The DNA methyltransferase inhibitor decitabine blunts the response to a high animal fat and protein diet in mice.

Increasing evidence hints that DNA hypermethylation may mediate the pathogenic response to cardiovascular risk factors. Here, we tested a corollary of that hypothesis, i.e., that the DNA methyltransferase inhibitor decitabine (Dec) ameliorates the metabolic profile of mice fed a moderately high-animal fat and protein diet (HAFPD), a proxy of cardiovascular risk-associated Western-type diet. HAFPD-fed mice were exposed to Dec or vehicle for eight weeks (8W set, 4-32/group). To assess any memory of past exposure to Dec, we surveyed a second mice set treated as 8W but HAFPD-fed for further eight weeks without any Dec (16W set, 4-20/group). In 8W, Dec markedly reduced HAFPD-induced body weight gain in females, but marginally in males. Characterization of females revealed that Dec augmented skeletal muscle lipid content, while decreasing liver fat content and increasing plasma non-esterified fatty acids, adipose insulin resistance, and -although marginally- whole blood acylcarnitines, compared to HAFPD alone. Skeletal muscle mitochondrial DNA copy number was higher in 8W mice exposed to HAFPD and Dec, or in 16W mice fed HAFPD only, relative to 8W mice fed HAFPD only, but Dec induced a transcriptional profile indicative of ameliorated mitochondrial function. Memory of past Dec exposure was tissue-specific and sensitive to both duration of exposure to HAFPD and age. In conclusion, Dec redirected HAFPD-induced lipid accumulation towards the skeletal muscle, likely due to augmented mitochondrial functionality and increased lipid demand. As caveat, Dec induced adipose insulin resistance. Our findings may help identifying strategies for prevention and treatment of lipid dysmetabolism.

Effects of paternal arachidonic acid supplementation on offspring behavior and hypothalamus inflammation markers in the mouse.

Arachidonic acid (AA) is involved in inflammation and plays a role in growth and brain development in infants. We previously showed that exposure of mouse sires to AA for three consecutive generations induces a cumulative change in fatty acid (FA) involved in inflammation and an increase in body and liver weight in the offspring. Here, we tested the hypothesis that paternal AA exposure changes the progeny's behavioral response to a proinflammatory insult, and asked whether tissue-specific FA are associated with that response. Male BALB/c mice were supplemented daily with three doses of AA for 10 days and crossed to non-supplemented females (n = 3/dose). Two-month-old unsupplemented male and female offspring (n = 6/paternal AA dose) were exposed to Gram-negative bacteria-derived lipopolysaccharides (LPS) or saline control two hours prior to open field test (OFT) behavioral analysis and subsequent sacrifice. We probed for significant effects of paternal AA exposure on: OFT behaviors; individual FA content of blood, hypothalamus and hypothalamus-free brain; hypothalamic expression profile of genes related to inflammation (Tnfa, Il1b, Cox1, Cox2) and FA synthesis (Scd1, Elovl6). All parameters were affected by paternal AA supplementation in a sex-specific manner. Paternal AA primed the progeny for behavior associated with increased anxiety, with a marked sex dimorphism: high AA doses acted as surrogate of LPS in males, realigning a number of OFT behaviors that in females were differential between saline and LPS groups. Progeny hypothalamic Scd1, a FA metabolism enzyme with documented pro-inflammatory activity, showed a similar pattern of differential expression between saline and LPS groups at high paternal AA dose in females, that was blunted in males. Progeny FA generally were not affected by LPS, but displayed non-linear associations with paternal AA doses. In conclusion, we document that paternal exposure to AA exerts long-term behavioral and biochemical effects in the progeny in a sex-specific manner.

Associations of fertility parameters with fatty acids and DNA methylation in Mexican women undergoing in vitro fertilization.

