Metabolic disorders linked to obesity represent a major health problem and a major part of the health costs worldwide. Obesity is a strong predictor of metabolic syndrome and insulin resistance. Insulin resistance is defined as inhibition of insulin stimulation of metabolic pathways, including lipid homeostasis. Both obesity (i.e. excess of adipose tissue) and ectopic fat deposition are closely associated with insulin resistance. The accumulation of excess lipids or their metabolites/derivatives (i.e. lipotoxicity hypothesis) in organs decreases insulin signalling and activate pro-inflammatory in liver, skeletal muscle and heart. On the other hand, insulin resistance is closely associated with dysregulation of fatty acid (FA) metabolism. The consequences of hepatic or cardiac steatosis are non-alcoholic fatty liver disease (NAFLD) and lipotoxic cardiomyopathy, respectively. How obesity promotes these visceral complications is not completely understood. The influence of excessive intake of macronutrients deserves much more attention than deficient intake of micronutrients in epidemiological and experimental studies, and the combined influence of global overnutrition and selective deficit in a category of micronutrient remains an open question. An increasing number of these studies have highlighted the influence of nutrients on the expression of genes regulating metabolism through a body of knowledge known as ‘nutrigenomics’.

As recently reviewed [48, 59], genetics, epigenetics, epigenomics and environment interact in defining the individual risk of obesity and related diseases. Epigenetics and epigenomics refer, respectively, to heritable and non-heritable (e.g. dependent from environment) changes of gene expression, which are caused by mechanisms such as DNA methylation, histone acetylation/methylation, dysregulation of gene transactivation and synthesis of miRNA. The influence of genetics is illustrated by the twofold to threefold increased disease risk in offspring from parents with obesity and type 2 diabetes mellitus (T2DM) in European populations [115]. Studies in twins undergoing overfeeding also suggest a strong influence of heritability [21]. Beside genetics, lifestyle and environmental factors clustered within families determine dietary preferences and food availability, two factors that may trigger epigenomic mechanisms. Barker’s concept of ‘foetal programming’ [10], also named developmental origins of health and disease, proposes that unfavourable intrauterine life, including intrauterine growth restriction (IUGR), predicts the risk of postnatal metabolic complex diseases through a relationship that may be further modified by the postnatal environment [179]. Environmental conditions of IUGR include nutritional stress such as protein and/or energy restriction [59, 88]. Recently, experimental and epidemiological studies have also highlighted the involvement of deficiencies in methyl donors during and after intrauterine life.

In this review, we will compare the epigenetic, epigenomic and nutrigenomic influences of three prevalent conditions of imbalanced intake of nutrients, overfeeding, dietary restriction (with a particular interest for proteins and amino acids) and deficit in methyl donors during pregnancy and early postnatal life and their consequences on the risk of obesity and related metabolic disorders in the liver and the heart. We will devote a specific interest for the mechanisms related to the influence of these imbalanced intakes on deregulation of FA metabolism.

The abnormal distribution and storage of fat influences the liver and heart complications of metabolic syndrome and insulin resistance

Obese individuals vary in their body fat distribution, their metabolic profile and the degree of associated cardiovascular and metabolic risks. As far as adipose tissue distribution is concerned, obesity consists of different subtypes or phenotypes with different metabolic profiles [141]. The mechanisms at the origin of the phenotype of metabolically healthy but obese subjects are poorly understood. Recent studies have confirmed that the combined phenotype of decreased lean body mass and central obesity reflects insulin resistance and indicates a high risk of diabetes mellitus [96] in Western and Asian countries. Metabolic syndrome related to central obesity may be viewed as ‘unhealthy obesity’ and the concerned individuals as metabolically abnormal obese patients [17, 140]. More recently, an acquired type of partial lipodystrophy closely linked with the severity of metabolic complications over a wide range of body mass index (BMI) values has been described in overweight adults, children, adolescents and elderly persons [162]. As described for the genetic type of partial lipodystrophy (e.g. Dunnigan syndrome), this phenotype is characterised by partial loss of peripheral fat and accumulation of fat in the trunk while the clinical presentation is less severe.

Many studies support the concept of a major role for intra-abdominal fat accumulation (visceral adipose tissue [VAT]) in the link between abdominal obesity and insulin resistance. Visceral hypertrophic adipocytes and VAT-resident macrophages produce pro-inflammatory cytokines like tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). VAT is also pathogenic because its venous blood drainage is direct via the portal vein to the liver. As a consequence of enlarged visceral adipocytes, the flux of free fatty acids (FFA) and pro-inflammatory cytokines contributes to hepatic insulin resistance and fat deposition in the liver (portal hypothesis of insulin resistance) [15, 92] and to NAFLD in obese patients [53]. FFAs promote insulin resistance by increasing hepatic glucose production. On the other hand, the production of pro-inflammatory cytokines by the VAT and circulating levels of IL-6 accelerate the subsequent transition to non-alcoholic steatohepatitis (NASH) and fibrosis [53]. Excess VAT could be also a surrogate marker of metabolic syndrome. The preferential accumulation of fat in the intra-abdominal cavity could be viewed as the consequence of an ectopic deposition [42]. According to this alternative hypothesis, increased visceral fat mass may represent a marker of the inability of patients with insulin-resistant obesity to store fat in subcutaneous adipose tissue (SAT). When the storage capacity of adipose tissue is exceeded, then a continuous exposure to FAs, not counterbalanced by FA oxidation, will lead to an accumulation of FFAs and induces a lipid ‘spill over’ to other tissues.

The impaired fatty acid oxidation is a major facet of the alterations of lipid metabolism in metabolic disorders related to obesity

FFAs meet all the criteria to be a metabolic factor related to insulin resistance in human subjects. Plasma FFA levels are elevated in most obese subjects and will inhibit the antilipolytic action of insulin, which will further increase FFAs release into the circulation [86]. Most studies have devoted attention to stressed adipose tissue releasing more FFAs, but a decreased rate of mitochondrial FA oxidation is another potential mechanism to be considered [127]. A substantial part of the insulin resistance-lowering activity of thiazolidinediones can be attributed to their lowering of plasma FFA levels through increasing FFA oxidation [18]. Several mechanisms grouped in the ‘lipid metabolite hypothesis’ have been proposed to explain how obesity and/or FFAs can interfere with insulin signalling. An increase in plasma FFA concentration results in intramyocellular and intrahepatic accumulation of triglycerides, long-chain acyl-CoAs and diacylglycerol (DAG) [18]. DAG is a potent activator of conventional and novel protein kinase C isoforms. In vivo, plasma FFA levels activate NF-κB in skeletal muscle and result in increased expression of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and MCP1. Possible mechanisms through which FFAs and the subsequent NF-κB activation and cytokine production can result in insulin resistance include activation of JNK by cytokines and induction of the suppressor of cytokine signalling, which can interfere with the binding of IRS1/2 to the insulin receptor [100, 101].

