Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia arising from insulin resistance combined with relative insulin deficiency. As hyperglycemia becomes chronic, it leads to oxidative stress, which is one of the central mechanisms for glucose toxicity. It is the proximate cause of retinopathy, kidney failure, neuropathies, and macrovascular disease in diabetes [1].
The genetic basis for developing T2DM has been recognized for a long time. The concordance of T2DM in monozygotic twins is ~70.0% compared with 20.0-30.0% in dizygotic twins and a sibling of an affected individual has about three times higher risk for developing the disorder than the general population [2].
The decades of research into the genetic causes of T2DM have culminated with a succession of large genome-wide association studies. Despite their power and cost, they have identified genetic variants that increase T2DM risk by only 10.0-30.0% [3-5].
The incidence of T2DM has increased dramatically over the past decades [6], which is a too short period for accumulation of considerable alterations in the human genome. Therefore, it is likely that environmental factors, such as diet and sedentary lifestyle, might play a significant role in the development of the disease.
The role of epigenetic factors in the gene environment interactions pointed to epigenetics as a possible molecular link between environmental factors and T2DM. Previous studies have shown that epigenetic mechanisms can predispose individuals to the diabetic phenotype. Conversely, the altered homeostasis in T2DM, such as prolonged hyperglycemia, dyslipidemia and increased oxidative stress could also cause epigenetic changes associated with the development of disease complications [7].
The operational definition of epigenetics proposes three successive signals for the establishment of the epi-genetic state: an environmental signal (i) that triggers a second intracellular signal, (ii) to establish the exact chromatin location where the modification will take place, and (iii) a third sustaining signal that helps maintain this modification. The epigenetic make-up changes during intrauter-ine and early postnatal development, as well as throughout adult life [8].
A classic example of epigenetic transcriptional regulation is the covalent post replicative methylation of DNA at the fifth position of the cytosine ring of CpG dinucleotides. DNA methylation is a highly dynamic process in development and disease. It is strongly associated with gene silencing and decreased gene expression [9]. The functional significance of DNA methylation alterations became apparent with their recognition as critical contributors to the pathogenesis of cancer, acting by silencing of tumor suppressor genes [10]. There is convincing evidence that DNA methylation is performed by DNA methyl-transferases (DNMTs). DNMT1 is essential for maintaining DNA methylation patterns in proliferating cells as it methylates CpG dinucleotides in the newly synthesized strand and DNMT3a and DNMT3b are necessary for
The biological consequences of DNA methylation are mediated by a family of methyl-CpG binding domain (MBD) proteins. Among these, only MBD2 seems to be specific for methyl-CpG sites alone. It directs a multi subunit complex containing nucleosome remodeling and histone deacetylase activities to methylated DNA, thus leading to gene silencing [12].
Taking into account the role of epigenetics in the pathogenesis of type 2 diabetes mellitus, we have per-formed analysis of
Based on the obtained results, we answered the following questions: i) are there any
We collected blood samples from healthy controls (
The participants were recruited at the Department of Diabetology, Clinical Centre of Endocrinology at the Medical University, Sofia, Bulgaria. All subjects provided written informed consent and were familiar with the aims, methods and risks of participating in the study in accordance with the Helsinki Declaration and rules of Good Clinical Practice. The study was approved by the Ethics Committee of the Medical University, Sofia, Bulgaria.
The patients were interviewed for T2DM duration, presence of arterial hypertension and other chronic diseases. Body mass index (BMI) was calculated. Arterial blood pressure was measured in standard conditions. Laboratory parameters were measured in all participants at fasting: Hb A1c in whole blood by immuneturbidimetric method (Roche Diagnostics Deutschland GmbH, Mannheim, Germany); total cholesterol, high density lipoprotein (HDL) cholesterol, and triglycerides by enzymatic colorimetric method (Roche Diagnostics); low density lipoprotein (LDL) cholesterol was calculated using Fridewald’s formula. The 2005 International Diabetes Federation (IDF) definition of the metabolic syndrome (MetS) was used. The main characteristics of the subgroups are summarized in Table 1.
Main characteristics (number, gender distribution, mean age, BMI, Hb A1c, diabetes duration, presence of MetS, arterial hypertension, chronic complications of diabetes of the participants in the subgroups according to diabetes duration: newly-diagnosed T2DM, T2DM duration of <5 years, and T2DM duration >5 years).
Parameters | Newly-Diagnosed T2DM | T2DM Duration <5 Years | T2DM Duration >5 Years |
---|---|---|---|
Number | 9 | 9 | 9 |
Sex | 6 males; 3 females | 5 males; 4 females | 5 males; 4 females |
Age (years) (mean + SD) | 62.0 + 7.5 | 51.3 + 14.0 | 55.7 + 7.7 |
BMI (kg/m2) | 34.8 + 8.4 | 38.1 + 13.7 | 34.0 + 2.7 |
Hb A1c (%) | 7.9 + 2.3 | 8.8 + 1.8 | 8.7 + 2.1 |
Duration (years) | 0.0 | 2.2 + 0.8 | 6.8 + 1.9 |
MetS (%) | 7/9 (77.8) | 6/9 (66.7) | 9/9 (100.0) |
Arterial hypertension (%) | 6/9 (66.7) | 8/9 (88.9) | 8/9 (88.9) |
Chronic complications of diabetes (%) | 2/9 (22.2) | 8/9 (88.9) | 8/9 (88.9) |
T2DM: type 2 diabetes mellitus; mean + SD: mean + standard deviation; BMI: body mass index; MetS: metabolic syndrome.
