The roles of IGF2 and DNMT methylation and elongase6 related fatty acids in metabolic syndrome.

Background: The prevalence of metabolic syndrome (MetS) has increased along with rapid socio-economic development in China in recent decades, aggravating the burden of the health care system. Both plasma levels of fatty acids (FAs) and aberrant DNA methylation profiles are associated with MetS risk. However, studies exploring the role of DNA methylation and FAs simultaneously in MetS etiology are sparse. Objective: We aimed to explore the association between the gene methylation levels of insulin-like growth factor II (IGF2), H19, DNA methyltransferases 1 (DNMT1), DNA methyltransferases 3a (DNMT3a), and DNA methyltransferases 3b (DNMT3b) and MetS risk, and the etiological role of elongation of very-long-chain fatty acid elongase 6 (ELOVL6) related fatty acids. Method: Plasma levels of FAs were measured using a Gas Chromatography-Flame Ionization Detector (GC-FID) after organic extraction, and gene methylation was quantified using a real-time Quantitative Polymerase Chain Reaction (Q-PCR) detecting system after bisulfite treatment. The C18/C16 ratio was used as the indicator of ELOVL6 activity. Odds Ratio (OR) and 95% Confidence Interval (CI) were estimated with logistic regression. Results: Methylation levels in IGF2 and DNMT3a were not significantly associated with MetS risk. However, when stratified by C18/C16 ratio (high vs. low), positive associations were observed between the risk of MetS and methylation levels (>median) of IGF2a3 (OR = 3.1, 95% CI = 1.3-7.5) and DNMT3a (OR = 2.5, 95% CI = 1.1-5.8) genes, in individuals with lower C18/C16 ratios, while no significant associations were observed in subjects with high C18/C16 ratios. Conclusion: Methylation levels in IGF2 and DNMT3a genes may affect the risk of MetS in an ELOVL6 activity-dependent way among Chinese adults. Further studies in other populations are needed to validate this finding.

Altered Indices of Fatty Acid Elongases ELOVL6, ELOVL5, and ELOVL2 Activities in Patients with Impaired Fasting Glycemia.

Background: Dysregulation of fatty acids (FA) seems to participate in the pathogenesis of disorders such as metabolic syndrome (MetS), cardiovascular diseases, or some cancers. Activities of enzymes FA desaturases and elongases [elongation of very long-chain fatty acid (ELOVL)] significantly influence FA profile in different body compartments. Although the impact of activities of desaturases on cardiometabolic diseases was broadly studied, relatively little attention was devoted to the role of elongases. Methods: Case-control study was carried out in 36 patients (18 men/18 women) with impaired fasting glycemia (IFG) without MetS and 36 age and gender-matched healthy controls. FA profiles in plasma phospholipids (PL) were assessed using gas chromatograph-flame ionization detector and indices of desaturase and elongase activities were calculated. Results: In the IFG group, we observed decreased estimated activities of ELOVL2 and ELOVL5, whereas higher estimated activities of elongase ELOVL6 were noted. IFG group was also characterized by altered composition of plasma PL FA, above all by lower percentage of cis-vaccenic acid (cVA; 18:1n-7) and of total polyunsaturated FA n-6, especially linoleic acid, and by higher proportion of stearic acid and gamma-linolenic acid. Concurrently, elevated estimated activities of desaturases delta-9-desaturase (D9D), D6D were found. Conclusions: Lower estimated activities of ELOVL2 and ELOVL5 with lowered proportion of PL cVA could be associated with disturbances of glucose homeostasis development and their corresponding indices could serve as biomarkers of such risk.

Higher Increase in Plasma DHA in Females Compared to Males Following EPA Supplementation May Be Influenced by a Polymorphism in ELOVL2: An Exploratory Study.