Influences of nutrition and metabolism on the epigenome: a facet of the mechanisms that may influence foetal programming and obesity

Foetal programming can produce heritable and non-heritable changes in adiposity through epigenetic and epigenomic mechanisms due to DNA methylation or histone modification of DNA in gene regulatory regions. The emerging level of evidence in human population studies contrasts with the number of arguments that sustain this hypothesis in experimental models [31].

DNA methylation by DNA (cytosine-5)-methyltransferases (DNMT) occurs at cytosine residues in CpG dinucleotides in vertebrates [140]. The methylated CG is symmetrically paired with the same sequence on the opposite DNA strand, which means that, after DNA replication, sites are transiently methylated in only one of the two strands. Although the pattern of 5-methylcytosines in the genome of differentiated somatic cells is quite stable, during early development, DNA methylation plays a critical role in the control of cell processes including embryonic development, transcription, X chromosome inactivation and genomic imprinting. Histone modifications influence DNA replication, repair and condensation and gene expression. The post-translational covalent modifications of histones include acetylation, methylation, phosphorylation and ubiquitination. The methylation and acetylation of histones are dynamic processes, which depend, respectively, on balances between histone methyltransferases (HMTs) and histone demethylases (HDMs) and between histone acetyltransferases and three classes of histone deacetylases (HDAC). The methylations and acetylations of histone residues obey to inter-histone and trans-histone regulatory pathways forming a ‘histone code’. Lysine methylation displays a high degree of complexity since each lysine residue can be mono-methylated, di-methylated or tri-methylated and each site of methylation can influence gene activity independently. Arginine residues can be mono-methylated or di-methylated symmetrically or asymmetrically [140].

Folate and vitamin B12 (cobalamins [Cbl]) are supposed to play a key role in epigenomic dysregulations of metabolism [64, 65]. These two nutritional methyl donors are needed for the endogenous synthesis of methionine by methionine synthase (MS). The homeostasis of methionine is crucial in cell life, since it is the amino acid required for the translation initiation and the immediate precursor of S-adenosyl methionine (SAM) [56]. They play a significant modulating role on DNA methylation through their influence on the cellular content of SAM, which is the universal methyl donor in transmethylation reactions [64, 65]. The cellular deficit in Cbl and Me-THF leads also to decreased synthesis of methionine and accumulation of homocysteine, which produces cellular stress by mechanisms largely documented in the literature [84, 177]. The role of SAM as methyl donor used by HMTs and DNMTs explains the influence of the deficiency in dietary methyl donors on histone and DNA methylation in rats [25, 132]. The importance of vitamin B12 and folate for maintaining an adequate cellular content of SAM is illustrated by the consequences of their deficiency on the cellular concentration of SAM, S-adenosyl homocysteine (SAH) and SAM/SAH ratio [61, 62]. The subsequent decreased SAM/SAH ratio impairs the cell capacity to ensure the transmethylation reactions of co-regulators of nuclear receptors (NRs), which play a key role in epigenetic and epigenomic mechanisms [65]

Beside dietary methyl donors, there are also nutrigenomic and epigenomic crosstalks between intermediate products of the metabolism of macronutrients and coenzymes derived from micronutrients [88]. The methylation and demethylation of DNA and histones depend on the cellular availability in coenzymes and substrates involved in energy metabolism and one-carbon metabolism (OCM). ATP is required for the synthesis of SAM. AMP-activated protein kinase (AMPK) senses nutrient deprivation through increased AMP levels, activating catabolic pathways to restore ATP levels and to increase mitochondrial biogenesis [33]. The macronutrient intake modifies the cellular content of nicotinamide dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), which act as metabolic sensors of epigenomic regulations through demethylation and deacetylation of histones [165]. There are two types of HDMs, the LSD1-containing domain demethylases and the JumomjiC (JmjC) domain containing demethylases [165]. The LSD1-containing domain HDM is regulated by FAD, while the activity of JmjC-containing domain HDM depends on alpha-ketoglutarate, which is produced from glutamate in the Krebs cycle [169] and from the vitamin B6-dependent cystathionine γ-lyase in the transsulfuration pathway of homocysteine, in the OCM [65]. The loss of function of Jhdm2a is associated with reduced expression of peroxisome proliferator-activated receptor alpha (PPAR-α) and medium-chain acyl-CoA dehydrogenase [164]. NAD+ is required for activation of the class III sirtuin (SIRT) HDACs. The deacetylation of histones catalysed by SIRT leads to the cleavage of NAD+ into nicotinamide and 1-O-acetyl-ADP-ribose [34]. Butyrate, a short-chain carboxylic acid (C4) produced by bacterial flora and the intermediate metabolism of macronutrients and, to a lesser extent, acetate (C2), propionate (C3), valerate (C5) and caproate (C6), inhibits the HDAC of classes I and II [43].

DNA methylation changes during lifetime in function of environmental factors that include nutrition. This is illustrated by studies in humans and in distinct animal models, such as rodents and insects. The methylome of monozygotic twins becomes divergent with age [57]. Both restriction and overnutrition diets are related to changes in DNA methylation in the few epigenome-wide association studies that have investigated foetal programming-related risk for T2DM and obesity. In particular, the study of individuals who were prenatally exposed to famine during the Dutch Hunger Winter in 1944 has provided pertinent findings in favour of epigenomic mechanisms [74]: Six decades later, these individuals exhibited obesity associated with decreased DNA methylation of the imprinted IGF2 gene, as compared to the same-sex siblings without exposure to prenatal malnutrition. Individuals with periconceptional exposure to famine had lower methylation of the INSIGF gene and higher methylation levels for several other genes, including IL10, LEP and ABCA1 [167]. The hypocaloric diet increases methylation in three loci of DNA from SAT in overweight or obese postmenopausal women [22]. This diet also increases methylation at two loci in DNA from peripheral blood mononuclear cells in overweight or obese men [118]. Recently, a meta-analysis of genome-wide association studies has highlighted the rs7202116 at the FTO gene locus to be correlated with BMI [185]. A role for epigenomics was suggested by the influence of methylation of a CpG site in the first intron on the association of the FTO risk allele with BMI. However, the causality role of this methylation remains unclear [3, 13]. The FTO protein has a demethylase activity on N 6-methyladenosine of nuclear RNA, but whether this metabolic function is related to the observed effects on BMI remains an open question [87]. The leptin-responsive preproopiomelanocortin (POMC) gene is a hypothalamic regulator of energy balance. A hypermethylation variant targeting individual CpGs at the intron 2–exon 3 boundary of POMC is associated to obesity in children, in another case–control study. The hypermethylation of exon3 reduces the expression of POMC through reduced binding of the transcription enhancer P300 [99]. The dynamic effect of obesity on the epigenome is also illustrated by an altered promoter methylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and pyruvate dehydrogenase kinase isozyme 4 in skeletal muscle from adult obese subjects, which is restored to non-obese levels after weight loss induced by by-pass surgery [11]. In this study, the genome-wide DNA methylation analysis of skeletal muscle showed also that obesity is associated with hypermethylation at CpG shores and exonic regions close to transcription start sites [11]. Consistently, sedentary lifestyle and lack of physical activity may modify epigenetic programming and predisposition to obesity and T2DM, as recently reviewed [94]. In particular, they result in decreased expression of the mitochondrial and glucoregulatory genes PGC-1α, hexokinase 2 and ATP synthase subunit beta through epigenomic mechanisms that involve the activation of HDAC family members in skeletal muscle [94].