A subset of the blood samples from the patients (
RNA was purified using QIAamp® RNA Blood Kit (Qiagen GmbH, Hilden, Germany). An amount of 250 ng of each RNA sample was reverse transcribed to cDNA (QuantiTect Reverse Transcription kit; Qiagen). The cDNAs were further examined using the quantitative polymerase chain reaction (qPCR) Primer Assay for Human MBD2 (Cat. #PPH08621A-200; Qiagen). The real-time PCR (RT-PCR) amplification was performed on an ABI PRISM® 7500 instrument and the results were analyzed with ABI PRISM® Sequence Detection Software, ver.1.4.0 (Applied Biosystems, Foster City, CA, USA). The total volume of each reaction was 50 µL with less than 100 ng cDNA per reaction. The amplification conditions of each RT-PCR cycle were as follows: denaturation at 94 °C for 15 seconds, primer annealing at 55 °C for 30 seconds, followed by primer extension at 72 °C for 30 seconds. The expression levels of
Whole genome DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen). The DNA samples were combined into four DNA pools each of nine healthy controls, newly-diagnosed T2DM patients, patients with T2DM duration of less and more than 5 years. DNA in each pool was adjusted to a final concentration of 50 ng/µL. Each DNA pool was analyzed for the promoter methylation status of 22 genes included in the Human Stress & Toxicity PathwayFinder EpiTect Methyl II Signature PCR Array (Qiagen Sciences Inc., Germantown, MD, USA) (Table 2). The method is based on detection of remaining input DNA after cleavage with a methylation-sensitive and/or a methylation-dependent restriction enzyme. These enzymes will digest unmethylated and methylated DNA, respectively. Following digestion, the remaining DNA in each individual enzyme reaction is quantified by RT-PCR using primers that flank a promoter (gene) region of interest. The relative fractions of methylated and unmethylated DNA are subsequently determined by comparing the amount in each digest with that of a mock (no enzymes added) digest using a ∆CT method. Unmethylated represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene. Methylated represents fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene. Data quality control parameters were strictly accomplished according to the protocol of the manufacturer.
List of 22 analyzed genes and the processes they mediate.
Genes | Processes Mediated by the Genes |
---|---|
Oxidative stress | |
DNA damage | |
Unfolded protein response | |
Hypoxia | |
Heat shock protein | |
Growth arrest and senescence | |
Inflammation | |
Anti-apoptosis | |
Proliferation and carcinogenesis |
The gene expression analysis of
Using EpiTect Methyl II Signature PCR Array (Qiagen Sciences Inc.), the methylated and non methylated fraction of 11 genes, connected to cellular response to stress and toxicity, were successfully analyzed in all DNA pools. The methylation fraction of these genes is represented in Table 3.
Methylated (M) fraction of the analyzed genes in four DNA pools.
Genes | Controls M Fraction (%) | Newly-Diagnosed T2DM M Fraction (%) | T2DM Duration <5 Years M Fraction (%) | T2DM Duration >5 Years M Fraction (%) |
---|---|---|---|---|
0.0 | 5.9 | 0.5 | 47.8 | |
0.0 | 13.6 | 14.7 | 34.1 | |
0.0 | 0.0 | 0.7 | 0.0 | |
0.0 | 4.9 | 4.8 | 0.0 | |
98.5 | 98.4 | 97.7 | 95.9 | |
1.4 | 23.6 | 13.1 | 9.5 | |
2.4 | 10.4 | 4.9 | 15.7 | |
7.3 | 22.3 | 22.4 | 32.0 | |
0.0 | 13.2 | 6.6 | 7.6 | |
3.5 | 8.1 | 20.2 | 23.3 | |
0.0 | 5.9 | 6.4 | 10.2 |
In the healthy controls, the methylated fraction was 0% in six of the analyzed genes, ranged from 1.4 to 7.3% in four genes and was 98.5% for the
The genes
The epigenetic status of certain genes in the human genome is affected by external environmental factors, pathological conditions, as well as during the normal processes of aging. Type 2 diabetes mellitus is a common disease, the pathogenesis of which involves factors such as genetic susceptibility, obesity, decreased physical activity, imbalanced nutrition and age. Age is an important factor that increases the risk of T2DM and furthermore, increasing age and T2DM both lead to decreased oxidation capacity and mitochondrial dysfunction.The mechanisms of these processes can be influenced by both genetic factors and by epigenetic processes. Literature data suggest that aging of the individual changes the epigenetic status of the respiratory chain genes [1,5,6,13].
Methylation of CpG dinucleotides is an important epigenetic mechanism used by vertebrate cells to repress transcription of many tissue-specific genes [14]. DNA methylation is the major modification of eukaryotic genomes and plays an essential function in mammalian progression. Methyl-CpG binding domain proteins are capable of binding specifically to methylated DNA and MBD1 and MBD2 can in addition repress transcription from methylated gene promoters [15]. In light of the functional significance of MBD proteins in the epigenomic landscape, we aimed to provide knowledge about the expression of the
In the present study, we detected a 10.4-times average increase in mRNA expression levels of
The present study also involved analysis of the methyl-ation status of 22 genes, connected to cellular stress and toxicity, in four DNA pools of patients with newly-diagnosed T2DM, patients with T2DM duration of less or more than 5 years and in healthy controls. Eleven of these genes were successfully analyzed in all DNA pools. We were able to evaluate the average methylated fraction for every gene in each DNA pool. Notably, most of the analyzed genes lacked methylated fractions in healthy individuals, thus indicating an active transcriptional state. The only exception was
Five genes:
In the course of T2DM, the methylated fraction of the
The methylated fraction of
The methylated fraction of
The methylated fraction of the universal tumor suppressor gene
The increasing methylated fraction of
In conclusion, the detected higher expression of
The results from the present study require broadening of the set of genes analyzed for epigenetic changes in T2DM. Furthermore, the results for the genes with consistently increasing methylation in T2DM should be further validated
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.