BACKGROUND: Fatty acids (FA) likely affect human fertility at multiple levels, as deviations from physiological FA profiles are obesogenic, and FA can modify DNA methylation (DNAm). Yet, the interplay of follicular fluid (FF) and serum FA with BMI and percentage body fat (PBF) in human fertility is not completely understood. Also, associations of DNAm with fertility are largely unexplored. METHODS: Reproductive parameters ranging from retrieved oocyte number to infant birth weight, were recorded in Mexican women undergoing in vitro fertilization (n = 88). Multiple regression analysis sought BMI-adjusted and age-adjusted associations. Receiver operating characteristic analysis tested for discrimination between outcomes. RESULTS: Associations of FF and serum FA were markedly distinct. While various FF FA (C16:1, C18:0, C20:2, C20:3, arachidonic acid) were significantly and inversely associated only with retrieved oocyte number, selected serum FA were associated with a broad range of pre-fertilization and post-fertilization parameters. Associations of BMI and FF FA were complex, as arachidonic acid was inversely associated with both BMI and retrieved oocyte number, while oleic acid (OA) was directly associated with BMI and PBF. Ultrasound-assessed clinical pregnancy outcome (CP) was directly associated with serum OA but inversely with its trans isomer elaidic acid (EA) and with BMI. Compounded BMI, serum EA and OA discriminated CP well (AUC = 0.74). Whole blood DNA methylation was significantly associated with and a moderate predictor (AUC = 0.66) of percent fertilized oocytes. CONCLUSIONS: Overall FF FA pool composition rather than FA identity may impact oocyte production and cellular memory of FF FA is lost as the oocyte exits the follicular environment. The contrasting associations of BMI, FF OA and arachidonic acid suggest that the control of oocyte homeostasis by FF FA is uncoupled from BMI. Further studies are warranted to assess the potential of compounding BMI with serum EA and OA to predict CP.

Dynamic epigenetic age mosaicism in the human atherosclerotic artery.

Accelerated epigenetic ageing, a promising marker of disease risk, has been detected in peripheral blood cells of atherosclerotic patients, but evidence in the vascular wall is lacking. Understanding the trends of epigenetic ageing in the atheroma may provide insights into mechanisms of atherogenesis or identify targets for molecular therapy. We surveyed DNA methylation age in two human artery samples: a set of donor-matched, paired atherosclerotic and healthy aortic portions, and a set of carotid artery atheromas. The well-characterized pan-tissue Horvath epigenetic clock was used, together with the Weidner whole-blood-specific clock as validation. For the first time, we document dynamic DNA methylation age mosaicism of the vascular wall that is atherosclerosis-related, switches from acceleration to deceleration with chronological ageing, and is consistent in human aorta and carotid atheroma. At CpG level, the Horvath epigenetic clock showed modest differential methylation between atherosclerotic and healthy aortic portions, weak association with atheroma histological grade and no clear evidence for participation in atherosclerosis-related cellular pathways. Our data suggest caution when assigning a unidirectional DNA methylation age change to the atherosclerotic arterial wall. Also, the results support previous conclusions that epigenetic ageing reflects non-disease-specific cellular alterations.

Cumulative Metabolic and Epigenetic Effects of Paternal and/or Maternal Supplementation with Arachidonic Acid across Three Consecutive Generations in Mice.

Apart from the known associations between arachidonic acid (AA), weight gain, and neurological and immune function, AA exposure leads to alterations in global and gene-specific DNA methylation (DNAm) and fatty acid (FA) content in human cultured cells. However, it is unknown as to whether the latter effects occur in vivo and are maintained over extended periods of time and across generations. To address this issue, we asked whether AA supplementation for three consecutive generations (prior to coitus in sires or in utero in dams) affected offspring growth phenotypes, in addition to liver DNAm and FA profiles in mice. Twelve-week-old BALB/c mice were exposed daily to AA dissolved in soybean oil (vehicle, VH), or VH only, for 10 days prior to mating or during the entire pregnancy (20 days). On average, 15 mice were supplemented per generation, followed by analysis of offspring body weight and liver traits (x average = 36 and 10 per generation, respectively). Body weight cumulatively increased in F2 and F3 offspring generations and positively correlated with milligrams of paternal or maternal offspring AA exposure. A concomitant increase in liver weight was observed. Notably, akin to AA-challenged cultured cells, global DNAm and cis-7-hexadecenoic acid (16:1n-9), an anti-inflammatory FA that is dependent on stearoyl-CoA desaturase 1 (SCD1) activity, increased with milligrams of AA exposure. In accordance, liver Scd1 promoter methylation decreased with milligrams of germline AA exposure and was negatively correlated with liver weight. Our results show that mice retain cellular memories of AA exposure across generations that could potentially be beneficial to the innate immune system.

