PRINCIPLES OF

NUTRIGENETICS

AND NUTRIGENOMICS

SMH Ghaderian MD,PhD

Human Genetics

1

Introduction

• The role of nutrition in the pathogenesis of metabolic diseases, such as type 2

diabetes and cardiovascular disease, is clearly recognized

• A new concept in nutrition research is the measurement of a wide range of

markers to characterize health, which is called “comprehensive phenotyping”

• Comprehensive phenotyping not only includes omics techniques but also requires

the measurement of classical markers and intermediary endpoint measures that

have been shown to be associated with disease.

• In one research, The ultimate goal was to develop a machine‐learning algorithm

that predicts personal postprandial glycemic responses to real‐life meals.

2

Regulation of transcription depends on the interplay between chromatin structure and transcription factor-DNA interactions.

3

Signals and general mechanisms in gene regulation

4

Overview of gene expression control by dietary factors. Dietary factors including specific food components, nutritional conditions and diet

types impact gene expression directly or indirectly and at multiple stages, from transcription to translation and protein activity. At the

transcriptional level, they affect the activity of trans acting factors as well as chromatin regulation in the short- and long-term. Changes in

gene expression induced by dietary factors can help the cell/organism to utilize or store the dietary nutrients, condition cell fate and function

(for instance, by adjusting cell growth and proliferation to nutrient availability), and be linked to health maintenance or the development of

dietrelated diseases.

5

Direct Control of TF Activity

Specific food compounds behave as ligands of NR superfamily members or as precursors of

endogenous nuclear receptor (NR) ligands.

For instance, retinoic acid receptors are activated by the vitamin A derivative retinoic acid,

the vitamin D receptor (VDR) is activated by active vitamin D (1,25-dihydroxycholecalciferol),

peroxisome proliferator activated receptors (PPARs) can be activated by certain dietary fatty

acids and other dietary compounds, and pregnane X receptor and constitutive androstane

receptor are both activated by xenobiotics in foods.

Upon ligand binding, these NRs gain affinity for coregulators that provide transcription

transactivation activities.

The following examples highlight the significance and therapeutic potential of the NR gene

regulatory circuitries controlled by dietary factors.

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In the enteric tract, NRs help to “absorb the good and neutralize the bad”: in the enterocytes,

vitamin D-activated VDR induces genes for proteins involved in the intestinal absorption of

dietary calcium, while xenobiotic-activated NRs induce genes for proteins involved in the

metabolism and detoxification of these potentially toxic compounds.

As another example, diet is the vehicle of PPARa activating molecules of potential interest for

health, such as certain polyunsaturated fatty acids (PUFA); 9-oxo-10(E),12(E)-

octadecadienoic acid (found in tomatoes); pterostilbene (a resveratrol analogue abundant in

blueberries); astaxanthin (a carotenoid abundant in seafood); and phytanic acid (a branched-

chain fatty acid found in dairy products).

PPARa transcriptional activity suppresses inflammatory pathways and favors mitochondrial

substrate oxidation and fatty acid catabolism, and preclinical studies support hypolipidemic

and antiobesity effects of PPARa activating ligands of dietary origin.

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Direct Control of TF Abundance

This mechanism is exemplified by the negative control by PUFA of the lipogenic TF sterol

regulatory elementebinding protein-1 (SREBP-1).

SREBP-1 is synthesized in a precursor form that is bound to the endoplasmic reticulum

(ER) membrane, and PUFAs inhibit the proteolytic processing of the precursor that renders

the active SREBP-1 form, by stabilizing an inhibitory protein in the process, Insig-1.

The direct target of PUFA is Ubxd8, an ER membrane-bound protein that normally facilitates

Insig-1 degradation: Ubxd8 is inhibited upon interaction with PUFA.

Additionally, PUFA potently lowers SREBP-1 mRNA levels: the PUFA-mediated inhibition of

SREBP-1 maturation disrupts an autostimulatory loop on SREBP-1 gene transcription, and,

in addition, PUFAs stimulate SREBP-1 mRNA decay and may function as antagonist ligands

of the liver X receptor (LXR), a nuclear receptor that normally transactivates the SREBP-1

gene.

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Besides repressing SREBP-1, PUFAs repress the nuclear import of a second important

lipogenic TF, carbohydrate response element binding protein (ChREBP).

Overall, by promoting SREBP-1 and ChREBP inhibition and PPARa activation, PUFAs promote

a shift in hepatic fatty acid metabolism, from synthesis and storage to oxidation, resulting in a

blood lipidelowering effect.

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Indirect Control of TFs Following Changes in Metabolic

Fluxes This mechanism is exemplified by glucose-mediated regulation of hepatic lipogenic capacity.

Highcarbohydrate low-PUFA diets increase liver lipogenic capacity independently of insulin, a

response that favors the storage as fat of excess carbohydrate-ingested energy.

When cellular glucose levels are high, increases in the flux through glucose-metabolizing

pathways such as glycolysis, the pentose phosphate pathway, and the hexosamine

biosynthetic pathway ensue the activation of the lipogenic TFs ChREBP, SREBP-1, and LXR.

These three TFs synergistically transactivate genes for enzymes involved in glucose

utilization and lipid synthesis, such as L-pyruvate kinase, fatty acid synthase, and acetyl-CoA

carboxylase, among others.

LXR transactivates SREBP-1 and ChREBP gene transcription..

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Mechanistically, three glucose-derived metabolites, glucose-6-phosphate (the first

intermediate in intracellular glucose metabolism), fructose-2,6-bisphosphate (the major

regulator of glycolysis), and xylulose 5- phosphate (a metabolite of the pentose phosphate

pathway), have been implicated as positive modulators of ChREBP translocation to the

nucleus and activation, through allosteric effects and effects on the ChREBP phosphorylation

status. gene and possibly other lipogenic targets including the ChREBP gene

The hexosamine biosynthetic pathway, in its turn, produces uridine diphosphate

Nacetylglucosamine (UDP-GlcNAc), which is the donor substrate for enzyme-catalyzed

regulatory OGlcNAcylation of many cellular proteins.