Epigenome-wide studies performed in insects and rodents under specific diets have also found changes in DNA methylation. An intriguing model is provided by honeybees. Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly, a mixture including proteins, sugars and FAs. Dnmt3 small interfering RNA-treated individuals emerged as queens with fully developed ovaries. DNA methylation by Dnmt3 triggers epigenetic information differentially altered by nutrition, with implications in development, reproduction and behavioural status of honeybees [55, 75, 98].

High-fat diet and foetal programming

The role of deacetylase sirtuin 1 and peroxisome proliferator-activated receptor-γ coactivator 1α in metabolic dysregulations

Maternal obesity and high-fat diet during pregnancy increase the risk of obesity in the offspring through complex mechanisms, which involve metabolic deregulation and alteration of food intake behaviour [82]. Paternal obesity has also to be considered. For example, paternal high-fat diet alters the expression of genes related to ATP binding and calcium-, MAPK- and Wnt-signalling pathways and to apoptosis and cell cycle in the pancreas of rat F1 female offspring. This suggests a role for epigenomic modifications of sperm produced by nutritional status, which continues after epigenetic reprogramming during the meiosis and first post-zygotic division [123].

Epidemiological studies have found an association between maternal overfeeding and chronic diseases in offspring, when they reach adulthood [174]. Worldwide prevalence of childhood obesity has increased greatly during the past three decades. The increasing occurrence of metabolic syndrome in children and adolescents and of type 2 diabetes in young adults is believed to be a consequence of this epidemic obesity. The pandemic obesity is largely related to easy access to energy-dense cheap foods and decreased energy requirements in daily life [175]. Sirtuin 1 (SIRT1), PGC-1α and the related downstream pathways play a major role in these deregulations [85, 146, 185]. The deacetylation of PGC-1α by SIRT1 increases, while its acetylation by general control non-derepressible 5 (GCN5) inhibits its activity [145]. High-fat feeding produces hyperacetylation of PGC-1α and a down-regulated expression of PGC-1α-dependent genes through increased expression of the two acetylases SRC-3 and GCN5 and decreased expression and activity of SIRT1 [32, 39]. These changes are consistent with the hyperacetylation of PGC-1α and the down-regulated expression of PGC-1α-dependent genes in mice lacking SRC-3 (Fig. 1) [39]. SIRT1 deacetylation also plays a key role in metabolic homeostasis through the regulation of the activity of Foxo transcription factors [26] and acetyl-CoA synthetases [70]. Consistently, the treatment of mice fed a high-fat diet with resveratrol and other SIRT1 agonists prevents weight gain and insulin resistance [100, 122].

Fig. 1
figure 1

Comparison of the effects of high fat/overfeeding and MDD. PGC-1α is a master regulator of lipid metabolism and FA oxidation. It is phosphorylated and acetylated under the control of AMPK, GCN5 acetylase and SIRT1 deacetylase. High-fat diet and overnutrition decrease the activity of AMPK through high intracellular ATP levels, leading to decreased phosphorylation of PGC-1α. It produces hyperacetylation of PGC-1α through increased expression of GCN5 and decreased expression and activity of SIRT1. MDD in rat pups born from mothers subjected to MDD during pregnancy and lactation produces similar consequences on SIRT1 expression and PGC-1α acetylation in the heart. In addition, MDD decreases the expression of PRMT1 and the level of SAM, leading to decreased methylation of PGC-1α in the heart and liver

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High-fat diet during pregnancy influences food intake behaviour in offsprings

Aberrant expression of genes that control the feeding behaviour may contribute to the development of obesity-related diseases. Maternal preference for macronutrient and energy intakes during pregnancy rather than paternal preference is associated with fat preference in the child [24]. The control of appetite and energy balance is regulated by peripheral signals, including glucose, leptin, insulin and gut hormones through their effects on hypothalamic neuropeptides mostly expressed in the arcuate nucleus. High-fat diet and/or maternal obesity increase neurogenesis and the production of hypothalamic neuropeptides that affect food intake, such as neuropeptide Y and POMC [36, 37, 124]. A recent experimental study performed with mice showed that maternal high-fat diet produces offsprings’ epigenetic marks with long-term alterations in expression of dopamine and opioids-related genes [173]. The neonatal overfeeding in rats also produces epigenomic DNA marks in the hypothalamus, with increased methylation of the promoters of POMC and insulin receptor [136, 137]. Leptin is another candidate, which is secreted almost exclusively by adipocytes and exerts a pivotal role in energy homeostasis and regulation of food intake. The high-fat diet produces changes of methylation at specific CpG sites within the leptin promoter, with consequences on circulating leptin levels [119]. Consequently, POMC is not up-regulated in these rats despite hyperinsulinaemia and hyperleptinaemia [136].

