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 (r² > 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.

Telomere length in blood and skeletal muscle in relation to measures of glycaemia and insulinaemia.

AIMS: Skeletal muscle is a major metabolic organ and plays important roles in glucose metabolism, insulin sensitivity and insulin action. Muscle telomere length reflects the myocyte's exposure to harmful environmental factors. Leukocyte telomere length is considered a marker of muscle telomere length and is used in epidemiologic studies to assess associations with ageing-related diseases where muscle physiology is important. However, the extent to which leucocyte and muscle telomere length are correlated is unknown, as are their relative correlations with glucose and insulin concentrations. The purpose of this study was to determine the extent of these relationships. METHODS: Leucocyte and muscle telomere length were measured by quantitative real-time polymerase chain reaction in participants from the Malmö Exercise Intervention (n = 27) and the Prevalence, Prediction and Prevention of Diabetes-Botnia studies (n = 31). Participants in both studies were free from Type 2 diabetes. We assessed the association between leucocyte telomere length, muscle telomere length and metabolic traits using Spearmen correlations and multivariate linear regression. Bland-Altman analysis was used to assess agreement between leucocyte and muscle telomere length. RESULTS: In age-, study-, diabetes family history- and sex-adjusted models, leucocyte and muscle telomere length were positively correlated (r = 0.39, 95% CI 0.15-0.59). Leucocyte telomere length was inversely associated with 2-h glucose concentrations (r = -0.58, 95% CI -1.0 to -0.16), but there was no correlation between muscle telomere length and 2-h glucose concentrations (r = 0.05, 95% CI -0.35 to 0.46) or between leucocyte or muscle telomere length with other metabolic traits. CONCLUSIONS: In summary, the current study supports the use of leucocyte telomere length as a proxy for muscle telomere length in epidemiological studies of Type 2 diabetes aetiology.

Unique splicing pattern of the TCF7L2 gene in human pancreatic islets.

AIMS/HYPOTHESIS: Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored. METHODS: RT-PCR and quantitative real-time PCR were employed to characterise TCF7L2 splicing in pancreatic islets, blood lymphocytes, skeletal muscle and subcutaneous and visceral adipose tissue from non-diabetic individuals. RESULTS: The mapping of TCF7L2 splice variants shows a specific pattern in pancreatic islets, with four predominant transcripts and high usage of the variable exons 4 and 15. The overall concentration of TCF7L2 mRNA is highest in islets and fat and lower in blood and muscle. No significant difference in overall amount or splicing pattern was observed between carriers and non-carriers of the rs7903146 risk (T) allele. However, incorporation of exon 4 in islets correlates positively with plasma HbA(1c) levels (r = 0.758; p = 0.018). CONCLUSIONS/INTERPRETATION: There were pronounced tissue-specific differences in the splicing of TCF7L2 with forms containing exon 4 and 15 being most abundant in islets. The incorporation of exon 4 in islets correlated with HbA(1c) levels. Further experiments will be needed to determine the direction of this correlation, and larger cohorts needed to unequivocally resolve whether there is a relationship between genotype and splicing in islets.

A common variant in MTNR1B, encoding melatonin receptor 1B, is associated with type 2 diabetes and fasting plasma glucose in Han Chinese individuals.

AIMS/HYPOTHESIS: Recently, three groups independently reported that variation in MTNR1B, the gene encoding melatonin receptor 1B, was associated with an increased risk of type 2 diabetes, increased fasting plasma glucose and impaired insulin secretion in populations of European ancestry. In the present study, we investigated whether a single MTNR1B polymorphism was associated with type 2 diabetes in Han Chinese individuals, to elucidate whether this is a cross-populational effect. METHODS: The MTNR1B variant rs10830963 was genotyped in 1,165 type 2 diabetic patients and 1,105 normoglycaemic control individuals of southern Han Chinese ancestry who were residents of the metropolitan area of Shanghai. The risk of developing type 2 diabetes was calculated using a logistic regression model adjusted for age, sex and BMI. A possible association with fasting plasma glucose was analysed in the normoglycaemic control individuals using a multiple linear regression analysis with adjustments for age, sex and BMI. RESULTS: The genetic variant rs10830963 was associated with an increased risk of type 2 diabetes in our Han Chinese cohort (OR 1.16, 95% CI 1.03-1.31, p = 0.015). As previously described, the risk variant was also associated with increased fasting plasma glucose, showing an increase of 0.068 mmol/l (95% CI 0.036-0.100, p = 4 x 10(-5)) per risk allele. CONCLUSIONS/INTERPRETATION: A common variant in the MTNR1B gene is associated with an increased risk of type 2 diabetes and increased fasting plasma glucose in Han Chinese, suggesting an important role for this polymorphism in populations of different ethnic and environmental backgrounds.