OGlcNAcylation of ChREBP stabilizes the ChREBP protein and increases its transcriptional

activity toward lipogenic genes.

Similarly, O-GlcNAcylation of LXR enhances its transcriptional activity toward the SREBP-1

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Indirect Control of TFs Following Changes in

Nutrient/Energy Sensing Signaling Pathways

Under conditions of caloric restriction, cellular levels of AMP and NADþ increase, leading to

the activation of two important intracellular energy sensors and master regulators of cell

metabolism, AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1).

AMPK and SIRT1 catalyze, respectively, regulatory phosphorylation and NADþ-dependent

deacetylation of target proteins, among them TF and coregulators involved in the

transcriptional control of energy, lipid and glucose metabolism.

AMPK and SIRT1 share many common target protein substrates and can regulate each

other.

In general, AMPK and SIRT1 activation favors a more efficient use of lipid energy sources

and respiratory metabolism.

Both sensors enhance mitochondrial biogenesis and mitochondria quality maintenance

mechanisms, owing to their action on PPAR gamma coactivator 1a (PGC1a) and on proteins

related to the autophagy of damaged mitochondria. 12

PGC-1a is a transcriptional coregulator that coactivates a constellation of TFsdsuch as

estrogen-related receptor a, nuclear respiratory factors 1 and 2, and PPARsdto induce

mitochondrial gene expression.

Interestingly, certain dietary chemicals such as resveratrol, other polyphenols, and the NADþ

precursor nicotinamide riboside have been shown to activate SIRT1 and/or AMPK and to

promote metabolic fitness in experimental animal models, independently of caloric restriction.

Another important nutrient/energy sensing pathway in cells is the mechanistic target of

rapamycin (mTOR) pathway.

Thus mTOR is a Ser/Thr protein kinase that is activated in response to insulin, growth factors,

and amino acids, and inhibited under conditions of cellular glucose deprivation or energetic

stress.

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mTOR drives cell growth by stimulating anabolic processes including protein, lipid, and

nucleotide synthesis, and by inhibiting degradative catabolic processes such as autophagy.

mTOR activity upregulates cellular protein synthesis and ribosome biogenesis by activating

translation factors and by inducing ribosomal RNA and ribosomal protein expression.

In addition, mTOR acts on specific TFs and coregulators such as signal transducer and

activator of transcription 3, SREBPs, hypoxia-inducible factor 1a, PGC-1a, PPARg, and PPARa

to favor cell growth, proliferation and survival, lipid synthesis, angiogenesis, mitochondria

biogenesis and adipogenesis, and to block hepatic fatty acid oxidation, among other responses.

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DIETARY FACTORS AND METABOLISM AS

CHROMATIN REGULATORS

Cellular levels of essential cofactors of chromatin remodelers are dependent on the cell’s

nutritional/ metabolic status, which allows the integration of metabolic information into

transcriptional control through chromatin changes.

Nutrient abundance boosts the nuclear acetyl-CoA pool, thus favoring histone acetylation

and gene transcription; interestingly, increased histone acetylation is predominantly observed

on cell growthe promoting genes and genes related to nutrient utilization and storage, which

in this way become upregulated.

The opposite condition, i.e., nutrient deprivation/ caloric restriction, also impacts the

chromatin landscape, in this case through increases in the cellular levels of NADþ and AMP.

The NADþ-dependent sirtuins may affect gene expression through direct deacetylation of

histones (sirtuins are class III HDACs), and AMPK activation regulates histone acetylation in

a complex manner, through a variety of mechanisms.

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Dietary factors may condition methylation reactions as well, because specific nutrients

(methionine, choline, folate, vitamin B12, vitamin B6, zinc, selenium) participate as substrates

or necessary cofactors of enzymes in the one-carbon metabolism pathway that produces

Sadenosylmethionine (SAM), the universal cellular methyl donor.

Dietary manipulations affecting the intake of these nutrients have been shown to affect the

methylation status of DNA in cells, with consequences on gene expression (DNA methylation

generally associates with transcriptional silencing).

Additionally, there are food compounds able to allosterically modulate chromatin remodelers in

cells.

For instance, dietary phytochemicals behave as DNA methyltransferases inhibitors (e.g.,

polyphenols from black raspberry, apples, and green tea) or as type I and II HDACs inhibitors

(e.g., curcumin, soy isoflavones, isothiocyanates from cruciferous vegetables, sulfur

compounds from garlic, green tea polyphenols).

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Butyrate and other short-chain fatty acids produced upon fermentation of some types of

dietary fiber by the colonic flora also inhibit type I and type II HDACs.

Inhibition of DNA methyltransferases and/or HDACs after exposure to such dietary

compounds has been shown to result in the reactivation of silenced tumorsuppressing genes

in cancer cell lines and animal models of cancer, suggesting that these activities may

contribute to anticancer action.

Exposure to certain dietary factors at sensitive periods such as the periconceptional period,

uterine life, and early postnatal life may trigger persistent epigenetic changes, particularly of

DNA methylation, affecting health outcomes in adulthood.

This is concluded from animal studies and sustained by the (so far still limited) evidence

available in humans.

Dietary factors that have been implicated in long-term metabolic programming include the

intake levels of one-carbon metabolism related nutrients (see above), overnutrition,

malnutrition, alcohol exposure, and milk leptin.

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DIETARY FACTORS AS MODULATORS OF MIRNAS

miRNAs are endogenous, small noncoding RNA molecules (of about 22 nucleotides) that

function in posttranscriptional regulation of gene expression by binding to complementary

sequences in target mRNAs, this resulting in mRNA cleavage, destabilization of the mRNA

through shortening of its poly(A) tail, or a less efficient translation by ribosomes.

