthesis and methylation. In agreement with this role for folic acid, maternal folate status has been associated with the methylation level and expression level of certain genes in the embryo, including those involved in neural tube closure (e.g., Alx3, Pax3). Folic acid has also been shown to act as an antioxidant by regulating the activity of the nicotinamide adenine dinucleotide phosphate oxidase, an enzyme that produces large quantities of reactive oxygen species (ROS). In rodent models of NTD, overt ROS production has been extensively documented. Besides folate, other antioxidants have been successfully used to prevent NTD in experimental models. In neural cells, oxidative imbalance has been shown not only to be toxic but also to regulate the expression of genes involved in migration and differentiation. For example, exposing mouse embryos to oxidative stress in vitro or in vivo leads to a reduction in Pax3 expression, whereas mutations in this gene are associated with NTD in mice and humans. After neural tube closure, the ventricles expand and the different areas of the brain form by the concerted migration and differentiation of neural progenitors. Nutrients such as long-chain polyunsaturated fatty acids (PUFA) have been suggested to modulate these processes. Maternal supplementation with the PUFA docosahexanoic acid (DHA) during pregnancy has been associated with improvements in specific cognitive functions in children aged 9 years or younger. Observational studies comparing high versus low DHA status have also supported the beneficial effect of DHA on cognitive tasks in infants. However, the long-term effects of DHA and the epigenetic mechanisms explaining its effects are still unknown.
Ying and Yang: Maternal Undernutrition and Overnutrition and the Epigenetic Programming of Chronic Diseases The idea that abnormal fetal growth resulting from undernutrition caused the 20th century epidemic of coronary heart disease in Western countries was pioneered by the English epidemiologist David Barker in his
studies on newborns in England around 1940. His hypothesis was strongly supported by posterior data provided by the Dutch Famine in 1944, when a military-occupied region of The Netherlands was subjected to limited rations of food for nearly 6 months. In those pregnant women, undernourishment from mid to late gestation was associated with reduced birth weight, whereas exposure to famine during early gestation did not affect birth weight but increased susceptibility to developing CVD, obesity, renal dysfunction, and type 2 diabetes. Studies on the methylation pattern of genes from individuals conceived during the Dutch famine demonstrated that embryonic undernutrition caused several epigenetic changes that persisted throughout life, including reduced DNA methylation of the insulin-like growth factor (IGF)2 (IGF2) gene and increased DNA methylation in interleukin-10, leptin, and ATP-binding cassette subfamily A member 1 genes. Similarly, individuals exposed to the Chinese Great Famine (1959e61) during the first trimester of pregnancy have higher risk for type 2 diabetes and hypertension in adulthood, although the epigenetic mechanisms explaining this phenomenon remain largely underexplored. Another important human study showing the influence of maternal nutrient consumption on perinatal outcomes examined pregnancies in Gambia, where seasonal food availability determines seasonal increase in the incidence of intrauterine growth restriction and prematurity. Studying methylation patterns in tissues derived from the three germ lines demonstrated that the embryonic epigenome is altered before gastrulation by periconceptional undernutrition in these women. Animal models, primarily rodents and sheep, revealed some information about the complex mechanisms of the programming of CVD by intrauterine nutrient deficiency. Gestational caloric restriction in different animal models has linked nutrient deficiency with altered expression of IGFs and abnormal fetal growth. In sheep, maternal global nutrient restriction during early pregnancy affected the offspring’s hypothalamusepituitarye adrenal axis response through downregulation of the adrenal glucocorticoid receptor (GR) expression. In pregnant rats, consumption of reduced protein diets leads to promoter hypomethylation in fetal liver genes such as peroxisome proliferator-activated receptor a and GR, key regulators of carbohydrate and lipid metabolism. Folic acid supplementation in that model can prevent some of the metabolic abnormalities in the offspring, which supports the idea that interventions with nutrients can reduce the long-term risk for diseases associated with undernutrition. Besides affecting gene methylation, prenatal undernutrition can induce epigenetic modifications in histones and in genes coding for miRNAs regulating metabolism.
