Investigating a personalised nutrition approach for modulating epigenetic profiles using over-expressed DNMTs in cell lines

Ph. D. Thesis.DNA methylation is an epigenetic mechanism that enables heritable changes in gene expression without changes in DNA sequence. Methyl groups are transferred from the methyl donor S-adenosyl methionine (SAM) to the 5-carbon of cytosine by DNA methyltransferases (DNMTs). The DNMT family comprises a set of DNA-modifying enzymes and uses a similar catalytic mechanism to form a covalent reaction intermediate between the substrate base and the enzyme. Food-derived bioactive compounds are among the exogenous factors that can modulate the DNA methylation patterns, either via generating SAM through one-carbon metabolism or by inhibiting the activity of DNMTs. In this study, cell lines with stable over-expression of each of 13 DNMTs isoform (DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMTΔ3B1, DNMTΔ3B2, DNMTΔ3B3, DNMTΔ3B4, DNMT3L, and DNMT1) were generated by lentiviral transduction of human embryonic kidney cells (HEK293T). DNA methylation patterns in these 13 cell lines were analysed by Illumina Infinium Methylation EPIC BeadChip, which interrogates more than 850,000 CpG sites across the genome. The sensitivity and specificity of each DNMT isoform to selected food constituents (caffeic acid (CA), (-)-Epigallocatechin-3-gallate (EGCG), curcumin, vitamin C, and theaflavin) were investigated by quantification of DNA methylation at specific CpG sites targeted by the DNMTs, using pyrosequencing. DNA methylation patterns for each DNMT isoform were obtained and the potential underlying biological mechanisms for DNMT-target CpGs were explored. At the selected CpG sites, DNA methylation was decreased with CA and vitamin C in DNMT∆3B4 and DNMT3A2 cells, respectively. In addition, the enzymatic activity of DNMT∆3B4 decreased after CA treatment. In summary, despite similarity of their protein structures, DNMT isoforms show regional specificity in the maintenance of DNA methylation patterns. This study also revealed that the activity of DNMT∆3B4 and DNMT3A2 can be specifically modulated by CA and vitamin C, respectively, in a dose-response manner. These observations further understanding of nutrition-epigenetic mechanisms, especially interactions with enzymatic activity, could be applied to modulate DNA methylation profiles using food-derived bioactive compounds in personalised nutrition

Can Vitamins Slow Down the Body’s Aging Process?

Some people look younger than their age, others older. Have you ever wondered why? Can we help our bodies age more slowly? Although there seems to be no way to reverse the process of aging, we may be able to slow it down. Improving our diets may help! Humans are born with an internal biological clock within our cells, which reflects the aging state of the body. This is called the epigenetic clock, and it can be changed by what we eat. In this study, we found that women who took supplements of folic acid and vitamin B12 had a slower biological aging. More studies on the effects of our diets on the epigenetic clock will help people to live longer and to stay in good health

Optimising blood glucose control with portioned meal box in type 2 diabetes mellitus patients: a randomised control trial

BackgroundThe impact of dietary factors on glycaemic control in type 2 diabetes mellitus (T2DM) is well established. However, the effectiveness of transforming portion control into a practical innovation for glycaemic control in T2DM has not yet been established for counselling in nutrition. The aim of this study was to compare the effect of general counselling in nutrition (GCN) and a portioned meal box (PMB) on fasting blood glucose, glycated haemoglobin (HbA1c) and body composition.MethodsA randomised, parallel intervention trial was conducted over 12 weeks, with GCN: carbohydrate portion control concept by using food exchange lists (n = 25) and PMB: portioned meal box was set by energy requirements (n = 25).ResultsBoth GCN and PMB demonstrated reductions in HbA1c levels at the 6th and 12th weeks compared to baseline. However, no significant difference in HbA1c was observed between GCN and PMB at either the 6th or 12th week. Using PMB at least four times a week significantly decreased HbA1c during the intervention period (p = 0.021 and p < 0.001 for weeks 6 and 12 when compared with baseline, respectively). Changes in body composition were observed: body weight decrease in PMB only, body fat decrease and constant muscle mass in both groups. Both methods tended to relieve hunger and increased satiety in both groups. The satisfaction evaluation showed that participants preferred to use PMB over GCN (p = 0.001). Additionally, participants consumed less energy, carbohydrate and fat in PMB (p = 0.001, p = 0.019, and p = 0.001, respectively) and less energy and fat in GCN (p = 0.006 and p = 0.001, respectively).ConclusionA better diet, either through GCN or PMB, can play an important role in improving dietary intake compliance and controlling blood glucose