It has been shown that dietary factors (e.g., macronutrients, minerals, trace elements,

vitamins, plant bioactives) alter the production of mammalian microRNAs.

Additionally, there is some initial evidence to suggest that, following absorption, secretion,

and tissue distribution, microRNAs contained in foods of plant (e.g., rice) and animal (e.g.,

cow milk, human breast milk) origin may affect gene expression in mammalian tissues.

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The Role of Nutrition in DNA

Replication, DNA Damage Prevention

and DNA Repair

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INTRODUCTION

Life as we know it depends entirely on the capacity of cells to utilize energy and molecules in

the environment for cellular function and reproduction.

Multicellular animal organisms, including humans, acquire energy and essential nutrients

from foods.

Some of these essential nutrients are required for DNA synthesis, maintenance of normal

chromosome structure, repair of DNA damage caused by nutrient deficiency and/or

environmental genotoxins, and for the control of gene expression by epigenetic mechanisms.

This review provides a brief outline of the role of nutrition in DNA replication, DNA damage

prevention, and DNA repair.

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The role of folate, as 10-formyl tetrahydrofolate (10-formyl THF) and 5,10-methylene tetrahydrofolate (5,10-meTHF) in de novo nucleotide synthesis.

5,10 meTHF, 5,10- methylenetetrahydrofolate; 10-formyl THF, 10-formyl tetrahydrofolate; AMP, adenosine monophosphate; ATP, adenosine

triphosphate; CTP, cytosine triphosphate; dTTP, deoxy-thymine triphosphate; dUMP, deoxy-uridine monophosphate; GMP, guanosine monophosphate;

GTP, guanosine triphosphate; IMP, inosine monophosphate; ribose-5-P, ribose-5-phosphate; UMP, uridine monophosphate; UTP, uridine triphosphate.

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Examples of the Role and the Effect of Deficiency of Specific Micronutrients on Genomic Stability.

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The various mechanisms by which micronutrient deficiency leads to DNA damage and chromosomal instability. BFB, chromosome breakage-fusion-bridge.

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Methods for Global Nutrigenomics

and Precision Nutrition

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From genotype to phenotype. Interacting “omics.” A depiction of the relations among genetic information (explored using genomic methods), gene

expression (transcriptome), protein expression (proteome), and metabolic consequences (metabolome). In addition, epigenetic regulation is involved in

gene expression and influences RNA levels and gene silencing.

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Methods in Nutrigenomics

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Main procedures to analyze locus-specific DNA methylation after sodium bisulfite treatment. HRM, high-resolution melt; MALDI-TOF, matrix-assisted

laser desorption ionizationetime of flight; MassARRAY, mass spectrometry methylation assay; PCR, polymerase chain reaction

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Next-Generation Sequencing Approaches for Nucleic Acid Modification Analysis.

5-hmC, 5-hydroxymethylcytosine; 5-mC, 5-methylcytosine; 6-mA, N6-methyladenosine; Aba-seq, DNA-modification-dependent restriction

endonuclease; anti-CMS, anti-cytosine-5-methylenesulfonate; CAB-seq, chemical modification-assisted bisulfite sequencing; CHARM,

comprehensive high-throughput arrays for relative methylation; DIP-seq, DNA immunoprecipitation and shotgun sequencing; fCAB-seq, 5fC

chemical modification-assisted bisulfite sequencing; fC-Seal, 5- formylcytosine selective chemical labeling (fC-Seal) approach for affinity

purification and genome-wide profiling of 5fC; GLIB, glucosylation, periodate oxidation and biotinylation; JBP1, J-binding protein ; LCM-RRBS,

laser-capture microdissection-reduced representation bisulfite sequencing; LHC-BS (pre- and postconversion), liquid hybridization capture-based

bisulfite sequencing; MBD-seq, methyl-CpG-binding domain protein sequencing; MeDIP-seq, methylation DNA immunoprecipitation sequencing;

MeKL-ChIP, methylated DNA, kinase pretreated ligation-mediated PCR amplification-chromatin immunoprecipitation; MeRIP-seq, methylation

RNA immunoprecipitation sequencing; MethylCap-seq, methylation DNA capture sequencing; MRE-seq, methylation restriction enzyme

sequencing; mRRBS, multiplexed reduced representation bisulfite sequencing; OxBS-MethylationEPIC, oxidative bisulfite Infinium

MethylationEPIC; oxBS-seq, oxidative bisulfite sequencing; PBAT, postbisulfite adaptor tagging; redBS-seq, reduced bisulfite sequencing;

RRBS, reduced representation bisulfite sequencing; RRHP, reduced representation 5-hmC profiling; scRRBS, single-cell reduced representation

bisulfite sequencing; TAB-seq, TET-assisted bisulfite sequencing; T-WGBS, transposase-based library construction; WGBS, whole-genome

bisulfite sequencing

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Metagenomics: Workflow for microbiome analysis. OTUs, operational taxonomic units; rRNA, ribosomal RNA.

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A Broader View on Omics and

Systems Biology

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Integration of omics technologies. Source: Badimon L., Vilahur G. and Padro T., Systems biology approaches to understand the effects of nutrition and

promote health. Br J Clin Pharmacol. 83 (1), 2017, 38e45

INTERINDIVIDUAL VARIABILITY AND OMICS TOOLS IN THE DEVELOPMENT OF NOVEL NUTRITIONAL

BIOMARKERS

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Role of foodomics in human health through the development of precision nutrition. Adapted from Arola-Arnal A., del Blas J.M., Caimari A., Crescenti A.,

Puiggro`s F., Sua´rez M., Arola L.l., 2013. How does foodomics impact optimal nutrition?. In: Cifuentes, A. (Ed.). Foodomics Advanced Mass Spectrometry

in Modern Food Science and Nutrition. Wiley, pp. 309.