Distinct Associations of BMI and Fatty Acids With DNA Methylation in Fasting and Postprandial States in Men.

We have previously shown that blood global DNA methylation (DNAm) differs between postprandial state (PS) and fasting state (FS) and is associated with BMI and polyunsaturated fatty acid (PUFA) (negatively and positively, respectively) in 12 metabolically healthy adult Mexican men (AMM cohort) equally distributed among conventional BMI classes. Here, we detailed those associations at CpG dinucleotide level by exploiting the Infinium methylation EPIC array (Illumina). We sought differentially methylated CpG (dmCpG) that were (1) associated with BMI (BMI-dmCpG) and/or fatty acids (FA) (FA-dmCpG) in FS or PS and (2) different across FS and PS within a BMI class. BMI-dmCpG and FA-dmCpG were more numerous in FS compared to PS and largely prandial state-specific. For saturated and monounsaturated FA, dmCpG overlap was higher across than within the respective saturation group. Several BMI- and FA-dmCpG mapped to genes involved in metabolic disease and in some cases matched published experimental data sets. Notably, SETDB1 and MTHFS promoter dmCpG could explain the previously observed associations between global DNAm, PUFA content, and BMI in FS. Surprisingly, overlap between BMI-dmCpG and FA-dmCpG was limited and the respective dmCpG were differentially distributed across functional genomic elements. BMI-dmCpG showed the highest overlap with dmCpG of the saturated FA palmitate, monounsaturated C20:1 and PUFA C20:2. Of these, selected promoter BMI-dmCpG showed opposite associations with palmitate compared to C20:1 and C20:2. As for the comparison between FS and PS within BMI classes, dmCpG were strikingly more abundant and variably methylated in overweight relative to normoweight or obese subjects (∼70-139-fold, respectively). Overweight-associated dmCpG-hosting genes were significantly enriched in targets for E47, SREBP1, and RREB1 transcription factors, which are known players in obesity and lipid homeostasis, but none overlapped with BMI-dmCpG. We show for the first time that the association of BMI and FA with methylation of disease-related genes is distinct in FS and PS and that limited overlap exists between BMI- and FA-dmCpG within and across prandial states. Our study also identifies a transcriptional regulation circuitry in overweight that might contribute to adaptation to that condition or to transition to obesity. Further work is necessary to define the pathophysiological implications of these findings.

Somatic Genetic Mosaicism in the Apolipoprotein E-null Mouse Aorta.

In addition to genetic and epigenetic inheritance, somatic variation may contribute to cardiovascular disease (CVD) risk. CVD-associated somatic mutations have been reported in human clonal hematopoiesis, but evidence in the atheroma is lacking. To probe for somatic variation in atherosclerosis, we sought single-nucleotide private variants (PVs) in whole-exome sequencing (WES) data of aorta, liver, and skeletal muscle of two C57BL/6J coisogenic male ApoE null/wild-type (WT) sibling pairs, and RNA-seq data of one of the two pairs. Relative to the C57BL/6 reference genome, we identified 9 and 11 ApoE null aorta- and liver-specific PVs that were shared by all WES and RNA-seq datasets. Corresponding PVs in WT sibling aorta and liver were 1 and 0, respectively, and not overlapping with ApoE null PVs. Pyrosequencing analysis of 4 representative PVs in 17 ApoE null aortas and livers confirmed tissue-specific shifts toward the alternative allele, in addition to significant deviations from mendelian allele ratios. Notably, all aorta and liver PVs were present in the dbSNP database and were predominantly transition mutations within atherosclerosis-related genes. The majority of PVs were in discrete clusters approximately 3 Mb and 65 to 73 Mb away from hypermutable immunoglobin loci in chromosome 6. These features were largely shared with previously reported CVD-associated somatic mutations in human clonal hematopoiesis. The observation that SNPs exhibit tissue-specific somatic DNA mosaicism in ApoE null mice is potentially relevant for genetic association study design. The proximity of PVs to hypermutable loci suggests testable mechanistic hypotheses.

Is Any Cardiovascular Disease-Specific DNA Methylation Biomarker Within Reach?