Consequences of high-fat diet during pregnancy on offspring’s liver

High-fat diet produces hepatic steatosis and NAFLD through complex mechanisms triggered by insulin resistance, which include the imbalance between uptake and de novo lipogenesis of non-esterified FAs and triglyceride, disposal by FA oxidation and secretion of triglyceride-rich lipoproteins [92]. The liver is subjected to epigenomic changes produced by high-fat diet during the in utero and postnatal periods, which participate in these mechanisms. In non-human primates, maternal high-fat diet during pregnancy increases H3K14 and H3K18 acetylation in offspring, with locus-specific effect on glutamic pyruvate transaminase 2 [1]. It increases the foetal H3K14ac, in relation with decreased SIRT1 expression and activity [159]. In rats, 2-day-old offspring of mothers fed a high-fat diet display hepatic cell cycle inhibition in relation with changes in DNA methylation and gene expression of the cyclin-dependent kinase inhibitor 1A [49], an alteration which may potentially contribute to NAFLD. Maternal high-fat diet impairs the activity of mitochondrial complexes I–IV and carnitine palmitoyltransferase 1A and up-regulates the hepatic lipogenesis in adult offspring. Consistently, high-fat diet during pregnancy and lactation alters hepatic expression of PPAR-α, insulin like growth factor-2 and microRNAs, including miR-122, miR-192, miR-194, miR-26a, let-7a, let7b and let-7c, miR-494 and miR-483, in adult offspring [186, 187]. High-fat feeding results in the up-regulation of miR-143 and down-regulation of miR-27a in mouse mesenteric adipose tissue through altered expression of PPARs [114]. High-fat diet produces also transgenerational and sex-dependent effects in mice. The male offspring of the F2 generation develop obesity and hepatic steatosis earlier and more severely than the F0 and F1 generations. The transgenerational changes in liver include enhanced lipogenesis and endoplasmic reticulum (ER) stress with increased expression of fatty acid synthase (FAS), SREBP, LXRα and β and ERO1-α. These effects play a key role in NAFLD and insulin resistance [52] and are related with epigenomic changes on histone methylations and reduced binding of methylated histones in LXRα and ERO1-α gene promoters [104].

Consequences of high-fat diet during pregnancy on offspring’s myocardium

Whether the deleterious effects of high-fat intake and caloric excess on the myocardium are independent from those related to the consequences of obesity and insulin resistance on endothelial dysfunction, hypertension and hyperlipidemia is a matter of debate in population studies and experimental models. The fat-enriched diet induces insulin resistance, inflammation and apoptosis, and these manifestations may also contribute to the cardiac hypertrophy. High-fat feeding of Wistar rats produces cardiac contractile dysfunction, which enhances CD36-mediated FA uptake and myocardium triacylglycerol accumulation [129]. Cardiac changes leading to cardiac lipotoxicity in rodents include mitochondrial dysfunction and impaired biogenesis, alteration of reactive oxygen species (ROS) production and increased lipid oxidation [188]. In obese Zucker rats, the high-fat ‘Western diet’ increases cardiac triglyceride content within a week, while long-term exposure promotes FA oxidation, which correlates with the prevention of further deterioration in cardiac function and triglyceride accumulation [30]. In response to high blood concentration of long-chain FAs, the heart is forced to increase FA uptake through increased expression of the FA transporter CD36, resulting ultimately to diabetic cardiomyopathy [45]. Then, this leads to the further increase of FAs and lipid intermediates such as DAG and ceramide and the increased production of ROS, with a profound impact on signalling pathways [45]. High-fat diet leads to glucose intolerance, elevated insulin plasma levels and reduced insulin-like growth factor 1 (IGF1) levels in mice. These changes may contribute to disrupting the phosphorylation of insulin receptor and post-receptor signalling molecules such as insulin receptor substrate 1 (tyrosine/serine phosphorylation), forkhead transcriptional factors and mTOR (target of rapamycin) [188].

PPARs play a master role in myocardium deregulations of FA uptake and mitochondrial β-oxidation [171, 176]. In particular, PPAR-α up-regulates the expression of FA transporters and induces the synthesis of metabolic enzymes of the β-oxidation pathway [171]. However, the role of the impaired expression PPAR-α in the metabolic effects of high-fat diet on the myocardium is questioned in part by the results obtained with KO mice. This animal model accumulates myocardial lipids through lower FA oxidation rate but does not produce high fat-induced insulin resistance, suggesting the contribution of other regulators [134, 177]. PPAR-γ seems to have a limited role in regulating cardiac metabolism, probably due to its low myocardial expression. Treatment of wild-type and PPAR-γ KO mice subjected to high-fat diet with PPARs agonists, rosiglitazone, fenofibrate and pioglitazone, indirectly protects the myocardium from lipid accumulation and lipotoxic damage by reducing FAs delivery to the heart. It also produces a shift of myocardial substrate utilisation towards greater contribution of glucose [9].

The influence of high-fat diet on PGC-1α down-regulation and the subsequent consequences in mitochondrial dysfunctions have also been proposed to play a major role in the mechanisms that relate obesity with heart failure [171]. High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction, up-regulates the forkhead transcription factor Foxo3a and suppresses mitochondrial aconitase activity without affecting the expression of SIRT1 [143]. However, SIRT1 seems to play an adaptive role since the myocardium stimulation of SIRT1 extends the life span of mice fed a high-fat diet and retards aging in the heart [183, 184]. Another deacetylase, HDAC3, may contribute to the epigenomic mechanisms underlying the deleterious influence of high-fat diet on the myocardium [158]. Embryonic deletion of myocardial Hdac3 in mice causes a major cardiomyopathy that reduces survival. When excision of Hdac3 in the heart is produced during late development, the switch to a high-fat diet leads to severe hypertrophic cardiomyopathy and heart failure, with down-regulation of myocardial mitochondrial genes, specifically those involved in lipid metabolism, suggesting that HDAC3 is important in maintaining proper mitochondrial function [158].

Protein restriction and foetal programming

The Dutch Hunger Winter of 1944 has provided a well-characterised population adapted for studying the relations between food restriction and foetal programming in humans. In the related Dutch cohort, pregnant mothers and foetuses have experienced malnutrition during the first, second or third trimesters of pregnancy. The mother’s food rations were known and the follow-up medical histories of foetuses could be collected in national population registers. This has permitted highlighting the associations between food restriction, IUGR and increased risks of metabolic syndrome, diabetes and cardiovascular diseases [110, 147, 148] and studying the epigenomic changes related to these associations [167]. Beside these population studies, animal models of food and protein restriction have been used to dissect the molecular mechanisms, which underlie the association between foetal growth retardation and metabolic and visceral consequences. In rodents, protein restriction during pregnancy and lactation results in reduced pancreatic β cell mass at birth and reduced insulin secretion in later life [44, 135, 163]. The transcriptional alterations induced by protein restriction during pregnancy rely mainly on transient transcription factor-dependent deregulations, with strong specific transcriptional changes in foetal tissue that cluster the liver with the heart [170]. The vast majority of studies have compared the 20–18 % casein diet (control situation) to 10–8 % casein diet. Methionine content is low in casein diets, and these experimental protocols raise the question of limiting the intake of methionine, which is insufficiently considered.

Dietary protein restriction in insect and rodent models has uncovered a likely role for key nutrient sensors, including mTOR, IGF1, insulin, AMPK, SIRT1 [152] and PGC-1α [51]. The effects of dietary restriction on PGC-1α regulation mirror those of the high-fat diet. Decreased acetylation of PGC-1α results from decreased expression of the two acetylases SRC-3 and GCN5 and increased expression of SIRT1 [32, 39]. The royal jelly diet induces queen bee differentiation through the influence of mTOR and IGF1/insulin pathways, with consequences on DNA methylation [125].