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The human metabolome. Adapted from Scalbert, A., Brennan, L., Manach, C., Andres-Lacueva, C., Dragsted LO, Draper, J., Rappaport, S.M., van der

Hooft, J.J., Wishart, D.S., 2014. The food metabolome: a window over dietary exposure. Am J Clin Nutr 99 (6), 1286e1308

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Schematic diagram for combining dietary questionnaires with biomarkers. Source: Garcia-Aloy, M., Rabassa, M., Casas-Agustench, P., Hidalgo-Liberona,

N., Llorach, R., Andres-Lacueva, C., 2017. Novel strategies for improving dietary exposure assessment: multiple-data fusion is a more accurate measure

than the traditional single-biomarker approach. Trends Food Sci Technol 69, pp. 220e229.

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Metabolic pattern analysis after a Mediterranean diet intervention in a nondiabetic population. Adapted from Va´zquez-Fresno, R., Llorach, R., Urpi-Sarda,

M., Lupianez-Barbero, A., Estruch, R., Corella, D., Fito´. M., Aro´s, F., Ruiz-Canela, M., Salas-Salvado´, J., Andres-Lacueva, C., 2015. Metabolomic pattern

analysis after mediterranean diet intervention in a nondiabetic population: a 1- and 3-year follow-up in the PREDIMED study. J Proteome Res 14 (1),

531e5400. The figure shows a two-way hierarchical clustering analysis (processed with PermutMatrix). Heat map representation of the clustered data matrix

in which each colored cell represents the intensity of appropriate NMR signals, according to the color scale at the bottom of the figure. Rows: NMR signals

(253, VIP > 1.5). Columns: urine samples baseline and Low-fat diet (1y þ 3y); Mediterranean diet þ Extra virgin olive oil (1y þ 3y), and Mediterranean diet þ

Nuts (1y þ 3y).

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Nutrimetabolomics fingerprinting to identify biomarkers of bread exposure. Adapted from Garcia-Aloy, M., Llorach, R., Urpisarda, M., Tulipani, S., Salas-

salvado´, J., Martı´nez-Gonza´lez, M.A., Corella, D., Fito´, M., Estruch, R., Serra-Majem, L., Andres-Lacueva, C., 2015. Nutrimetabolomics fingerprinting to

identify biomarkers of bread exposure in a free-living population from the PREDIMED study cohort. Metabolomics 11, 155

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Discovery of biomarkers by nutritional systems biology. Adapted from van Ommen B., van den Broek T., de Hoogh I., van Erk M., van Someren E.,

Rouhani-Rankouhi T., Anthony J.C., Hogenelst K., Pasman W., Boorsma A. and Wopereis S., Systems biology of personalized nutrition, Nutr Rev 75 (8),

2017, 579e599. Topol, E.J., 2014. Individualized medicine from pre-womb to tomb. Cell 157 (1), 241e253.

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Nutrigenetics

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Nutrigenetics and the Early Life Origins of Health

and Disease: Effects of Protein Restriction“Nutrigenetics” describes how human genetic variation results in distinct nutritional

requirements.

Interindividual differences in genetics, resulting in different effects of nutrients on metabolism,

were recognized early in nutrition research.

As the interface between the mother and fetus, the placenta plays a key role in developmental

programming.

This may result from nutrition-driven changes in maternofetal endocrine cross-talk, through

alterations in the capacity to supply the fetus with substrates and changes in the expression of

signaling molecules.

Protein restriction alters the expression of placental amino acid transporters, increases fetal

glucocorticoid exposure, and remodels placental vasculature.

The impact of maternal protein restriction on the placental transcriptome at day 13 gestation

was investigated using RNASeq as a means of capturing the impact of the dietary insult upon

the tissue. with obesity) in human placenta.41

The main pathways and processes in placenta that were associated with these differentially

expressed genes were atherosclerosis signaling, LXR/FXR activation of RXR, and other

processes strongly linked to atherosclerosis including inflammation.

Follow-up PCR studies of genes from these pathways showed major upregulation (18e32-

fold) with protein restriction.

The strongest effects were noted for cubilin, retinol binding protein-4, microsomal

triglyceride transfer protein, and the apolipoproteins A2 and C2.

Interestingly, the same genes were shown in a separate study to be sensitive to maternal fat

intake in rats (upregulated gene expression as with LP) and maternal body mass index

(upregulated gene expression

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The concept of tissue remodeling. Organs and tissues develop according to a genetically determined pattern during embryonic and fetal

life. Programming of long-term tissue function will occur due to environmental impacts on the genetic pattern, with disruption of

proliferation and differentiation resulting in tissues with fewer functional units.

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The impact of protein restriction on the rat placental transcriptome. RNASeq showed that maternal protein restriction altered expression of

91 genes in rat placenta and that pathways associated with cholesterol metabolism and transport were highly enriched in the differentially

expressed set of genes. Data drawn from Daniel, Z., Swali, A., Emes, R., Langley-Evans, S.C., 2016. The effect of maternal undernutrition

on the rat placental transcriptome: protein restriction up-regulates cholesterol transport. Genes Nutr 11, 27.

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The gatekeeper approach to explore mechanisms of early life programming. Exposure of rat fetuses to diverse nutritional insults in

development results in a common phenotype comprising high blood pressure, renal impairment, and metabolic disturbance. Using a

transciptomics approach to identify genes that are differentially expressed in response to all three insults, it is possible to identify sets

of gatekeeper genes, proteins, and pathways that play an integral role in the nutritional programming effect. Adapted from McMullen,

S., Langley-Evans, S.C., Gambling, L., Lang, C., Swali, A., McArdle, H.J., 2012. A common cause for a common phenotype: the

gatekeeper hypothesis in fetal programming. Med Hypotheses 78, 88e94

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PROTEIN AND THE DNA METHYLOME

The view that fetal programming of adult disease is a consequence of resetting of epigenetic

marks is highly favored in the field, and an array of evidence showing that DNA methylation or

histone modifications at specific gene loci are responsive to maternal dietary insults supports

this hypothesis.