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The population in most developing countries faces the opposite of malnutrition, caused by consumption of diets with low-quality nutrients and excessive calories. Maternal overweight and obesity are independent risk factors for maternal and fetal pregnancy complications, including gestational diabetes mellitus, embryonic malformations, and fetal macrosomia. Women who are obese before conception or gain excess weight during pregnancy are more likely to have babies who develop obesity, insulin resistance and early markers of CVD during their infancy or adult life, including higher blood pressure and inflammation. Patients with obesity and metabolic syndrome exhibit different global DNA methylation, histone acetylation, and miRNA expression marks than do healthy patients, which suggests that epigenetic modifications on key genes for metabolism could be a mechanism explaining metabolic abnormalities in the offspring of obese mothers. A major target of epigenetic influence is peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a), a transcriptional coactivator that regulates mitochondrial biogenesis and integrates different metabolic signals. Hypermethylation of the PGC-1a promoter has been linked to a reduction in PGC-1a expression in skeletal muscle and pancreatic islets from patients with type 2 diabetes. A variety of animal models have been established to study developmental programming by overnutrition. Pregnant rodents fed highly palatable, hypercaloric diets are being used as experimental models of gestational obesity. Their offspring exhibit hyperphagia, insulin resistance, hypertension, and glucose intolerance during adulthood. Several different, yet mutually interacting epigenetic mechanisms have been proposed to explain intrauterine programming by maternal obesity. Different metabolically relevant fetal tissues are epigenetically modified in response to obesity. In the hypothalamus, specific neuropeptides involved in energy homeostasis (e.g., Neuropeptide Y and proopiomelanocortin) and receptors for metabolites such as insulin and estrogen are differentially expressed in offspring from obese mice compared with controls. In livers of mice exposed to maternal obesity, high triglyceride content is associated with changes in histone marks at the liver X receptor, an important regulator of cholesterol, fatty acid, and glucose metabolism.
THE ROLE OF THE FATHER Evidence described previously showed how different nutrients consumed by females can affect embryonic and fetal development and modulate the offspring’s health. Besides the intrauterine influence on embryos and fetuses, studies in both humans and
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animal models support the idea that maternal prepregnancy and even paternal nutrition can also contribute to reprogramming of the offspring. Germ cells resulting in sperm and eggs start differentiation during fetal life, so DNA damage, mutations, and epigenetic marks on those embryonic germ cells can also affect the following generation. Existing evidence demonstrates that feeding male mice a suboptimal diet (e.g., lowprotein, high-fat, or folate deficient) can alter the global methylation and miRNA content in germ cells, which persist in sperm and may influence the offspring phenotype. Thus, epigenetic changes resulting from malnutrition in both mothers and fathers might have important roles in the transgenerational persistence of metabolic diseases. Different studies show that exposure to a high-fat diet (HFD) in utero causes metabolic disturbances in the offspring and that epigenetic modifications in certain genes, i.e., those coding for adiponectin and leptin, may persist for several generations. Paternal body mass index (BMI) also affects the offspring’s BMI via epigenetic marks, and these effects are additive to the influence of maternal BMI. Interestingly, the transgenerational reprogramming effects are reversible: a study showed that consumption of a normal diet by the offspring during subsequent generations after maternal or paternal HFD exposure first diminished and then completely abolished the effect of HFD exposure on the metabolic traits and the epigenetic changes in the offspring.
CONCLUDING REMARKS: UNDERSTANDING NUTRIGENOMICS TO IMPROVE PREGNANCY OUTCOME Nutrients encompass a large, diverse, and complex family of molecules with many known roles, and probably more yet to be discovered. These molecules can serve as energy sources, substrates, or cofactors of other physiologically relevant molecules, antioxidants, methyl donors, and enzymes, among many functions. In this chapter, we focused on nutrients affecting epigenetics and gene expression during intrauterine development. Pregnancy is a critical period when maternal and paternal nutrition choices can influence the embryonic, fetal, and postnatal health of the offspring. Inadequate levels of nutrients during critical stages of intrauterine development can lead to birth defects, suboptimal fetal development, and reprogramming of fetal tissues, predisposing the individual to chronic conditions later in life. In adolescent and young women, consuming a healthy, balanced diet and keeping a BMI within the recommended range for the gestational stage are required to achieve a healthy pregnancy. Consumption of insufficient,
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excessive, or poor-quality diets, or increased body requirements (e.g., caused by maternal infections, intense physical activity, or growth in adolescents) may promote malnutrition and negatively affect the offspring’s health. Appropriate and timely nutrient supplementation policies, nutritional counseling, and education can aid in preventing some consequences of malnutrition. Our knowledge of nutrigenomics during pregnancy will help us understand how the environment tailors diversity and susceptibility to disease in human beings and provide us with the necessary information to improve the health of future generations globally.