DNA methylation patterns of LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 μg/day) and vitamin B12 (500 μg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD

Genomic targets and selective inhibition of DNA methyltransferase isoforms

Background: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. Results: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δβ > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (β: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δβ > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. Conclusions: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome

Novel Zinc-Related Differentially Methylated Regions in Leukocytes of Women With and Without Obesity

INTRODUCTION: Nutriepigenetic markers are predictive responses associated with changes in “surrounding” environmental conditions of humans, which may influence metabolic diseases. Although rich in calories, Western diets could be linked with the deficiency of micronutrients, resulting in the downstream of epigenetic and metabolic effects and consequently in obesity. Zinc (Zn) is an essential nutrient associated with distinct biological roles in human health. Despite the importance of Zn in metabolic processes, little is known about the relationship between Zn and epigenetic. Thus, the present study aimed to identify the epigenetic variables associated with Zn daily ingestion (ZnDI) and serum Zinc (ZnS) levels in women with and without obesity. MATERIALS AND METHODS: This is a case-control, non-randomized, single-center study conducted with 21 women allocated into two groups: control group (CG), composed of 11 women without obesity, and study group (SG), composed of 10 women with obesity. Anthropometric measurements, ZnDI, and ZnS levels were evaluated. Also, leukocyte DNA was extracted for DNA methylation analysis using 450 k Illumina BeadChips. The epigenetic clock was calculated by Horvath method. The chip analysis methylation pipeline (ChAMP) package selected the differentially methylated regions (DMRs). RESULTS: The SG had lower ZnS levels than the CG. Moreover, in SG, the ZnS levels were negatively associated with the epigenetic age acceleration. The DMR analysis revealed 37 DMRs associated with ZnDI and ZnS levels. The DMR of PM20D1 gene was commonly associated with ZnDI and ZnS levels and was hypomethylated in the SG. CONCLUSION: Our findings provide new information on Zn's modulation of DNA methylation patterns and bring new perspectives for understanding the nutriepigenetic mechanisms in obesity

14-weeks combined exercise epigenetically modulated 118 genes of menopausal women with prediabetes

Background: Pre-diabetes precedes Diabetes Mellitus (DM) disease and is a critical period for hyperglycemia treatment, especially for menopausal women, considering all metabolic alterations due to hormonal changes. Recently, the literature has demonstrated the role of physical exercise in epigenetic reprogramming to modulate the gene expression patterns of metabolic conditions, such as hyperglycemia, and prevent DM development. In the present study, we hypothesized that physical exercise training could modify the epigenetic patterns of women with poor glycemic control. Methods: 48 post-menopause women aged 60.3 ± 4.5 years were divided according to their fasting blood glucose levels into two groups: Prediabetes Group, PG (n=24), and Normal Glucose Group, NGG (n=24). All participants performed 14 weeks of physical exercise three times a week. The Infinium Methylation EPIC BeadChip measured the participants’ Different Methylated Regions (DMRs). Results: Before the intervention, the PG group had 12 DMRs compared to NGG. After the intervention, five DMRs remained different. Interestingly, when comparing the PG group before and after training, 118 DMRs were found. The enrichment analysis revealed that the genes were related to different biological functions such as energy metabolism, cell differentiation, and tumor suppression. Conclusion: Physical exercise is a relevant alternative in treating hyperglycemia and preventing DM in post-menopause women with poor glycemic control

ChREBP Regulates Itself and Metabolic Genes Implicated in Lipid Accumulation in β-Cell Line.