For example, Bogdarina et al. (2010) showed that methylation of the angiotensin II type 1b

receptor in adrenal, and subsequently the expression of the gene, was sensitive to maternal

LP feeding in a glucocorticoid-sensitive manner.

The epigenetic hypothesis is attractive as epigenetic marks can be passed on to subsequent

generations through both male and female gametes.

A number of studies have shown that the programming effects of maternal undernutrition,

including LP, are persistent across two or even three generations.

Moreover, research in which pregnant animals are fed diets that are lacking in methyl donors

required to maintain epigenetic marks typically finds that offspring are programmed for

adverse cardiovascular and metabolic phenotypes.46

Mismatch between DNA methylation and gene expression. Analysis of DNA methylation across the whole genome of neonatal rat livers following exposure

to maternal protein restriction in utero showed 555 differentially methylated loci. In the same tissue, protein restriction was associated with differential

expression of 577 genes. There was little overlap between the differentially methylated and differentially expressed sets. Data redrawn from Altobelli, G.,

Bogdarina, I.G., Stupka, E., Clark, A.J.L., Langley-Evans, S., 2013. Genome-wide methylation and gene expression changes in newborn rats following

maternal protein restriction and reversal by folic acid. PLoS One 8.

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Maternal Perinatal Nutrition and Offspring

ProgrammingThe suitable interactions between nutrient intake, in both quality and quantity, and genetic

background are crucial for maintaining a healthy metabolic and physiologic condition across

different lifetime periods.

However, nutritional requirements, parallel to physiological condition, are adapted to the

different biological necessities from each lifetime stage and environmental situation.

It is understood, for example, that the requirements of an adolescent during fast-growth

stages are not the same as those of adults or elderlies as well as during special situations or

diseases.

Thus, during perinatal periods, including preconception, pregnancy, and lactation, the

physiological and metabolic adaptations lead to changes of normal macro- and micronutrient

requirements.

During pregnancy there are circulatory system modifications whose main objective is to

provide adequate placental circulation and fetal nutrient supply.

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Moreover, there is increased blood volume, higher iron requirements, basal metabolism and

body weight, together with decreased hematocrit due to hemodilution and hormonal alterations

(cortisol, thyroid hormones T3 and T4, adrenocorticotropic hormone or prolactin).

During lactation, besides endocrine changes such as increased prolactin and oxytocin

hormones in plasma, there is also increased basal metabolism and calcium lost, among other

physiometabolic modifications.

These maternal transformations require nutritional adjustments.

During pregnancy, the interactions between maternal nutrition and genetic background

determine fetus nutrient supply, which also interacts with fetal genetic information.

Therefore, there is a double nutrient-gene interaction between the mother and the fetus.

Furthermore, the term perinatal encompasses different time frames and, thus, different

physiological requirement in each one of them.

First, during a preconception period, maternal nutrition may affect the oocyte and its

fecundation, with possible consequences on the future development of the offspring.

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Thus, novel research has highlighted that dietary polyphenols appear to inhibit oocyte

apoptosis and follicle atresia during ovarian development.

Other investigators have shown that the maturation of gametes and early embryonic

development can be compromised in malnutrition situations leading to early pregnancy failure

and postnatal increased risks of offspring cardiovascular, metabolic, immune, and

neurological morbidities.

Obesity has been also related with impaired oocyte meiosis, failure of spindle assembly, and

increased oxidative stress and abnormal mitochondrial distribution.

After fertilization, fetal development depends on the maternal dietary intake, metabolism, and

physiologic features.

During this phase of in utero environment, the availability of triglycerides, amino acids,

cholesterol, glucose, vitamins, and other metabolites in the maternal plasma leads the

development of the fetus.

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For example, maternal fatness and/or obesogenic nutrition exposes the offspring to a diseased

environment (hyperinsulinemia, hyperleptinemia, hypercholesterolemia, etc.) associated with

obesity comorbidities, programming offspring physiology and increasing the susceptibility to

develop metabolic diseases during adulthood.

Finally, during early stages after birth, mainly during the lactation period, there is also an effect

of nutrient supply in the offspring development and risk of suffering later metabolic diseases

during adulthood.

In this sense, there is a substantial volume of experimental and observational data

demonstrating the importance of lactation for the development of metabolic diseases such as

cardiovascular disease, insulin resistance, or nonalcoholic fatty liver disease (NAFLD), among

others.

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Nutritional interactions during pregnancy

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MECHANISMS OF PERINATAL

PROGRAMMINGNutrigenetics and nutrigenomics study how the information contained in the genome affect

the response to nutrients and how nutrients can regulate gene expression, respectively.

These dual interactions are maintained during the life course and, therefore, during

pregnancy and other perinatal periods.

Furthermore, as previously commented, during these stages, there is a combinatory effect of

maternal nutrition and genetic background affecting offspring nutrient availability and genome.

However, beyond the genome nucleotide sequence, maternal and offspring genetic material

is conditioned by their epigenetic profiles.

Epigenetics involves a molecular machinery around the gene sequence, which regulated its

transcription without altering the nucleotide sequence.

The main epigenetic modifications are DNA methylation, histone modifications, microRNAs,

and long noncoding RNAs (lncRNAs).

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The most studied, understood, and potentially therapeutically targeted is the DNA methylation,

which is based in the addition of a methyl group to a cytosine having a guanine as next

nucleotide (CpG site).

This covalent reaction is mediated by the DNA methyltransferases (Dnmts).

These CpG sites are abundant in the promoter region of the genes, where transcription

machinery joins the DNA sequence in order to regulate genome reading.

