The role of genetic variation and DNA methylation in human glucose metabolism and type 2 diabetes

The incidence of diabetes is increasing worldwide, with the most prevalent form being type 2 diabetes. Two fundamental processes contribute to the development of type 2 diabetes: insulin resistance in target organs and insufficient insulin secretion from the pancreatic beta-cells. The aim of this thesis was to explore the role of DNA methylation and common genetic variation on glucose metabolism and the pathogenesis of type 2 diabetes. Reduced oxidative capacity of the mitochondria in skeletal muscle has been suggested to play a role in insulin resistance and type 2 diabetes. In studies I and II, we investigated the regulation of COX7A1 and ATP5O, which encode two subunits of the mitochondrial respiratory chain. We found that genetic variation and age were associated with skeletal muscle mRNA expression in both studies. mRNA levels were also positively correlated with the expression of the transcriptional co-activator PPARGC1A and insulin-stimulated glucose uptake, i.e., elderly individuals had reduced mRNA expression levels and reduced in vivo glucose uptake. Additionally, DNA methylation of the COX7A1 promoter was increased in elderly individuals concordant with the decrease in COX7A1 mRNA expression, suggesting a role for genetic, epigenetic and non-genetic factors in gene regulation. In study III, we investigated a common genetic variant in MTNR1B that has previously been found to be associated with increased risk of type 2 diabetes, increased fasting plasma glucose and impaired insulin secretion in populations of European ancestry. We aimed to replicate these findings in a type 2 diabetes case-control cohort of Han Chinese ancestry. We confirmed the association between rs10830963 and both the risk of type 2 diabetes and increased fasting plasma glucose levels, suggesting a relatively ancient origin for this variant. In study IV, common genetic variants that introduce or remove potential DNA methylation sites were selected based on their association with the risk of type 2 diabetes and changes in gene expression in blood. These genetic variants were analysed together with the level of DNA methylation and gene expression in human skeletal muscle, adipose tissue, blood and pancreatic islets. We found that 18 of the 19 sites that we analysed were associated with a difference in DNA methylation related to genotype, and for 11 of these sites this finding was consistent in all four tissues. Additionally, our data suggested a tissue-specific pattern of DNA methylation. Our results confirm an interaction between genetic and epigenetic mechanisms, which introduces a new level of complexity to our knowledge of gene regulation in type 2 diabetes

Identification of CpG-SNPs associated with type 2 diabetes and differential DNA methylation in human pancreatic islets.

AIMS/HYPOTHESIS: To date, the molecular function of most of the reported type 2 diabetes-associated loci remains unknown. The introduction or removal of cytosine-phosphate-guanine (CpG) dinucleotides, which are possible sites of DNA methylation, has been suggested as a potential mechanism through which single-nucleotide polymorphisms (SNPs) can affect gene function via epigenetics. The aim of this study was to examine if any of 40 SNPs previously associated with type 2 diabetes introduce or remove a CpG site and if these CpG-SNPs are associated with differential DNA methylation in pancreatic islets of 84 human donors. METHODS: DNA methylation was analysed using pyrosequencing. RESULTS: We found that 19 of 40 (48%) type 2 diabetes-associated SNPs introduce or remove a CpG site. Successful DNA methylation data were generated for 16 of these 19 CpG-SNP loci, representing the candidate genes TCF7L2, KCNQ1, PPARG, HHEX, CDKN2A, SLC30A8, DUSP9, CDKAL1, ADCY5, SRR, WFS1, IRS1, DUSP8, HMGA2, TSPAN8 and CHCHD9. All analysed CpG-SNPs were associated with differential DNA methylation of the CpG-SNP site in human islets. Moreover, six CpG-SNPs, representing TCF7L2, KCNQ1, CDKN2A, ADCY5, WFS1 and HMGA2, were also associated with DNA methylation of surrounding CpG sites. Some of the type 2 diabetes CpG-SNP sites that exhibit differential DNA methylation were further associated with gene expression, alternative splicing events determined by splice index, and hormone secretion in the human islets. The 19 type 2 diabetes-associated CpG-SNPs are in strong linkage disequilibrium (r (2) > 0.8) with a total of 295 SNPs, including 91 CpG-SNPs. CONCLUSIONS/INTERPRETATION: Our results suggest that the introduction or removal of a CpG site may be a molecular mechanism through which some of the type 2 diabetes SNPs affect gene function via differential DNA methylation and consequently contributes to the phenotype of the disease

Two common genetic variants near nuclear-encoded OXPHOS genes are associated with insulin secretion in vivo