PURPOSE OF REVIEW: A detailed understanding of the epigenome of cardiovascular disease (CVD) should broaden current insights into mechanisms of atherogenesis and help identify suitable biomarkers for disease risk and progression. This review addresses the question whether a consensus has been reached on identifying the main aberrant DNA methylation profile in CVD. Additionally, it presents advances and setbacks in the search for specific CVD biomarkers. RECENT FINDINGS: Although the literature points to DNA hypermethylation as an epigenetic landmark of CVD, inconsistencies are significant. In particular, the DNA methylomes of peripheral blood cells and the vascular wall do not show a consistent direction of change in all studies. An additional significant hurdle is the relatively low study-to-study reproducibility and the difficulty to assess specificity for CVD. Nonetheless, a number of biologically plausible markers have been proposed that warrant further studies. An integrated model for dynamic changes of DNA methylation during the natural history of atherosclerosis predisposition and progression is presented, that might reconcile conflicting findings. Cohort design and technical criteria for DNA methylation analysis need to be further homogenized to allow for meaningful validation. As stable DNA methylation profiles are likely determined by genetic variants, many of which might control a range of diseases, it is anticipated that CVD biomarker discovery will be a delicate balancing act between reproducibility and specificity.

Associations between atherosclerosis and neurological diseases, beyond ischemia-induced cerebral damage.

Neurodegeneration is traditionally viewed as a consequence of peptide accumulation in the brain, stroke and/or cerebral ischemia. Nonetheless, a number of scattered observations suggest that neurological disease and atherosclerosis may be linked by more complex mechanisms. Understanding the intricate link between atherosclerosis and neurological conditions may have a significant impact on the quality of life of the growing ageing population and of high cardiovascular risk groups in general. Epidemiological data support the notion that neurological dysfunction and atherosclerosis coexist long before any evident clinical complications of cardiovascular disease appear and may be causally linked. Baffling, often overlooked, molecular data suggest that nervous tissue-specific gene expression is relaxed specifically in the atheromatous vascular wall, and/or that a systemic dysregulation of genes involved in nervous system biology dictates a concomitant progression of neurological disease and atherosclerosis. Further epidemiological and experimental work is needed to clarify the details and clinical relevance of those complex links.

Liver X Receptor-Binding DNA Motif Associated With Atherosclerosis-Specific DNA Methylation Profiles of Alu Elements and Neighboring CpG Islands.

BACKGROUND: The signals that determine atherosclerosis-specific DNA methylation profiles are only partially known. We previously identified a 29-bp DNA motif (differential methylation motif [DMM]) proximal to CpG islands (CGIs) that undergo demethylation in advanced human atheromas. Those data hinted that the DMM docks modifiers of DNA methylation and transcription. METHODS AND RESULTS: We sought to functionally characterize the DMM. We showed that the DMM overlaps with the RNA polymerase III-binding B box of Alu short interspersed nuclear elements and contains a DR2 nuclear receptor response element. Pointing to a possible functional role for an Alu DMM, CGIs proximal (<100 bp) to near-intact DMM-harboring Alu are significantly less methylated relative to CGIs proximal to degenerate DMM-harboring Alu or to DMM-devoid mammalian-wide interspersed repeat short interspersed nuclear elements in human arteries. As for DMM-binding factors, LXRB (liver X receptor ß) binds the DMM in a DR2-dependent fashion, and LXR (liver X receptor) agonists induce significant hypermethylation of the bulk of Alu in THP-1 cells. Furthermore, we describe 3 intergenic long noncoding RNAs that harbor a DMM, are under transcriptional control by LXR agonists, and are differentially expressed between normal and atherosclerotic human aortas. Notably, CGIs adjacent to those long noncoding RNAs tend to be hypomethylated in symptomatic relative to stable human atheromas. CONCLUSIONS: Collectively, the data suggest that a DMM is associated with 2 distinct methylation states: relatively low methylation of in cis CGIs and Alu element hypermethylation. Based on the known atheroprotective role of LXRs, we propose that LXR agonist-induced Alu hypermethylation, a landmark of atherosclerosis, is a compensatory rather than proatherogenic response.

Array probe density and pathobiological relevant CpG calling bias in human disease and physiological DNA methylation profiling.