According to the histone marks, covalent reactions (e.g., acetylation, phosphorylation,

methylation, ubiquitination) in their amino acidic tails regulate the chromatin conformation and

accessibility of the transcriptional machinery to the DNA sequence.

MicroRNAs and lncRNAs are short chains of nucleotides and nonprotein coding transcripts

longer than 200 nucleotides, respectively, which are able to modulate posttranscriptional

regulation of gene expression.

The combination of these reactions will determine the cellular physiology and metabolism

through affecting gene expression patterns in a cell-specific manner, whilst preserving the

nucleotide sequences. 54

Nutriepigenetic and nutriepigenomic regulation.

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Nutritional insults affect phenotypical characteristics by epigenetic modifications (C, carbon; Ca, calcium; CHO, carbohydrates; Cr,

chromium; FA, fatty acids; Fe, iron; Mg, magnesium; Se, selenium; TG, triglycerides; Vit, vitamin).

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Epigenetics

The extent of DNA methylation varies between organisms and tissues.

In humans, 5% of cytosines are methylated at the global level, with hypermethylation

observed in areas of heterochromatin and hypomethylation observed in euchromatin.

Across the genome, CpG sites localized to repetitive DNA, such as Alu repeats, are typically

methylated, whereas high densities of CpG sites localized more closely to genes are

unmethylated.

The effect of DNA methylation on gene expression depends on the genomic context,

specifically the localization of methyl-cytosines in relation to the gene and the density of

CpGs.

Although most DNA methylation occurs at CpG sites, the genome has a lower proportion of

CpG sites than expected by chance.

According to evolution, this results from the mutability of a methylated cytosine, because

spontaneous deamination can yield a thymine and cause a mutation.57

Whereas most of the genome is CpG poor, there are segments of 300e3000 bp with a 65%

observed-to-expected ratio of CpG sites that are called CpG islands (CGIs).

CGIs are localized to the promoter region of 70% of human genes, including nearly all

housekeeping genes, tissue-specific genes, and developmentally regulated genes.

Approximately 50% of CGIs in the human genome are associated with transcription start sites

of genes, whereas the remaining 50% are considered orphan CGIs that are intragenic or

intergenic.

Nearly all promoter-associated CGIs are unmethylated in healthy somatic cells; only about

3% f promoter-associated CGIs are methylated.

Special cases of promoter-associated DNA methylation in a subset of developmental and

tissue specific-genes become methylated during embryonic development and subsequently

silence gene expression.

Tissue differences in DNA methylation are also localized outside promoter-associated CGIs,

specifically within 2 kb of CGIs that are denoted CGI shores.

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There is an inverse relation between CGI shore methylation and gene expression;

hypermethylation of the CGI shore is associated with decreased gene expression and

hypomethylation is associated with increased gene expression.

This supports the notion that DNA methylation differences have a role in differentiating

gene expression profiles between tissues.

By comparison, approximately 20%e34% of intragenic CGIs are methylated.

The underlying function of intragenic DNA methylation is not well-understood, although it is

hypothesized that this methylation may regulate alternative splicing, the rate of

transcriptional elongation, or expression of noncoding RNAs.

Each histone protein is wrapped by 147 bp of DNA to form a nucleosome.

The spacing between each nucleosome varies between 30 and 100 bp and can be

modified by nucleosome repositioning complexes.

The interaction between nucleosome positioning, histone modifications and DNA

methylation determines local chromatin packaging.

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Highly compacted chromatin is designated as heterochromatin, yielding DNA that is

inaccessible to transcription factors and thus associated with gene repression.

Gene transcription activation requires the euchromatin state in which DNA is loosely wrapped

enabling accessibility to transcriptional regulators.

The effect of histone PTMs on gene expression depends on which chemical modification has

been added to specifically which amino acid on a particular histone subunit.

The best studied chemical modifications are acetylation, phosphorylation and methylation;

though ongoing research is continuing to characterize the effect of other modifications such

as SUMOlyation, butyrylation, propionylation, crotonylation and addition of Olinked b-N-

acetylglucosamine sugar moieties.

Gene expression effects of the beststudied histone PTMs are summarized in Table 17.1 and

discussed briefly next.

Generally, the addition of acetyl groups to histones is associated with a loosening of the

chromatin structure and transcriptional activation.

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Acetylation of lysine 9 or 27 on histone 3 (H3K9 and H3K27) and lysine 16 of histone 4

(H4K16) are all associated with open, accessible euchromatin and active gene expression.

Specifically, H3K27ac and H3K9ac are implicated in transcriptional initiation at active gene

promoters.

Histone acetylation is catalyzed by a family of histone acetyl transferase (HAT) enzymes and

removed by histone deacetylase (HDAC) enzymes.

The acetylation status of histones is dynamically regulated by a balance of HAT and HDAC

activity to transition between active gene expression and gene repression.

The gene activation effects of histone acetylation are thought to be twofold.

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Map of histone marks. Map of posttranslational histone modifications at specific amino acids within the tails of the histone octamer subunits. From Pandian,

G.N., Sugiyama, H., 2013. Strategies to modulate heritable epigenetic defects in cellular machinery: lessons from nature. Pharmaceuticals 6(1), 1e24.

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Epigenetic Marks and Their Effect on Gene Expression.

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Dynamic balance of histone acetyl transferases (HATs) and histone deacetylase (HDACs) on gene transcription. Addition of acetyl

groups to histones by HAT is associated with an accessible chromatin structure, transcription factors (TF) poised with coactivators

including activating epigenetic writers such as HATs, specific histone methyl transferases (HMTs), and specific histone demethylases

(HDMs) yielding active gene expression. The removal of acetyl groups from histones by HDAC is associated with a condensed

chromatin structure, transcription factors poised with corepressors including HDACs, DNA methyl-transferases (DNMTs), and specific

HMTs yielding repressed gene expression.