Protein or calorie restriction during pregnancy favours hyperphagia in offspring

The protein or calorie restriction during gestation and/or suckling of rodents produces hyperphagia. Low-protein diet throughout pregnancy and lactation reduces the anorexic effects of serotonin (5-HT) in malnourished rats through a desensitisation of 5-HT(1B) receptors. Pups born from protein-restricted dams and treated with dl-fenfluramine exhibit hyperphagia, at least partially related to a reduced regulatory function of 5-HT [108]. The hypothalamus is one of the brain areas affected by the programming effect of protein restriction. Perinatal protein restriction permanently alters the expression of two gene clusters, the first one regulating insulin signalling and nutrient sensing and the second a functional network of NRs and co-regulators of transcription involved in the use of lipids as fuel and in metabolic regulation by the circadian clock [128]. Consistently, giving the restricted diet at the same time each day changes the daily profiles of PERIOD1 and PERIOD2 protein expression in the brain nucleus of rats [172]. Mice born to mothers fed a low-protein diet have lower body weight and adiposity and higher food intake than animals born to mothers fed a control diet. These modifications persist throughout life and are associated with methylation changes in leptin gene, as observed in rats subjected to high-fat diet. This nutritional stress results in lesser methylation of CpGs located in the promoter of leptin, causing a permanent modification in the dynamics of leptin expression, with stronger induction in response to a meal [90]. The low-protein diet in pregnant rats may have subsequent effects on feeding behaviour of offspring through alteration in the expression level of the genes encoding Dnmt1 and Dnmt3a and in methylation status of promoters of genes encoding orexigenic and anorexigenic hypothalamic neuropeptides, including neuropeptide Y, proopiomelanocortin and leptin [41].

Consequences of calorie or protein restriction during pregnancy on offspring’s liver

Caloric restriction causes epigenetic and epigenomic changes that contribute to an increased risk of insulin resistance and NAFLD. It produces changes in acetylation and methylation of histones and hypomethylation of gene promoters with consequences that include reduced expression of the GLUT4 glucose transporter in rat skeletal muscle [142] and it increases foetal liver phosphoenolpyruvate carboxykinase (PEPCK) in primate [126]. The increased expression and activity of PEPCK in the liver promote gluconeogenesis and, consequently, the induction of insulin resistance. In contrast, hepatic PEPCK activity is reduced in female offspring of dams fed a protein restriction diet [143]

The effects of caloric restriction are targeted by deacetylases [155]. Low birth weight foetuses from pregnant dams fed a restricted diet have an increased prenatal hepatic SIRT1 activity. Low birth weight newborn offspring undergo postnatal suppression of hepatic SIRT1 and AMPK activities in conjunction with increased lipogenesis, decreased lipolysis and increased fat stores [180]. Moreover, transgenic mice overexpressing Sirt1 exhibit metabolic and physiological effects mimicking those induced by dietary restriction [8]. Moderate energy restriction seems to produce distinct results influenced by sex, with lower hepatic expression of SIRT1, sterol response element binding protein 1c (SREBP1c) and stearoyl-CoA desaturase-1 in males and unchanged levels in females, compared to controls [130]. SIRT3 is also a prominent regulator of the mitochondrial adaptation to caloric restriction by coordinately deacetylating proteins involved in mitochondrial maintenance [73]. SIRT6 is significantly decreased following dietary restriction, and this may contribute to the increased acetylation at lysine residue 9, 27 and 56 in H3 [91].

The low-protein diet in maternal rats produces epigenomic hallmarks of NAFLD in foetuses and offsprings [144]. However, the deregulation of imprinting through a general effect on the methylation of differentially methylated regions (DMRs) is unlikely to be involved in developmental programming. Indeed, although expression of some imprinted genes in the liver of offspring is sustainably changed, the methylation of DMRs that control their monoallelic expression remains largely unaltered [83]. Protein restriction during pregnancy induces hypomethylation of PPAR-α and glucocorticoid receptor (GR) promoters and increases expression of GR and PPAR-α in liver of juvenile and day 80 adult offspring [27, 29, 106]. The altered expression of GR, PPAR-α and hepatic nuclear factor 4 (HNF-4) deregulates lipid oxidative metabolism and promotes liver steatosis and insulin resistance. These alterations have an epigenetic characteristic with transmission to future generations [29]. Protein restriction alters the epigenetic regulation of GR through reduced Dnmt1 expression in the offsprings [107]. Other changes include hypomethylation of specific cytosines in the PPAR-α promoter and in offspring’s liver [107] and repressive changes in histone modifications at the cholesterol 7alpha-hydroxylase promoter through decreased hepatic expression of Jmjd2a demethylase in foetal liver [154]. Transgenerational effects of protein restriction have been studied in rats and mice. Offspring from protein-restricted F0 rats show altered fasting glucose homeostasis and changes in PEPCK promoter methylation and expression in the three F0, F2 and F3 generations [79]. The protein restriction in F0 produces also a lower promoter methylation of PPAR-α and GR in the liver from F1 and F2 generations [27]. Similarly to what has been observed on high-fat models, the father’s restriction diet may also produce transgenerational effects, with elevated hepatic expression of genes involved in lipid and cholesterol biosynthesis in offspring. Epigenomic profiling of offspring livers revealed changes in cytosine methylation depending on paternal diet, including changes in methylation over a likely enhancer for PPAR-α [35].

Consequences of caloric or protein restriction during pregnancy on offspring’s myocardium

In contrast to the deleterious effects of high-fat intake and overnutrition, long-term caloric restriction leads to decreased cardiac triglyceride content in obese type 2 diabetic subjects [53, 71] and protects healthy individuals against age-associated decline in diastolic function [117]. However, little is known about the long-term effects of the intrauterine exposure to protein and/or caloric restriction in population studies and, in particular, the molecular and metabolic changes in the myocardium [54, 151]. Long-term caloric restriction decreases left ventricular wall thickness and cardiomyocyte cell length and increases cardiac autophagy in C57BL/6 mice. At the molecular level, it increases the phosphorylation of Akt/glycogen synthase kinase-3β and AMPK/acetyl-CoA carboxylase [72]. Long-term caloric restriction of mice reduces triglyceride content in the heart, with a more dynamic pool in females than in males [7]. It maintains mitochondrial protein synthesis in the heart and skeletal muscle, an observation, which is consistent with an increased somatic maintenance [120]. In contrast, long-term caloric restriction of mice decreases ubiquinone Q9 and COQ transcript levels in heart [131].