Less pronounced response to exercise in healthy relatives to type 2 diabetic subjects compared with controls.

Healthy first-degree relatives with heredity of type 2 diabetes (FH+) are known to have metabolic inflexibility compared with subjects without heredity for diabetes (FH-). In this study, we aimed to test the hypothesis that FH+ individuals have an impaired response to exercise compared with FH-. Sixteen FH+ and 19 FH- insulin-sensitive men similar in age, peak oxygen consumption (V̇o2 peak), and body mass index completed an exercise intervention with heart rate monitored during exercise for 7 mo. Before and after the exercise intervention, the participants underwent a physical examination and tests for glucose tolerance and exercise capacity, and muscle biopsies were taken for expression analysis. The participants attended, on average, 39 training sessions during the intervention and spent 18.8 MJ on exercise. V̇o2 peak/kg increased by 14%, and the participants lost 1.2 kg of weight and 3 cm waist circumference. Given that the FH+ group expended 61% more energy during the intervention, we used regression analysis to analyze the response in the FH+ and FH- groups separately. Exercise volume had a significant effect on V̇o2 peak, weight, and waist circumference in the FH- group, but not in the FH+ group. After exercise, expression of genes involved in metabolism, oxidative phosphorylation, and cellular respiration increased more in the FH- compared with the FH+ group. This suggests that healthy, insulin-sensitive FH+ and FH- participants with similar age, V̇o2 peak, and body mass index may respond differently to an exercise intervention. The FH+ background might limit muscle adaptation to exercise, which may contribute to the increased susceptibility to type 2 diabetes in FH+ individuals.

Extensive changes in the transcriptional profile of human adipose tissue including genes involved in oxidative phosphorylation after a 6-month exercise intervention.

AIM: Adipose tissue has an important function in total energy homeostasis, and its dysregulation may contribute to lifestyle-related diseases such as type 2 diabetes, cancer and cardiovascular diseases. The aim of this study was to investigate genome-wide mRNA expression in adipose tissue in healthy men before and after an exercise intervention to identify genes or pathways that mediate the beneficial effect of regular exercise. We also investigated the difference in adipose tissue mRNA expression between individuals with or without a family history of type 2 diabetes. METHODS: The 6-month supervised exercise intervention was conducted in 47 healthy men (age 37.8 ± 4.3 years, BMI 28.5 ± 3.6 kg m(-2) ) with a previous low level of physical activity. RNA was analysed using GeneChip Human Gene 1.0 ST arrays (Affymetrix) before and after the exercise. RESULTS: We identified 2,560 significant transcripts differentially expressed before vs. after exercise with a false discovery rate (FDR) < 0.1%, including genes encoding the respiratory chain, histone subunits, small nucleolar RNAs and ribosomal proteins. Additionally, pathways enriched in response to exercise include the ribosome, oxidative phosphorylation, proteasome and many metabolic pathways, whereas the WNT and MAPK signalling pathways were down-regulated (FDR < 5%) after exercise. There were no significant differences in mRNA expression between individuals with or without a family history of type 2 diabetes. CONCLUSION: Exercise increased the expression of genes involved in oxidative phosphorylation, which is the opposite of what has been seen in adipose tissue from elderly or obese individuals with low physical fitness, and our study thereby demonstrates a mechanism for the beneficial effect of exercise.

First-degree relatives of type 2 diabetic patients have reduced expression of genes involved in fatty acid metabolism in skeletal muscle.