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Chromatin remodeling entails large multisubunit protein complexes that alter the local

chromatin architecture to regulate accessibility to transcriptional machinery.

Subunits of these protein complexes can simultaneously catalyze multiple levels of

epigenetic reactions and/or reposition nucleosomes to transition between euchromatin and

heterochromatin states.

Two well-studied chromatin remodeling complexes include the geneactivating

switch/sucrose nonfermentable (SWI/SNF) complex and the transcriptionally silencing

polycomb group (PcG) proteins. SWI/SNF functions in adenosine triphosphate (ATP)-

dependent nucleosome repositioning.

Originally discovered in Saccharomyces cerevisiae, its name was based on its functions in

altering yeast mating type and metabolism.

SWI/SNF contains a core ATPase/remodeling domain that harnesses the energy of ATP to

destabilize the DNAehistone interaction to reposition the nucleosome effectively.

Binding of transcription factors is inhibited at DNA sequences that are wrapped around

histones; therefore, SWI/SNF nucleosome repositioning enables accessibility of DNA to

transcriptional regulators. 65

Additional subunits of the SWI/SNF complex include bromodomains that are specific to

acetylated histones and subunits with specificity to transcriptional regulators and

coregulators.

Biochemical models indicate that transcriptional regulators bind sequence-specifically to a

region of DNA and then subsequently recruit SWI/SNF to alter the local chromatin

architecture to promote transcription.

In contrast, the PcG proteins are a large protein complex containing enzymatic subunits that

catalyze the writing of repressive histone marks to silence gene expression.

Originally discovered in D. melanogaster for their role in Hox gene silencing in anteroposterior

morphogenesis, PcG proteins are implicated in epigenetic silencing during development.

In mammals, there are two well-characterized members of the PcG family including PRC1

and PRC2, respectively.

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PRC1 complexes catalyze the addition of the transcriptionally silencing monoubiquitination

mark on lysine 119 of H2A (H2AK119Ub1) by the Bmi1 subunit.

PRC2 complexes include the EZH2 subunit, which is responsible for the trimethylation of

H3K27.

PcG proteins are critical in working with DNA methylation for X inactivation and to maintain

somatic cell identity through gene repression.

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RNA INTERFERENCE The continually developing field of epigenetics has expanded with the discovery of RNA

interference in 1998 by Andrew Fire and Craig C. Mello while working with Caenorhabditis

elegans.

MicroRNAs (miRNA) are endogenously expressed short RNAs that do not encode for

proteins. Initially transcribed as single-stranded premiRNAs, they fold over on themselves to

form a stem loop structure that is then subsequently processed by RNA-induced silencing

complexes to yield 18e22 nucleotideelong, single-stranded mature miRNA transcripts.

miRNAs silence gene expression by sequence-specifically binding to messenger RNAs

(mRNAs) to promote degradation or inhibit translation.

RNA interference can also be induced by exogenous small interfering RNAs (siRNAs).

siRNAs can be introduced by viruses or inserted synthetically in the laboratory.

Currently, siRNAs are used experimentally to modify gene expression and are investigated for

possible targeted therapeutic approaches in disease.68

Environmental influences on epigenetics. Factors that can alter epigenetic patterns through development and aging and in response

to environmental influences. Ongoing research is characterizing how these epigenetic changes can alter phenotype.

69

Genetics of Chrononutrition

Our physiology changes during the day. It is wellknown that several hormones that are

related to obesity, such as cortisol, leptin, and adiponectin, among others, display circadian

rhythmicity.

Alteration of this normal pattern is called chronodisruption(CD) and is related to several

disturbances in many systems and organs of our body.

CD can be defined as a serious disruption of the internal temporal order of the biochemical,

physiological, and behavioral circadian rhythms.

In our modern society, CD may be produced by external situations that are relatively

common, such as jet lag, shift work, night light pollution, and overnight recreational activities

(social jet lag).

Other factors are internal and may produce CD by altering the core machinery of the

molecular circadian clock.

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Brain- and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1),

Period 2, and Circadian Locomotor Output Cycles Kaput (CLOCK), among others clock

proteins, have a specific role in our physiology as well as in the circadian molecular clock.

Indeed, several studies performed in mutant animals demonstrated that mutations in clock

genes are related to obesity, aging, and other metabolic alterations implicated in several

chronic diseases.

For example, studies performed in experimental animals showed that animals that have

mutations in Clock are more obese and display metabolic disturbances.

Clock genes are not only associated with obesity, they may interact with several obesogenic

behaviors for obesity or weight control parameters.

One clear example is the interaction between CLOCK 3111T/C and emotional eating

behaviors.

71

Our results demonstrate that during a dietary treatment to lose weight, subjects who were

very emotional eaters and carriers of the risk allele C in CLOCK 3111T/C lost less weight

compared with (1) C carriers who were nonemotional eaters and (2) TT carriers, independent

of their emotional status.

Many other examples of interactions between clock genes and several behaviors will be

explained subsequently.

Thus, through nutrigenetics we know that our behaviors may interact with our genes and thus

decrease the deleterious effect of one specific risk variant.

In other words, we can change our obesity predisposition: although we cannot change our

genetic code, we can definitely change our behaviors.

From epigenetics, the message is even more positive: by changing our behaviors, we can

even change our genome.

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For example, we demonstrated that DNA methylation levels at different CpG sites of CLOCK

are higher in obese than in nonobese women, and these methylation levels were associated

with several obesogenic behaviors such as snacking frequently, eating when bored, and eating

from large packages.

Therefore, we presume from these data that through some small but stable changes in eating

behaviors, we can change DNA superstructure and consequentially gene expression, thus

changing our destiny.