Food and protein restriction deregulates fat and energy metabolism in the heart of pregnant rat. The methylation of PPAR-α promoter is decreased and mRNA expression of PPAR-α and carnitine palmitoyltransferase-1 is increased by maternal protein restriction diet in adult, but not in neonatal offspring [153]. Maternal protein restriction of rats during gestation increases the myocardial expression and deposition of collagens in day 70 offspring, compared to controls, in relation with changes in the expression patterns of pro-fibrotic genes and discrete structural abnormalities of heart [116].

Consequences of amino acid restriction during pregnancy

Beside the large number of studies devoted to the foetal programming effects of protein restriction, a lesser attention has been paid to the global or specific deficit in amino acids. Amino acid homeostasis depends on dietary intake of proteins and on the endogenous turnover of protein synthesis and degradation. Part of the mechanisms of foetal programming related to protein restriction may be, therefore, related to this altered homeostasis. Some of them may be explained by specific functions of amino acids [60]. For example, 5-HT is needed for the biosynthesis of tryptophan and glutamate is a neurotransmitter. The deficit in amino acids may also produce consequences through their role as intermediate substrates in many pathways of metabolism as exemplified by the role of methionine, glycine and serine in the OCM [65] and the role of alanine in neoglucogenesis [60].

Interestingly, tryptophan and methionine are the two least abundant essential amino acids in casein, the protein commonly used in the protein restriction diets used in experiments with rodents. Thus, the regimens using casein as the sole source of protein could produce effects related to inadequate intake of these two amino acids, including reduced adiposity, extended maximal longevity, increased resistance to acetaminophen toxicity in the liver and reduced insulin, IGF1 and thyroid hormone levels [121]. Methionine restriction contributes to adiposity resistance by altering the lipogenic/lipolytic balance [133], while leucine deprivation improves insulin sensitivity [180].

Two signal transduction mechanisms are involved in sensing intracellular deficit in amino acids [60]. GCN2 senses the absence of many amino acids, while the TOR pathway senses the presence of branched amino acids (leucine, isoleucine and valine), arginine and glutamine. Both repress general translation through distinct mechanisms. The signal-transducing kinase GCN2 binds to uncharged tRNAs, resulting in kinase activation and phosphorylation of eIF2α in the brain and liver, which leads to slow down translation of most mRNAs and to favour the translation of mRNAs containing specific regulatory elements in their 5′-untranslated regions [60]. TOR is a serine/threonine kinase that controls cell proliferation in response to adequate energy, nutrients and growth factors. The activation of TOR does not involve its binding to amino acids or surrogates such as tRNAs. Rag family of small GTPases is required for amino acid sensing by mTORC1 [60].

Part of the effects of protein restriction diet on food intake could be explained by the influence of amino acid deprivation in the brain. Leucine reduces food intake through activation of mTOR in the hypothalamus [40]; mTORC1 activation by amino acid restriction also results in changes in metabolic pathways such as the stimulation of glycolysis, pentose phosphate pathway and de novo lipid biosynthesis through the activation of hypoxia-inducible factor-1α, SREBP1 and SREBP2 [50]. Aversion to food intake can be produced by diets lacking essential amino acids through activation of GCN2 in anterior piriform cortex [63]. GCN2 can also influence fat metabolism. Expression of lipogenic genes and activity of FAS in the liver are repressed and lipid stores in adipose tissue are mobilised upon leucine deprivation in mice. In contrast, GCN2-deficient mice develop liver steatosis and exhibit reduced lipid mobilisation [69]. Leucine deprivation improves hepatic insulin sensitivity by sequentially activating GCN2 and decreasing mTOR/S6K1 signalling and by activating AMPK [180].

Methyl donors, foetal programming and manifestations of metabolic syndrome

Methyl donors could play a modulating role in complex metabolic diseases, according to epidemiological and experimental data. Deficiencies in folate and vitamin B12, and to a lesser extent in choline, are common during the gestational period in Western countries. The animal models with methyl donor deficiency (MDD) have a small birth weight and undergo altered expression of key regulators of metabolism and obesity-related diseases, such as SIRT1, PGC-1α and Mir-124 [2, 34, 65, 93]. Methyl donors play an essential role in the epigenetic and epigenomic mechanisms (methylation, acetylation) that influence gene expression and cell signalling. They produce IUGR with long-lasting consequences during development and throughout life [46, 65]. In this context, it is noteworthy to note that experimental cellular and animal models of MDD produce molecular hallmarks similar than those associated with overfeeding and obesity, such as decreased expression of SIRT1 and inactivation of PGC-1α through imbalanced acetylation/methylation (Fig. 1).

Methyl donors and foetal programming: the population-based studies

Recent large epidemiological studies have shown that vitamin B12 deficit during pregnancy is the most powerful micronutrient predictor for the risk of small birth weight and subsequent events related to foetal programming, including metabolic syndrome and insulin resistance. In contrast, the influence of folate status during pregnancy on foetal programming manifestations is less consistent. In India, babies are thin but fat, compared to Europe [182] and the high prevalence of mothers with low serum vitamin B12 is associated with IUGR. In a rural area from South India, the most insulin resistant children were born to mothers who had the lowest serum vitamin B12 and the highest erythrocyte folate concentrations [181]. Low maternal vitamin B12 blood level in early pregnancy predicts an elevated risk of insulin resistance, while folic acid maternal supplementation has a weak protective effect on incidence of metabolic syndrome, in school-aged offsprings from rural Nepal [156]. Population studies focused on the influence of folate in foetal programming have also produced contrasted results in Western countries. Antenatal supplementation with folic acid increases birth weight, while high homocysteine and low folate concentrations in early pregnancy are associated with both low placental and birth weights, and higher risk of adverse pregnancy outcomes, in the Netherlands [14, 166]. Folate intake estimated by questionnaire does not influence childhood body composition, in a cohort from South UK [103], while it is associated with birth weight and insulin resistance in French obese adolescents [58]. Methylenetetrahydrofolate reductase (MTHFR) 677 C > T polymorphism is not associated with obesity, BMI or metabolic syndrome in three cohorts from UK, Danemark and Italy [102, 149]. In contrast, in France, this polymorphism was associated with low birth weight and high insulin resistance in morbidly obese adolescents [58].

Several studies suggest that these associations could involve epigenomic mechanisms [157]. A variant of adenosyl-methionine decarboxylase is associated with childhood obesity, in India [161]. Studies conducted in Gambia, have found changes in methylation of metastable epi-alleles [177] when comparing birth exposure to seasonal variations of food-consumption patterns, which produce also significant changes in methyl-donor biomarkers [47].