The HumanMethylation450 BeadChip array (450K; Infinium) is a widely used tool in epigenomics. A recognized concern in the 450K platform is the potential effect of the number of probes/gene (PG) on ranking differentially methylated (DM) CpGs (DM-CpGs) before testing for enrichment of gene ontology categories. We previously showed in a fatty acid (FA)-induced DNA methylation profiling study that when DM-CpGs are ranked by the number of called DM-CpGs-to-PG ratio, the 150 top-ranking gene list is enriched in pathways that overlap with the corresponding Affymetrix array-based expression data. In this study, a comparative analysis of thirteen 450K-based studies representing FA-stimulated cellular models, aging, diseased and normal tissues, revealed that the 150 top-ranking DM-CpGs are in high PG genes. This points to a significant false-negative rate in the low PG gene set when delta-beta-based ranking is performed. We show that PG is not related to the density of methylation-prone sites, as it does not follow gene length or GC content. Conversely, ranking genes by the number of DM-CpGs-to-PG ratio and analysing the 150 top-ranking entries yields significantly enriched gene disease- or tissue-specific function categories that are increased both in number and in the degree of overlap with expression data compared with delta-beta-only ranking or to the previously published gometh-based pipeline. The 15 top-ranking loci list is also significantly enriched in non-coding RNAs, a greatly underrepresented transcript type in 450K. In summary, the proposed simple normalization method yields pathobiologically relevant DM-CpGs. This method is relevant for the newly developed MethylationEPIC (Infinium) microarray.

Connecting the Dots Between Fatty Acids, Mitochondrial Function, and DNA Methylation in Atherosclerosis.

PURPOSE OF REVIEW: The quest for factors and mechanisms responsible for aberrant DNA methylation in human disease-including atherosclerosis-is a promising area of research. This review focuses on the role of fatty acids (FAs) as modulators of DNA methylation-in particular the role of mitochondrial beta-oxidation in FA-induced changes in DNA methylation during the progression of atherosclerosis. RECENT FINDINGS: Recent publications have advanced the knowledge in all areas touched by this review: the causal role of lipids in shaping the DNA methylome, the associations between chronic degenerative disease and mitochondrial function, the lipid composition of the atheroma, and the relevance of DNA hypermethylation in atherosclerosis. Evidence is beginning to emerge, linking the dynamics of FA type abundance, mitochondrial function, and DNA methylation in the atheroma and systemically. In particular, this review highlights mitochondrial beta-oxidation as an important regulator of DNA methylation in metabolic disease. Despite the many questions still unanswered, this area of research promises to identify mechanisms and molecular factors that establish a pathological gene expression pattern in atherosclerosis.

11 beta-hydroxysteroid dehydrogenase 2 promoter methylation is associated with placental protein expression in small for gestational age newborns.

Small for gestational age infants have greater risk of developing metabolic diseases in adult life. It has been suggested that low birth weight may result from glucocorticoid excess in utero, a key mechanism in fetal programming. The placental enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11ß-HSD2, HSD11B2 gene) acts as a barrier protecting the fetus from maternal corticosteroid deleterious effects. Low placental 11ß-HSD2 transcription and activity have been associated with low birth weight, yet the mechanism regulating its protein expression is not fully understood. In the present study we aimed to analyze 11ß-HSD2 protein expression in placentas of adequate and small for gestational age (AGA and SGA, respectively) newborns from healthy mothers, and to explore whether 11ß-HSD2 protein expression could be modulated by DNA methylation. 11ß-HSD2 protein levels were measured by western blot in placental biopsies from term AGA and SGA infants (n=10 per group). DNA methylation was profiled both globally and in the HSD11B2 promoter by liquid chromatography with UV detection and methylation-specific melting curve analysis, respectively. We found lower placental 11ß-HSD2 protein expression and higher HSD11B2 promoter methylation in SGA compared to AGA. Promoter methylation was inversely correlated with both protein expression and, importantly, birth weight. No changes in global placental methylation were found. In conclusion, lower 11ß-HSD2 protein expression is associated with higher HSD11B2 promoter methylation, correlating with birth weight in healthy pregnancy. Our data support the role of 11ß-HSD2 in determining birth weight, providing evidence of its regulation by epigenetic mechanisms, which may affect postnatal metabolic disease risk.