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How several impairments in the central clock are related directly with obesity. In humans, mutations are rare; these impairments are

caused by human genetic variants (SNPs). Thus, these human genetic variants may be associated directly with obesity or different behaviors. Moreover, these

genetic variants may interact with different behaviors to influence obesity. We can consider several solutions: (A) changes in behaviors directly related to a decrease

in obesity and an increase in weight loss; or (B) through genetics, by changes in our behaviors that may interact with SNPs to produce a decrease in obesity and

weight loss. Two typical examples are shown of how behaviors may influence genetic variants. Panel A shows an example of genetic association between the genetic

variant of CLOCK rs37499474 and energy intake, where TT carriers exhibit a greater energy intake than AA carriers. Panel B represents an example of geneediet

interaction for insulin resistance. It is remarkable that when subjects are CC carriers of CRY1 rs2287161 and also eat high amounts of carbohydrates, their insulin

resistance (HOMA) is higher. However, among G carriers (GG þ GC), values of HOMA are independent of carbohydrate intake and remain constant. CH,

cholesterol; CLOCK, Circadian Locomotor Output Cycles Kaput; MUFA, monounsaturated fatty acids; PER2, Period 2; SFA, saturated fatty acids.

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Advice to Prevent Genetic and Behavioral Associations and Interactions for Obesity that may be Crucial for Genetically Informed

Personalized Nutrition in Obesity Treatment.

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Advice to Prevent Genetic and Behavioral Associations and Interactions for Obesity that may be Crucial for Genetically Informed

Personalized Nutrition in Obesity Treatment.dcont’d

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Effect of Different Behaviors in Methylation Levels of CLOCK CpG1, Showing Importance of how we eat.

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How eating late affects the metabolic characteristics of lean women. After 1 week of eating late, their metabolic pattern was similar to that

of obese women.

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Specific Recommendations for Preventing Obesity and Metabolic Syndrome by Improving Circadian System Health, Based on Available

Scientific Evidence Proved in (A) Epidemiological, Clinical, and Experimental Animals (A); and Based on (B) Interventional Studies in

Humans or in Animal Models and (C) Preliminary Studies or a Low Number of Studies.

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Gene-Diet Interactions and Cardiovascular

Diseases: Saturated and Monounsaturated Fat

Nowadays, the cholesterol in the diet has been almost exonerated as a significant driver of

circulating blood cholesterol.

Concomitantly, the once apparently stable connection between dietary fat and mainly SFAs

and CVD appears to be deteriorating as the result of mounting evidence casting doubt about

previous beliefs.

Thus, whereas the Dietary Guidelines for Americans 2015e20, and several organizations,

steadily recommend limiting intake of SFAs to approximately and sometimes, unexpected

effects.

A classic example of this exchange took place a few decades ago with the replacement of

high trans fatty acids margarine for the traditional butter.

It is humbling to recognize that concerns similar to those present in contemporary nutrition-

related literature were already being articulated by some of the most knowledgeable nutrition

scientists of the late 1940s and early 1950s. 80

Of particular interest is the recognition by some that the association between dietary factors

such as cholesterol or SFAs and plasma cholesterol levels and CVD may be present in some,

but not in all, individuals.

Monounsaturated fatty acids (MUFAs) have endured through history a more lenient judgment

than SFAs.

Whereas it is true that MUFAs were found guilty by association during the prosecution against

total dietary fat, in general, they were accepted as a health-neutral or even health-positive fat

as a component of the Mediterranean diet.

Nevertheless, similar to SFAs, their effect on lipid metabolism and other cardiovascular risk

factors show significant interindividual variability.

These observations highlight the need for more personalized or precision nutrition.

Therefore, some of the nutrition research of the last 2 decades has shifted toward the

identification of the factors underlying those interindividual differences in response to dietary

fats and cholesterol, with greater emphasis placed on the search for genetic factors.

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Progress in this area has been made possible by the increased understanding of the genetic

architecture of complex, common diseases.

However, despite the knowledge accumulated, many of the questions remain unanswered,

and some of them may be concealed as part of gene-environment (GxE) interactions.

In this regard, a few years back, we mined the scientific literature to collect information about

such interactions from almost 400 publications for CVDrelated traits (e.g., blood lipids,

glycemic traits, obesity anthropometrics, vascular measures, inflammation, and metabolic

syndrome (MetS)), and generated the CardioGxE.

The synthesis of this information revealed that the CardioGxE SNPs showed little overlap with

variants identified by main-effect genome-wide association studies (GWAS), indicating the

importance of environmental interactions with genetic factors on cardiometabolic traits.

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Moreover, comparison to gene networks responding to plasma cholesterol-lowering or

regression of atherosclerotic plaques showed that GxE genes have a more significant role in

those responses, mainly through high-energy diets and fat intake, than do GWAS-identified

genes for the same traits.

In this work, the focus will be on the interaction between genetic variants, SFAs, MUFAs, and

cardiovascular risk factors.

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Windows of susceptibility to nutritional exposures during intrauterine development and consequences in the offspring.

84

Dietary factors that affect gene expression and regulate some of the molecular mechanisms involved in controlling body weight and

insulin sensitivity.

85

Integration of different “omics” technologies that, in addition to more conventional biomarkers, will be useful in implementing

precision nutrition.

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Epigenomic mechanisms involved in the promotion and prevention of carcinogenesis. Nutrients and biofactors may contribute to the

prevention of carcinogenesis by regulating the epigenetic machinery. This regulation can modify the activity of

procarcinogenicrelated gene transcription. Therefore, the analysis of epigenomic or transcriptomic biomarkers provides insights for

precision medicine in counteracting cancer onset and progression. CpG, cytosine-phosphate-guanine; lncRNA, long noncoding

RNA; miRNA, microRNA.

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References1. Principles of Nutrigenetics and Nutrigenomics: Fundamentals of

Individualized Nutrition: by Raffaele De Caterina, J. Alfredo Martinez,

Martin Kohlmeier 2020

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