Consequences of methyl donor deficiency on birth weight, food intake and digestive mucosa

The mother-progeny rat model of deficiency in folate, vitamin B12 and choline contributes to understand the complex links between metabolic syndrome and birth weight, according to the foetal programming hypothesis [16, 61, 89, 139]. MDD produces a dramatic injury of digestive mucosa that may participate to the pathomechanisms of IUGR, small birth weight and abnormal food intake, but little is known on its effects on food intake [20]. Ghrelin is a gastric peptide involved in foetal growth through its dual role as growth hormone-releasing factor and appetite stimulating peptide [95]. MDD affects gastric cell organisation and impairs the release of ghrelin in blood [20]. It also produces an over-expression of Cox-2, PLA2 and TNF-α pro-inflammatory pathways in gastric and intestine mucosa and alters mucosal differentiation and function in rat pups from dams subjected to deficiency during gestation and lactation [23, 78].

The link between the one-carbon metabolism and non-alcoholic fatty liver disease

Vitamin B12, folate, homocysteine and genetic determinants of the OCM are associated with NAFLD severity in operated and non-operated obese subjects [68, 77, 97]. Hepatic steatosis is also observed in patients with genetic disorders of OCM [38, 150]

A restriction of the periconceptional diet in cobalt and sulphur in sheep produces low vitamin B12 and methionine and leads to heavier and fatter adult offspring, with insulin resistance and elevated blood pressure that are most obvious in males. The altered methylation status of CpG islands examined by restriction landmark genome scanning in the foetal liver revealed that more than half of the affected loci are specific to males [152]. The normal mitochondrial proteome function relies on the maintenance of methylation in rodents [5]. Impaired mitochondrial metabolism has been reported in genetically altered mice exhibiting SAM deficiency in the liver [112], suggesting that SAM plays a critical role in maintaining proper mitochondrial function. Consistently, supplementation with methyl donors and SAM at concentrations that maintain methylation reactions preserves mitochondrial proteome as well as prevents ethanol-dependent defects in mitochondrial respiration [4, 6, 160]

In the mother–progeny rat model of MDD, fatty liver and fatty heart can be regarded as two early predisposing manifestation of metabolic syndrome through deregulation of lipid fuel homeostasis and mitochondrial metabolism [91, 139]. The deficiency yields microvesicular liver steatosis in pups [16]. The steatosis results from a deficit in carnitine synthesis and epigenomic deregulations, with hypomethylation and decreased expression of PPAR-α, oestrogen-related receptor alpha (ERR-α), ER-α and liver-specific NR HNF-4α and hypomethylation of PGC-1α coactivator through decreased activity of arginine methyltransferase 1 (PRMT1) [139]. The hypomethylation of PGC-1α in the liver participates in the epigenomic deregulation of FA β-oxidation by reducing its partnership with PPAR-α, ERR-α and HNF-4α (Fig. 2) [61, 139]. Consistently, other data show that the lack of PPAR-α enhances steatohepatitis and that the PPAR-α agonist has the opposite effect in mice fed a methionine- and choline-deficient diet [109]. Mice fed a methionine- and choline-deficient diet develop steatohepatitis, with an inflammatory infiltrate prominent in the hepatic acinar zone [178]. Feeding mice a choline- and folate-deficient diet for 12 weeks causes NAFLD with a down-regulation of PPAR-α-regulated lipid catabolic pathway genes [168]. These results have to be considered in regard to the central role of mitochondrial dysfunction in insulin resistance [109]. MDD produces a decreased expression of SIRT1 [136], which is also observed in high-fat diet [81]. Male inbred C57BL/6J and DBA/2J mice fed a methyl-deficient diet develop liver injury similar to NASH. The livers of DBA/2J mice had initially a lower degree of methylation of repetitive elements and lower extent of histone H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) trimethylation, compared to those in the C57BL/6J mice. They developed subsequently more prominent NASH-specific pathomorphological changes [138].

Fig. 2
figure 2

The molecular links between MDD and impaired FA oxidation. The deficiency in folate and vitamin B12 of the rat ‘dam–progeny’ model impairs FA oxidation and complexes I and II of the mitochondrion respiratory chain through decreased expression of PPAR-α, ERR-α and HNF-4. It impairs the co-regulation of these NRs by PGC-1α through its decreased methylation and/or increased acetylation. This imbalanced methylation/acetylation is the consequence of the decreased expression of arginine methyltransferase PRMT1, the decreased cellular ration of SAM/SAH ratio (which directly influence PRMT1 activity as substrate and inhibitor, respectively) and the decreased expression of deacetylase SIRT1 (adapted from refs. [61, 63, 68])

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Consequences of MDD on the heart

MDD during pregnancy and lactation of rodents induces heart hypertrophy, with impaired mitochondrial FA oxidation and decreased activity of complex I and II of the respiratory chain in rat pups [61]. The role of the deficiency in producing myocardium hypertrophy has been ascertained by a significant correlation of heart/body weight with folate, vitamin B12 and Hcy. The diet produces a decreased concentration of folate, a decreased SAM/SAH ratio and an increased immunohistochemical detection of Hcy in the myocardium of the deficient animals. The decreased SAM/SAH ratio is the consequence of a trap whereby the impaired remethylation pathway and the absence of cystathionine-β-synthase produced a conversion of homocysteine into SAH by the reversible enzyme SAH hydrolase [61]. The proteomic analysis of the myocardium of pups from rat dams fed a methyl donor-deficient diet shows altered abundance of proteins associated with developmental disorder, cellular compromise and lipid metabolism [111]. Concurrently, increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins [111]. The main proteomic changes are observed for proteins regulated by PGC-1α, PPARs and ERR-α. These proteins, namely, trifunctional enzyme subunit α-complex, short-chain acyl-CoA dehydrogenase, acyl-CoA thioesterase 2, fatty acid-binding protein-3, NADH dehydrogenase (ubiquinone) flavoprotein 2, NADH dehydrogenase (ubiquinone) 1α-subunit 10 and Hspd1 protein, are involved in FA oxidation and mitochondrial respiration. These changes are related to the decreased expression of PPAR-α and ERR-α and to the hypomethylation and hyperacetylation of PGC-1α through decreased expression and activity of PRMT1 and SIRT1 and decreased SAM/SAH ratio (Fig. 2). It is noticeable that the decreased expression of SIRT1 is also a molecular hallmark of overnutrition (Fig. 1) [19, 32, 51, 61, 80].