TAKE-HOME MESSAGES • Embryonic development is a complex and dynamic process requiring the well-controlled regulation of gene expression to sustain cell division and differentiation. • Embryonic nutrition can be histotrophic, hemotrophic, or both. In humans, histotrophic nutrition takes place before the establishment of a mature placenta, the specialized organ that allows the maternal fetal exchange of blood-borne nutrients. • Altered nutrient availability (by defect or excess) can induce embryonic lethality, teratogenesis, or organ malformations. In some cases, maternal nutrient consumption determines the phenotype of the offspring via epigenetic marks. These can influence fetal growth, organ maturation, and the postnatal susceptibility to disease. • Epigenetic marks in the embryo (DNA methylation, histone methylation, or acetylation) provide mechanisms to ensure the spatial and temporal organization of gene expression and confer adaptability to the intrauterine environment. • Maternal nutrient intake during pregnancy can modify (enhance or reduce) epigenetic marks in the embryo, changing the expression of critical genes associated with development.
• Both maternal preconceptional and paternal nutrient inadequacies may affect the epigenetic status of germ cells and their derived gametes, explaining the transgenerational effect of nutrition.
Suggested Reading Fleming, T.P., Watkins, A.J., Velazquez, M.A., Mathers, J.C., Prentice, A.M., Stephenson, J., Barker, M., Saffery, R., Yajnik, C.S., Eckert, J.J., Hanson, M.A., Forrester, T., et al., 2018. Origins of lifetime health around the time of conception: causes and consequences. Lancet 391, 1842e1852. Gilbert, S.F., 2006. Developmental Biology, eighth ed. Sinauer Associates, Sunderland, MA. Greene, N.D., Copp, A.J., 2014. Neural tube defects. Annu Rev Neurosci 37, 221e242. Hanson, M.A., Bardsley, A., De-Regil, L.M., Moore, S.E., Oken, E., Poston, L., et al., 2015. The international federation of gynecology and obstetrics (FIGO) recommendations on adolescent, preconception, and maternal nutrition: "think nutrition first". Int J Gynaecol Obstet 131 (Suppl. 4), S213eS253. Jirtle, R.L., Skinner, M.K., 2007. Environmental epigenomics and disease susceptibility. Nat Rev Genet 8 (4), 253e262. Masuyama, H., Mitsui, T., Eguchi, T., Tamada, S., Hiramatsu, Y., 2016. The effects of paternal high-fat diet exposure on offspring metabolism with epigenetic changes in the mouse adiponectin and leptin gene promoters. Am J Physiol Endocrinol Metab 311, E236eE245. Masuyama, H., Mitsui, T., Nobumoto, E., Hiramatsu, Y., 2015. The effects of high-fat diet exposure in utero on the obesogenic and diabetogenic traits through epigenetic changes in adiponectin and leptin gene expression for multiple generations in female mice. Endocrinology 156, 2482e2491. Nilsson, E.E., Sadler-Riggleman, I., Skinner, M.K., 2018. Environmentally induced epigenetic transgenerational inheritance of disease. Environ Epigenet 4, dvy016. Rasmussen, E.M., Amdam, G.V., 2015. Cytosine modifications in the honey bee (Apis mellifera) worker genome. Front Genet 6, 8. Vanhees, K., Vonhogen, I.G., van Schooten, F.J., Godschalk, R.W., 2014. You are what you eat, and so are your children: the impact of micronutrients on the epigenetic programming of offspring. Cell Mol Life Sci 71 (2), 271e285. Warner, M.J., Ozanne, S.E., 2010. Mechanisms involved in the developmental programming of adulthood disease. Biochem J 427 (3), 333e347.
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