CONTEXT: First-degree relatives of patients with type 2 diabetes (FH+) have been shown to have decreased energy expenditure and decreased expression of mitochondrial genes in skeletal muscle. In previous studies, it has been difficult to distinguish whether mitochondrial dysfunction and differential regulation of genes are primary (genetic) or due to reduced physical activity, obesity, or other correlated factors. OBJECTIVE: The aim of this study was to investigate whether mitochondrial dysfunction is a primary defect or results from an altered metabolic state. DESIGN: We compared gene expression in skeletal muscle from 24 male subjects with FH and 26 without FH matched for age, glucose tolerance, VO(2peak) (peak oxygen uptake), and body mass index using microarrays. Additionally, type fiber composition, mitochondrial DNA content, and citrate synthase activity were measured. The results were followed up in an additional cohort with measurements of in vivo metabolism. RESULTS: FH+ vs. FH- subjects showed reduced expression of mitochondrial genes (P = 2.75 × 10(-6)), particularly genes involved in fatty acid metabolism (P = 4.08 × 10(-7)), despite similar mitochondrial DNA content. Strikingly, a 70% reduced expression of the monoamine oxidase A (MAOA) gene was found in FH+ vs. FH- individuals (P = 0.0009). Down-regulation of the genes involved in fat metabolism was associated with decreased in vivo fat oxidation and increased glucose oxidation examined in an additional cohort of elderly men. CONCLUSIONS: These results suggest that genetically altered fatty acid metabolism predisposes to type 2 diabetes and propose a role for catecholamine-metabolizing enzymes like MAOA in the regulation of energy metabolism.

Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion.

AIMS/HYPOTHESIS: Insulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1alpha; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion. METHODS: The PPARGC1A Gly482Ser polymorphism was genotyped in human pancreatic islets from 48 non-diabetic and 12 type 2 diabetic multi-organ donors and related to PPARGC1A mRNA expression. DNA methylation of the PPARGC1A promoter was analysed in pancreatic islets from ten type 2 diabetic and nine control donors. Isolated human islets were transfected with PPARGC1A silencing RNA (siRNA). RESULTS: PPARGC1A mRNA expression was reduced by 90% (p<0.005) and correlated with the reduction in insulin secretion in islets from patients with type 2 diabetes. After downregulation of PPARGC1A expression in human islets by siRNA, insulin secretion was reduced by 41% (p

Age influences DNA methylation and gene expression of COX7A1 in human skeletal muscle.

AIMS/HYPOTHESIS: Reduced oxidative capacity of the mitochondria in skeletal muscle has been suggested to contribute to insulin resistance and type 2 diabetes. Moreover, a set of genes influencing oxidative phosphorylation (OXPHOS) is downregulated in diabetic muscle. Here we studied whether genetic, epigenetic and non-genetic factors influence a component of the respiratory chain, COX7A1, previously shown to be downregulated in skeletal muscle from patients with type 2 diabetes. The specific aims were to: (1) evaluate the impact of genetic (single nucleotide polymorphisms [SNPs]), epigenetic (DNA methylation) and non-genetic (age) factors on the expression of COX7A1 in human skeletal muscle; and (2) investigate whether common variants in the COX7A1 gene are associated with increased risk of type 2 diabetes. METHODS: COX7A1 mRNA expression was analysed in muscle biopsies from young (n = 110) and elderly (n = 86) non-diabetic twins and related to measures of in vivo metabolism. Genetic variants (three SNPs) from the COX7A1 locus were genotyped in the twins and in two independent type 2 diabetes case-control cohorts (n = 1466 and 6380, respectively). DNA methylation of the COX7A1 promoter was analysed in a subset of twins (ten young, ten elderly) using bisulphite sequencing. RESULTS: While DNA methylation of the COX7A1 promoter was increased in muscle from elderly compared with young twins (19.9 +/- 8.3% vs 1.8 +/- 2.7%; p = 0.035), the opposite was found for COX7A1 mRNA expression (elderly 1.00 +/- 0.05 vs young 1.68 +/- 0.06; p = 0.0005). The heritability of COX7A1 expression was estimated to be 50% in young and 72% in elderly twins. One of the polymorphisms investigated, rs753420, influenced basal COX7A1 expression in muscle of young (p = 0.0001) but not of elderly twins. The transcript level of COX7A1 was associated with increased in vivo glucose uptake and VO(2max) (p = 0.009 and p = 0.001, respectively). We did not observe any genetic association between COX7A1 polymorphisms and type 2 diabetes after correcting for multiple testing. CONCLUSIONS/INTERPRETATION: Our results provide further evidence for age as a factor influencing DNA methylation and expression of OXPHOS genes, and thereby in vivo metabolism.