Folate- and vitamin B12-dependent pathways are needed for the proper foetal development of the heart. Indeed, the c.56 + 781 A > C (rs326119) variant of intron 1 of MS reductase results in functionally reduced gene transcription and significantly increases the risk of congenital heart disease in the Han Chinese population [188]. The link between the OCM, cardiomyopathies and impaired mitochondrial FA oxidation has been confirmed in two contrasting populations, patients undergoing coronarography and ambulatory elderly subjects [67]. Folate deficit was more frequent in the coronarography population than in the elderly ambulatory volunteers and produced a higher concentration of homocysteine. Homocysteine and NT-pro-BNP were positively correlated with short-chain, medium-chain and long-chain acylcarnitines and with acylcarnitine ratios indicative of decreased mitochondrial acyldehydrogenase activities. This can contribute to explaining the association of homocysteine with left ventricular mass and left ventricular systolic dysfunction [66, 76].

Comparison of the tissue, metabolic, genomic and epigenomic effects of high fat/overfeeding, protein/caloric restriction and methyl donor deficiency

The pups deprived of methyl donors had a decreased birth weight and no change in insulin concentration, as observed in the protein restriction and food restriction rat models of foetal programming [24–26]. However, comparing the heart and liver manifestations in the three diets and the underlying molecular mechanisms shows intriguing similarities between high fat/overfeeding and MDD. The methyl donor-deficient rats have a dramatic increase of plasma FFA, which is similar to that observed in high-fat diet animals and contrasts to the mild increased level reported in pups under protein and/or food restriction [24–26]. MDD produces a cardiac hypertrophy with myocardium FA accumulation, a phenotype, which is due to impaired FA β-oxidation and possibly to an increased import of FFA from plasma.

Intriguingly, the deficit in methyl donors may produce a central lipodystrophy, with similar molecular hallmarks than those observed following overfeeding and high-fat diet. These data and epidemiological data on the methyl donor status of mothers during pregnancy suggest considering with particular interest the effects of MDD during pregnancy and overnutrition in later life as part of the explanation of the links between foetal programming and metabolic disorders related to obesity. It is noteworthy that several studies on cell and experimental models found a strong imbalance in energy metabolism similar to that observed in metabolic syndrome, with molecular hallmarks similar to those produced by nutrition overload and obesity, including the decreased expression and activity of SIRT1, the related hyperacetylation of PGC-1α and the subsequent disruption of mitochondrial energy metabolism [65]. High-fat diet and overfeeding impairs the AMPK-dependent phosphorylation of PGC-1α, a change to be documented in MDD. AMPK is a key metabolic sensor, which behaves as a control switch to maintain metabolic homeostasis in function of energy supply. It is decreased by calorie restriction and increased by overfeeding, with a mirrored influence that depends on the cellular level of ATP and on AMP/ATP ratio (Fig. 1). The expression and influence of AMPK in MDD needs to be better evaluated, considering the possible inhibitory effect of SAM on its activity [113]. PP2A, one of the four major classes of eukaryotic serine/threonine phosphoprotein phosphatases, is also a metabolic sensor to be considered, since it is activated by methylation and up-regulated by vitamin B12 deficiency [12]. The visceral manifestations of metabolic syndrome and insulin resistance are under the influence of ER stress in overnourished animal models [52]. These mechanisms should also deserve attention in the foetal programming effects of MDD since vitamin B12 influences ER stress through impaired SIRT1 deacetylation of heat shock factor protein 1 (HSF1) (Fig. 3) [62].

Fig. 3
figure 3

Influence of vitamin B12 on cell proliferation through SIRT1-dependent activation of ER stress. The decreased cellular availability in B12 activates ER stress pathways and apoptosis by hyperacetylation of HSF1 through decreased expression of SIRT1. The dramatic reduction in both protein expression and acetylation of HSF1 leads to critical reduction of the molecular chaperons needed for a functional proteome in normal cellular growth. In contrast, B12 supplementation protects the cells from the activation of ER stress and apoptosis in cells subjected to thapsigargin, an ER stress inducer. Thus, B12 will favour fully adapted unfolded protein response (UPR) against any downstream attacks in non-deficient cells, while in deficient cells, activated UPR will likely be shifted from adaptive toward apoptotic maladaptive end point. SIRT1 sirtuin (silent mating type information regulation 2 homolog) 1, HSF1 heat shock factor protein 1

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The mirrored influence of MDD and protein restriction on heart and liver manifestations of foetal programming is consistent with the reversal of the metabolic and epigenomic phenotype induced by maternal nutritional restriction when supplementing the mothers with folic acid, in rats [28]. Intriguingly, giving folate supplementation during the juvenile–pubertal period of offspring produced opposite results, with hepatosteatosis, impaired liver FA β-oxidation and decreased liver expression of PPAR-α through promoter hypermethylation [28, 105]. As highlighted by Burdge et al., these findings may reflect the fact that puberty is one of the four periods of increased instability of the epigenome [28]. Giving a high-fat diet to pups from mother fed a protein-restricted diet after weaning exacerbates their obesity [145]. Similar studies should be performed to test the consecutive or contemporary influence of MDD and high-fat diet, in regard to the potential synergic effects of the two diets at the tissue, metabolic and genomic levels.

Conclusion

A limited number of population studies have evaluated the metabolic, epigenomic and genomic effects of foetal programming on liver and heart manifestations. This probably reflects the difficulty to design follow-up and interventional studies adapted to the tested hypotheses, despite the remarkable experimental attention, which has been given to animal models and, particularly, rodents. In contrast to overfeeding and MDD, protein and/or food restriction does not assess a situation, which reproduces the more frequent causes of abnormal nutritional status of mothers during pregnancy and lactation, at least in Western countries. High-fat intake/overfeeding and MDD are two situations, which are frequent during pregnancy, in adolescents and in elderly subjects, in countries, which are not subjected to folate fortification. Their consecutive and/or contemporary influence during pregnancy and later life should be, therefore, investigated in population studies and experimental animal models.

In the liver and heart, experimental consequences include increased influx and storage produced by overfeeding/high fat and impaired FA β-oxidation by MDD. Beside the influence of high fat and overfeeding on insulin resistance and metabolic syndrome, several large epidemiological studies have shown that vitamin B12 deficit during pregnancy is a powerful predictor for the risk of small birth weight, lipodystrophy, metabolic syndrome and insulin resistance in India and Nepal. Paradoxically, its effects are more important in newborns and children from mothers supplemented with folate. Meanwhile, experimental data have shown that the deficiency in Cbl and folate produces central fat accumulation and epigenomic dysregulations, which lead to ER stress and disruption of mitochondrial energy metabolism. Whether methyl donor status and the related dysregulations are involved in the pathomechanisms of metabolic syndrome and insulin resistance in humans needs further attention using integrative genomics, proteomics and metabolomics approaches.