Context Mitochondrial ATP production is important in the regulation of glucose-stimulated insulin secretion. Genetic factors may modulate the capacity of the β-cells to secrete insulin and thereby contribute to the risk of type 2 diabetes. OBJECTIVE: The aim of this study was to identify genetic loci in or adjacent to nuclear encoded genes of the oxidative phosphorylation (OXPHOS) pathway that are associated with insulin secretion in vivo. DESIGN AND METHODS: To find polymorphisms associated with glucose-stimulated insulin secretion, data from a genome-wide association study (GWAS) of 1467 non-diabetic individuals, the Diabetes Genetic Initiative (DGI), was examined. 413 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) ≥0.05 located in or adjacent to 76 OXPHOS genes were included in the DGI GWAS. A more extensive population based study of 4323 non-diabetics, the PPP-Botnia, was used as a replication cohort. Insulinogenic index during an oral glucose tolerance test (OGTT) was used as a surrogate marker of glucose-stimulated insulin secretion. Multivariate linear regression analyses were used to test genotype-phenotype associations. RESULTS: Two common variants were indentified in the DGI, where the major C-allele of rs606164, adjacent to NDUFC2 (NADH dehyrogenase (ubiqinone) 1 subunit C2), and the minor G-allele of rs1323070, adjacent to COX7A2 (cythochrome c oxidase subunit VIIa polypeptide 2), showed nominal associations with decreased glucose-stimulated insulin secretion (p=0.0009 respective p=0.003). These associations were replicated in PPP-Botnia (p=0.002 and p=0.05). CONCLUSION: Our study shows that genetic variation near genes involved in oxidative phosphorylation may influence glucose-stimulated insulin secretion in vivo

Investigation of Type 2 Diabetes Risk Alleles Support CDKN2A/B, CDKAL1, and TCF7L2 As Susceptibility Genes in a Han Chinese Cohort

Background: Recent genome-wide association studies (GWASs) have reported several genetic variants to be reproducibly associated with type 2 diabetes. Additional variants have also been detected from a metaanalysis of three GWASs, performed in populations of European ancestry. In the present study, we evaluated the influence of 17 genetic variants from 15 candidate loci, identified in type 2 diabetes GWASs and the metaanalysis, in a Han Chinese cohort. Methodology/Principal Findings: Selected type 2 diabetes-associated genetic variants were genotyped in 1,165 type 2 diabetic patients and 1,136 normoglycemic control individuals of Southern Han Chinese ancestry. The OR for risk of developing type 2 diabetes was calculated using a logistic regression model adjusted for age, sex, and BMI. Genotype-phenotype associations were tested using a multivariate linear regression model. Genetic variants in CDKN2A/B, CDKAL1, TCF7L2, TCF2, MC4R, and PPARG showed a nominal association with type 2 diabetes (P <= 0.05), of whom the three first would stand correction for multiple testing: CDKN2A/B rs10811661, OR: 1.26 (1.12-1.43) P = 1.8* 10(-4); CDKAL1 rs10946398, OR: 1.23 (1.09-1.39); P = 7.1* 10(-4), and TCF7L2 rs7903146, OR: 1.61 (1.19-2.18) P = 2.3* 10(-3). Only nominal phenotype associations were observed, notably for rs8050136 in FTO and fasting plasma glucose (P = 0.002), postprandial plasma glucose (P = 0.002), and fasting C-peptide levels (P = 0.006) in the diabetic patients, and with BMI in controls (P = 0.033). Conclusions/Significance: We have identified significant association between variants in CDKN2A/B, CDKAL1 and TCF7L2, and type 2 diabetes in a Han Chinese cohort, indicating these genes as strong candidates conferring susceptibility to type 2 diabetes across different ethnicities

Epigenetic markers to further understand insulin resistance

Epigenetic variation in human adipose tissue has been linked to type 2 diabetes and its related risk factors including age and obesity. Insulin resistance, a key risk factor for type 2 diabetes, may also be associated with altered DNA methylation in visceral and subcutaneous adipose tissue. Furthermore, linking epigenetic variation in target tissues to similar changes in blood cells may identify new blood-based biomarkers. In this issue of Diabetologia, Arner et al studied the transcriptome and methylome in subcutaneous and visceral adipose tissue of 80 obese women who were either insulin-sensitive or -resistant (DOI 10.1007/s00125-016-4074-5). While they found differences in gene expression between the two groups, no alterations in DNA methylation were found after correction for multiple testing. Nevertheless, based on nominal p values, their methylation data overlapped with methylation differences identified in adipose tissue of individuals with type 2 diabetes compared with healthy individuals. Differential methylation of these overlapping CpG sites may predispose to diabetes by occurring already in the insulin-resistant state. Furthermore, some methylation changes may contribute to an inflammatory process in adipose tissue since the identified CpG sites were annotated to genes encoding proteins involved in inflammation. Finally, the methylation pattern in circulating leucocytes did not mirror the adipose tissue methylome of these 80 women. Together, identifying novel molecular mechanisms contributing to insulin resistance and type 2 diabetes may help advance the search for new therapeutic alternatives