Carbohydrate response element binding protein (ChREBP) is an important transcription factor that regulates a variety of glucose-responsive genes in hepatocytes. To date, only two natural isoforms, Chrebpα and Chrebpβ, have been identified. Although ChREBP is known to be expressed in pancreatic β cells, most of the glucose-responsive genes have never been verified as ChREBP targets in this organ. We aimed to explore the impact of ChREBP expression on regulating genes linked to accumulation of lipid droplets, a typical feature of β-cell glucotoxicity. We assessed gene expression in 832/13 cells overexpressing constitutively active ChREBP (caChREBP), truncated ChREBP with nearly identical amino acid sequence to Chrebpβ, or dominant negative ChREBP (dnChREBP). Among multiple ChREBP-controlled genes, ChREBP was sufficient and necessary for regulation of Eno1, Pklr, Mdh1, Me1, Pdha1, Acly, Acaca, Fasn, Elovl6, Gpd1, Cpt1a, Rgs16, Mid1ip1,Txnip, and Chrebpβ. Expression of Chrebpα and Srebp1c were not changed by caChREBP or dnChREBP. We identified functional ChREBP binding sequences that were located on the promoters of Chrebpβ and Rgs16. We also showed that Rgs16 overexpression lead to increased considerable amounts of lipids in 832/13 cells. This phenotype was accompanied by reduction of Cpt1a expression and slight induction of Fasn and Pklr gene in these cells. In summary, we conclude that Chrebpβ modulates its own expression, not that of Chrebpα; it also regulates the expression of several metabolic genes in β-cells without affecting SREBP-1c dependent regulation. We also demonstrate that Rgs16 is one of the ChREBP-controlled genes that potentiate accumulation of lipid droplets in β-cells

Exogenous ChREBP regulates <i>Chrebpβ</i> expression.

<p>(A) Amino acid alignment among rat ChREBPβ, mouse ChREBPβ, caChREBP, and dnChREBP. GRACE, glucose response activation conserved element; bHLH, basic Helix-Loop-Helix-Leucine. (B) Schematic diagram of tetracycline-inducible vectors for overexpression of <i>caChREBP</i>, <i>dnChREBP</i>, and <i>eYFPnuc</i>. LTR, long terminal repeat; TRE, tetracycline-responsive promoter element; UBC, human ubiquitin C promoter; <i>rtTA3</i>, reverse tetracycline-transactivator 3; IRES, internal ribosomal entry site; <i>Puro</i>^<i>R</i>, puromycin resistance gene; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; SIN LTR, self-inactivating long terminal repeat. (C) caChREBP induces <i>Chrebpβ</i> expression. We incubated caChREBP cells for 48h in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. We isolated the RNA and performed RT-qPCR using ChREBP isoform-specific primers. The histograms are the means of relative RNA levels normalized to <i>Eef1g</i> and <i>Rpl13a</i> and expressed as fold activation over the activity seen in eYFPnuc cells incubated in RPMI with 11 mmol/l D-glucose in the presence of doxycycline 1 μg/mL. *, p< 0.05 compared with eYFPnuc cells. (D) dnChREBP reduces the <i>Chrebpβ</i> expression. We preincubated dnChREBP cells for 24h in RPMI with 5.5 mmol/l D-glucose in the presence of doxycycline 1 μg/mL to minimize expression of endogenous nuclear ChREBP and let the induced dnChREBP occupy ChoREs and switched to in RPMI with 25 mmol/l D-glucose in the presence of doxycycline 1 μg/mL for 48h to induce strong expression and activity of endogenous nuclear ChREBP. We isolated the RNA and performed RT-qPCR using ChREBP isoform-specific primers. The histograms are the means of relative RNA levels normalized to <i>Rns18</i> and <i>Hprt1</i> and expressed as fold activation over the activity seen in eYFPnuc cells incubated under the same condition. *, p< 0.05 compared with eYFPnuc cells. (E) Alignment of ChoRE sequence presents in <i>Chrebpβ</i> promoter among rat (at the position -109 to -93), mouse, dog, horse, rhesus, and human. Sequence assemblies and coordinates are as follows: Rat: Mar.2012 Chr12(-): 26638089–26638073, Mouse: Jul.2007 Chr5(+): 135565651–135565667, Rhesus: Jan.2006 Chr3(-):51178915–51178899, Horse: Jan.2007 ChrUn(-):175282430–175282414, Dog: May 2005 Chr6(+): 9619423–9619439, Human: Mar.2006 Chr7(-):72700300–72700284. Color coding: light grey, identical residues; dark grey, unconserved residues. (F) Functional analysis of putative ChoRE sequences at the position -109 to -92 on <i>Chrebpβ</i>. We co-transfected luciferase reporter driven by two copies of rat <i>Chrebpβ</i> ChoRE upstream of minimal TATA promoter with caChREBP in 832/13 cells in RPMI with 5.5 mmol/l D-glucose. <i>Gaussia</i> luciferase activity was measured at 48h and normalized to <i>Cypridina</i> luciferase, and expressed as fold activation over the activity seen in cells transfected with two copies of ChoRE with minimal TATA promoter and empty vector. *, p< 0.05.</p