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Title:
DNA methylation profile associated with metabolic complications in obese individuals
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DNA methylation ; obesity ; hypercholesterolemia ; fibroblasts growth factor FGF21 ; microRNAs
Abstract:
Obesity is a chronic disease that affects over 650 million people worldwide. The causes for the development of the disease are increased consumption of fat and carbohydrates, low physical activity and individual genetic predisposition. Only a small percentage of obesity is monogenic. The remaining impact on the development of obesity is considered in the epigenetic regulations as DNA methylation, post-translational modifications of histones and synthesis of non-coding microRNAs that cause changes in genes activities. DNA methylation occurs by attaching a methyl group to the fifth carbon of the cytosine in gene promoters and important regulatory sites. DNA methylation is cell and tissue specific, sensitive to environmental influence, and may alter in response to changes in physical activity, caloric restrictions and bariatric surgery. It is postulated that DNA methylation may be the cause of development of obesity and its accompanying complications, but it also arises in response to disease. The assessment of the DNA methylation profile in obese patients' leukocytes, carried out as part of this doctoral dissertation, was associated with high levels of LDL cholesterol and FGF21 in the serum. OBJECTIVES The overall aim of the dissertation was to investigate whether there are epigenetic regulations related to metabolic complications i ; n obese patients. Specific objectives included: Checking if there is a DNA methylation profile associated with hypercholesterolaemia in the cohort of obese patients. Finding genes regulated by DNA methylation and associated with high levels of fibroblast growth factor 21 (FGF21) in obese subjects. To test if there is a specific miRNA expression profile associated with high levels of FGF21. RESEARCH METHODOLOGY AND RESULTS 1. Original article: Płatek T. et al.: DNA methylation microarrays identify epigenetically regulated lipid related genes in obese patients with hypercholesterolemia. Mol Med. 2020 Oct 7;26(1):93. The study was conducted in a group of 137 people (99 women and 38 men) with BMI over 27 (min. 27 - max. 45 kg/m2) and aged 25 to 65 years. The comparison of biochemical parameters was carried out in 2 subgroups: in the hypercholesterolaemia group with the LDL-CH level ≥ 3,4 mmol / l (n = 68) and in the control group with the LDLCH level <3,4 mmol / l (n = 69) in the serum. The concentration of glucose, total cholesterol, HDL cholesterol, TG, insulin, leptin, adiponectin, FGF19, FGF21, GIP in the serum and total fatty acid content in the plasma were determined. The study group showed higher levels of LDLCH, total cholesterol, TG, non-HDL cholesterol and TG/HDL ratio compared to the control group. Whole genome DNA methylation ; screening was performed on Agilent Technologies arrays (Human DNA Methylation Microarray: G4495A, Design ID, 023795) in ten randomly selected DNA samples from the entire cohort. In the differential DNA methylation analysis, 7480 statistically significant probes were identified comparing the high LDLCH group to the low LDL-CH group. The 190 probes related to lipid metabolism pathways were selected from this list. The selected genes have been involved in the following metabolic pathways: metabolism and clearance of LDL and VLDL lipoproteins, regulation of lipid metabolism by PPARα, SREBP and NR1H2, metabolism and betaoxidation of fatty acids, and metabolism of TG. New epigenetically regulated genes and associated with dyslipidemia have been demonstrated: ABCG4, ANGPTL4, AP2A2, AP2M1, AP2S1, CLTC, FGF19, FGF1R, HDLBP, LIPA, LMF1, LRP5, LSR, NR1H2 and ZDHHC8. 2. Original article: Płatek T. et al.: Epigenetic Regulation of Processes Related to High Level of Fibroblast Growth Factor 21 in Obese Subjects. Genes (Basel). 2021 Feb 21;12(2):307. The study group included overweight and obese adults (n = 136; 100 women and 36 men) with a BMI of 27 to 45 kg/m2. Patients were divided into 2 subgroups based on fasting serum levels of FGF21 (FGF21 <213 pg / ml and ≥ 213 pg / ml). Glucose, insulin, GIP, lipids, selected adipokines, myokines and cyt ; okines were measured and compared in the high FGF21 group (n = 68) to the low FGF21 (n = 68) group in the serum. The group with high circulating FGF21 levels had a higher WHR, increased levels of fasting insulin and HOMA-IR, free fatty acids and triglycerides. Increased levels of GIP as well as markers of liver damage (ALT, GGT) were also found. Obese patients with high FGF21 had elevated levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein (MCP1), and decreased levels of adiponectin. Analysis of DNA methylation was performed on methylation microarrays (Human DNA Methylation Microarray: G4495A, Agilent Technologies) and miRNA expression on TLDA microarrays (TaqMan® Array Human MicroRNA A + B Cards Set v3.0, Thermo Scientific) in leukocytes of randomly selected samples (8 from each group). We identified 11198 differentially methylated CpG probes (p <0,01) in the high FGF21 group compared to the low FGF21 group. The identified genes were associated with glucose transport, insulin secretion and signaling, lipid transport and cellular metabolism, response to nutrient levels, thermogenesis, browning of adipose tissue and bone mineralization. We showed a statistically significant differences in the expression of four miRNAs in peripheral blood leukocytes that were associated with high serum levels of FGF21 ; . Increased expression of hsa-miR-875-5p and decreased expression of hsa-miR-133a-3p, hsa-miR185-5p and hsa-miR-200c-3p were found in the group with high serum FGF21. The detected regulations were associated with high levels of FGF21, VEGF and low levels of adiponectin in the serum. CONCLUSIONS 1. A specific DNA methylation profile has been demonstrated in peripheral blood leukocytes associated with high levels of LDL cholesterol in obese individuals. 2. The presented DNA methylation pattern were in genes related to lipid metabolism. 3. We showed novel epigenetically regulated genes: ABCG4, ANGPTL4, AP2A2, AP2M1, AP2S1, CLTC, FGF19, FGF1R, HDLBP, LIPA, LMF1, LRP5, LSR, NR1H2 and ZDHHC8 and associated with high serum LDL cholesterol. 4. We demonstrated the metabolic profile associated with high level of FGF21 in the studied group of obese patients. 5. The DNA methylation profile associated with high levels of FGF21 in obese subjects were presented in peripheral blood leukocytes. 6. Altered expression of four miRNAs was demonstrated: decreased for hsa-miR-133a-3p, hsa-miR-185-5p and hsamiR-200c-3p and increased for hsa-miR-875-5p in peripheral blood leukocytes in obese subjects with high serum levels of FGF21. These findings may have a potential clinical significance as it has been shown that there is a regulation of epigenetic pr ; ocesses related to high levels of LDL cholesterol and the FGF21 in obesity. The reversible nature of DNA methylation makes it possible to develop new methods of preventing and treating lipid disorders accompanying obesity.
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Rada Dyscypliny Nauki medyczne