DNA methylation as a diagnostic and therapeutic target in the battle against Type 2 diabetes.

Type 2 diabetes (T2D) develops due to insulin resistance and impaired insulin secretion, predominantly in genetically predisposed subjects exposed to nongenetic risk factors like obesity, physical inactivity and ageing. Emerging data suggest that epigenetics also play a key role in the pathogenesis of T2D. Genome-wide studies have identified altered DNA methylation patterns in pancreatic islets, skeletal muscle and adipose tissue from subjects with T2D compared with nondiabetic controls. Environmental factors known to affect T2D, including obesity, exercise and diet, have also been found to alter the human epigenome. Additionally, ageing and the intrauterine environment are associated with differential DNA methylation. Together, these data highlight a key role for epigenetics and particularly DNA methylation in the growing incidence of T2D

Genome-Wide DNA and Histone Modification Studies in Metabolic Disease

The last decade has witnessed a revolution in genetic technology, where genome-wide analyses, covering the majority of genetic variation, were thought to explain disease-causing mechanisms in common metabolic disorders. However, these genetic data only explain a modest proportion of the estimated heritability of type 2 diabetes and obesity and hence suggest a potential role for epigenetic variation in the etiology of metabolic disease. Indeed, recent genome-wide epigenetic studies have identified altered DNA methylation patterns in human pancreatic islets, adipose tissue, skeletal muscle, and blood from subjects with type 2 diabetes compared with normal subjects. Also, measures of obesity, such as increased body mass index (BMI), have been associated with epigenetic modifications in humans. It should also be noted that environmental risk factors for metabolic disease, for example, energy-rich diets, physical inactivity, and aging have been found to alter the epigenetic pattern genome-wide and in candidate genes for type 2 diabetes and obesity in human tissues. Additionally, interactions between genetic and epigenetic variations seem to contribute to the risk for metabolic disease. Together, genome-wide epigenetic studies highlight the importance of altered DNA methylation and histone modifications in the pathogenesis of metabolic disease. This chapter aims at summarizing current knowledge in the field of metabolic disease and genome-wide epigenetic analyses in humans

Epigenetics in Human Obesity and Type 2 Diabetes

Epigenetic mechanisms control gene activity and the development of an organism. The epigenome includes DNA methylation, histone modifications, and RNA-mediated processes, and disruption of this balance may cause several pathologies and contribute to obesity and type 2 diabetes (T2D). This Review summarizes epigenetic signatures obtained from human tissues of relevance for metabolism—i.e., adipose tissue, skeletal muscle, pancreatic islets, liver, and blood—in relation to obesity and T2D. Although this research field is still young, these comprehensive data support not only a role for epigenetics in disease development, but also epigenetic alterations as a response to disease. Genetic predisposition, as well as aging, contribute to epigenetic variability, and several environmental factors, including exercise and diet, further interact with the human epigenome. The reversible nature of epigenetic modifications holds promise for future therapeutic strategies in obesity and T2D. Epigenetic factors are suggested to contribute to metabolic dysfunctions. In this Review, Ling and Rönn summarize evidence for altered DNA methylation, both as a cause and a consequence of human obesity and type 2 diabetes. As epigenetic alterations are dynamic in nature, they may also provide targets for drug development

Epigenetic adaptation to regular exercise in humans.

Regular exercise has numerous health benefits, for example, it reduces the risk of cardiovascular disease and cancer. It has also been shown that the risk of type 2 diabetes can be halved in high-risk groups through nonpharmacological lifestyle interventions involving exercise and diet. Nevertheless, the number of people living a sedentary life is dramatically increasing worldwide. Researchers have searched for molecular mechanisms explaining the health benefits of regular exercise for decades and it is well established that exercise alters the gene expression pattern in multiple tissues. However, until recently it was unknown that regular exercise can modify the genome-wide DNA methylation pattern in humans. This review will focus on recent progress in the field of regular exercise and epigenetics