Young adult females have higher blood docosahexaenoic acid (DHA), 22:6n-3 levels than males, and this is believed to be due to higher DHA synthesis rates, although DHA may also accumulate due to a longer half-life or a combination of both. However, sex differences in blood fatty acid responses to eicosapentaenoic acid (EPA), 20:5n-3 or DHA supplementation have not been fully investigated. In this exploratory analysis, females and males (n = 14-15 per group) were supplemented with 3 g/day EPA, 3 g/day DHA, or olive oil control for 12 weeks. Plasma was analyzed for sex effects at baseline and changes following 12 weeks' supplementation for fatty acid levels and carbon-13 signature (δ13 C). Following EPA supplementation, the increase in plasma DHA in females (+23.8 ± 11.8, nmol/mL ± SEM) was higher than males (-13.8 ± 9.2, p < 0.01). The increase in plasma δ13 C-DHA of females (+2.79 ± 0.31, milliUrey (mUr ± SEM) compared with males (+1.88 ± 0.44) did not reach statistical significance (p = 0.10). The sex effect appears driven largely by increased plasma DHA in the AA genotype of females (+58.8 ± 11.5, nmol/mL ± SEM, n = 5) compared to GA + GG in females (+4.34 ± 13.5, n = 9) and AA in males (-29.1 ± 17.2, n = 6) for rs953413 in the ELOVL2 gene (p < 0.001). In conclusion, EPA supplementation increases plasma DHA levels in females compared to males, which may be dependent on the AA genotype for rs953413 in ELOVL2.

Age prediction in living: Forensic epigenetic age estimation based on blood samples.

DNA methylation analysis in a variety of genes has brought promising results in age estimation. The main aim of this study was to evaluate DNA methylation levels from four age-correlated genes, ELOVL2, FHL2, EDARADD and PDE4C, in blood samples of healthy Portuguese individuals. Fifty-three samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for CpG dinucleotide methylation status. Linear regression models were used to analyze relationships between methylation levels and chronological age. The highest age-associated CpG in each locus was chosen to build a multi-locus age prediction model (APM), allowing to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 5.35 years, explaining 94.1% of age variation. Validation approaches demonstrated the accuracy and reproducibility of the proposed multi-locus APM. Testing the APM in 51 blood samples from deceased individuals a MAD of 9.72 years was obtained. Potential differences in methylation status between samples from living and deceased individuals could exist since the highest age-correlated CpGs were different in some genes between both groups. In conclusion, our study using the bisulfite PCR sequencing method is in accordance with the high age prediction accuracy of DNA methylation levels in four previously reported age-associated genes. DNA methylation pattern differences between blood samples from living and deceased individuals should be taken into account in forensic contexts.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Effects of different dietary regimes alone or in combination with standardized Aronia melanocarpa extract supplementation on lipid and fatty acids profiles in rats.

This study investigated different dietary strategies, high-fat (HFd), or standard diet (Sd) alone or in combination with standardized Aronia melanocarpa extract (SAE), as a polyphenol-rich diet, and their effects on lipids and fatty acids (FA) in rats with metabolic syndrome (MetS). Wistar albino rats were randomly divided into two groups: healthy and rats with MetS, and then depending on dietary patterns on six groups: healthy rats fed with Sd, healthy rats fed with Sd and SAE, rats with MetS fed with HFd, rats with MetS fed with HFd and SAE, rats with MetS fed with Sd, and rats with MetS fed with Sd and SAE. 4 weeks later, after an overnight fast (12-14 h), blood for determination of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), index of lipid peroxidation (measured as TBARS), and FA was collected. Increased FA and lipid concentration found in MetS rats were reduced when changing dietary habits from HFd to Sd with or without SAE consumption. Consumption of SAE slightly affects the FA profiles, mostly palmitoleic acid in healthy rats and PUFA in MetS + HFd rats. Nevertheless, in a high-fat diet, SAE supplementation significantly decreases n-6/n-3 ratio, thereby decreasing systemic inflammation. Further researches are warranted to confirm these effects in humans.