Leptin and its Receptors in Human Placenta of Small, Adequate, and Large for Gestational Age Newborns.

Alterations in birth weight impact postnatal outcome and adult metabolic health. Therefore, fetal growth regulation is crucial for preventing chronic metabolic diseases. Leptin has been suggested to play an important role in placental and fetal growth, albeit its specific mechanisms of action have not been elucidated. The aim of this study was to analyze leptin concentrations in placenta, cord blood, and maternal blood of SGA, AGA, and LGA (small, adequate and large for gestational age, respectively) newborns, as well as placental leptin receptor (LEPRa and LEPRb) protein expression. We performed a cross-sectional comparative study in 3 groups of healthy mothers and their term newborns at delivery (SGA, AGA, and LGA, n=20 per group). Placental, maternal blood, and cord blood leptin content were measured by ELISA. Placental LEPRa and LEPRb protein expression were determined by Western Blot. Maternal leptin concentrations correlated positively with maternal weight before and at the end of gestation, without differences between groups. Cord leptin is higher in LGA and lower in SGA, whereas placental leptin is higher in SGA. Placental leptin was inversely correlated with placental weight, independently from maternal weight and gestational age. Both LEPRa and LEPRb expression are lower in SGA, while LEPRa positively correlated with placental weight and birthweight. The current findings indicate that placental leptin and its receptors are differentially expressed in SGA, AGA, and LGA newborns. We suggest that placental leptin and LEPR protein expression may influence placental growth and thus, birth weight.

Inter-individual variation in DNA methylation is largely restricted to tissue-specific differentially methylated regions in maize.

BACKGROUND: Variation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP). The latter represents a useful alterative when sample size is large, or when analysing methylation changes in genomes that have yet to be sequenced. In this study we compared variation in methylation across ten individual leaf and endosperm samples from maize hybrid and inbred lines using MSAP. We also addressed the methodological implications of analysing methylation variation using pooled versus individual DNA samples, in addition to the validity of MSAP compared to WGBS. Finally, we analysed a subset of variable and non-variable fragments with respect to genomic location, vicinity to repetitive elements and expression patterns across leaf and endosperm tissues. RESULTS: On average, 30% of individuals showed inter-individual methylation variation, mostly of leaf and endosperm-specific differentially methylated DNA regions. With the exception of low frequency demethylation events, the bulk of inter-individual methylation variation (84 and 80% in leaf and endosperm, respectively) was effectively captured in DNA from pooled individuals. Furthermore, available genome-wide methylation data largely confirmed MSAP leaf methylation profiles. Most variable methylation that mapped within genes was associated with CG methylation, and many of such genes showed tissue-specific expression profiles. Finally, we found that the hAT DNA transposon was the most common class II transposable element found in close proximity to variable DNA regions. CONCLUSIONS: The relevance of our results with respect to future studies of methylation variation is the following: firstly, the finding that inter-individual methylation variation is largely restricted to tissue-specific differentially methylated DNA regions, underlines the importance of tissue-type when analysing the methylation response to a defined stimulus. Secondly, we show that pooled sample-based MSAP studies are methodologically appropriate to study methylation variation. Thirdly, we confirm that MSAP is a powerful tool when WGBS is not required or feasible, for example in plant species that have yet to be sequenced.

Activity of Extracts from Submerged Cultured Mycelium of Winter Mushroom, Flammulina velutipes (Agaricomycetes), on the Immune System In Vitro.

Extracts from submerged cultured mycelium of two strains of Flammulina velutipes, a popular culinary mushroom, were obtained by ultrasound and tested in vitro to determine their activity in innate immunity (monocytes/ macrophages). In addition, polyclonal antibodies against the extracts were produced. Both extracts have similar glycoproteins that contain mannose and glucose but have different glycoproteins with galactoseamine units. Two novel immunogenic glycoproteins with molecular weights of 32 and 25 kDa have been revealed. It is thought that these proteins are produced only by submerged cultured mycelium. Both extracts show immune-enhancing activity based on the significant modification of various parameters such as cytokine production, phagocytosis, and reactive oxygen species production.

Associations between whole peripheral blood fatty acids and DNA methylation in humans.

Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage.