Circulating triglycerides are associated with human adipose tissue DNA methylation of genes linked to metabolic disease.

Dysregulation of circulating lipids is a central element for the metabolic syndrome. However, it is not well established whether human subcutaneous adipose tissue is affected by or affect circulating lipids through epigenetic mechanisms. Hence, our aim was to investigate the association between circulating lipids and DNA methylation levels in human adipose tissue. DNA methylation and gene expression were analysed genome-wide in subcutaneous adipose tissue from two different cohorts, including 85 men and 93 women, respectively. Associations between DNA methylation and circulating levels of triglycerides, low-density lipoprotein, high-density lipoprotein and total cholesterol were analysed. Causal mediation analyses tested if adipose tissue DNA methylation mediates the effects of triglycerides on gene expression or insulin resistance. We found 115 novel associations between triglycerides and adipose tissue DNA methylation, e.g. in the promoter of RFS1, ARID2 and HOXA5 in the male cohort (P ≤ 1.1 × 10-7), and 63 associations, e.g. within the gene body of PTPRN2 and COL6A3 in the female cohort. We further connected these findings to altered mRNA expression levels in adipose tissue (e.g. HOXA5, IL11 and FAM45B). Interestingly, there was no overlap between methylation sites associated with triglycerides in men and the sites found in women, which points towards sex-specific effects of triglycerides on the epigenome. Finally, a causal mediation analysis provided support for adipose tissue DNA methylation as a partial mediating factor between circulating triglycerides and insulin resistance. This study identified novel epigenetic alterations in adipose tissue associated with circulating lipids. Identified epigenetic changes seem to mediate effects of triglycerides on insulin resistance.

Serum aromatic and branched-chain amino acids associated with NASH demonstrate divergent associations with serum lipids.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has been associated with multiple metabolic abnormalities. By applying a non-targeted metabolomics approach, we aimed at investigating whether serum metabolite profile that associates with NAFLD would differ in its association with NAFLD-related metabolic risk factors. METHODS & RESULTS: A total of 233 subjects (mean ± SD: 48.3 ± 9.3 years old; BMI: 43.1 ± 5.4 kg/m2 ; 64 male) undergoing bariatric surgery were studied. Of these participants, 164 with liver histology could be classified as normal liver (n = 79), simple steatosis (SS, n = 40) or non-alcoholic steatohepatitis (NASH, n = 45). Among the identified fasting serum metabolites with higher levels in those with NASH when compared to those with normal phenotype were the aromatic amino acids (AAAs: tryptophan, tyrosine and phenylalanine), the branched-chain amino acids (BCAAs: leucine and isoleucine), a phosphatidylcholine (PC(16:0/16:1)) and uridine (all FDRp < 0.05). Only tryptophan was significantly higher in those with NASH compared to those with SS (FDRp < 0.05). Only the AAAs tryptophan and tyrosine correlated positively with serum total and LDL cholesterol (FDRp < 0.1), and accordingly, with liver LDLR at mRNA expression level. In addition, tryptophan was the single AA associated with liver DNA methylation of CpG sites known to be differentially methylated in those with NASH. CONCLUSIONS: We found that serum levels of the NASH-related AAAs and BCAAs demonstrate divergent associations with serum lipids. The specific correlation of tryptophan with LDL-c may result from the molecular events affecting LDLR mRNA expression and NASH-associated methylation of genes in the liver.

Epigenome- and Transcriptome-wide Changes in Muscle Stem Cells from Low Birth Weight Men.

Background: Being born with low birth weight (LBW) is a risk factor for muscle insulin resistance and type 2 diabetes (T2D), which may be mediated by epigenetic mechanisms programmed by the intrauterine environment. Epigenetic mechanisms exert their prime effects in developing cells. We hypothesized that muscle insulin resistance in LBW subjects may be due to early differential epigenomic and transcriptomic alterations in their immature muscle progenitor cells.Results: Muscle progenitor cells were obtained from 23 healthy young adult men born at term with LBW, and 15 BMI-matched normal birth weight (NBW) controls. The cells were subsequently cultured and differentiated into myotubes. DNA and RNA were harvested before and after differentiation for genome-wide DNA methylation and RNA expression measurements.After correcting for multiple comparisons (q ≤ 0.05), 56 CpG sites were found to be significantly, differentially methylated in myoblasts from LBW compared with NBW men, of which the top five gene-annotated CpG sites (SKI, ARMCX3, NR5A2, NEUROG, ESRRG) previously have been associated to regulation of cholesterol, fatty acid and glucose metabolism and muscle development or hypertrophy. LBW men displayed markedly decreased myotube gene expression levels of the AMPK-repressing tyrosine kinase gene FYN and the histone deacetylase gene HDAC7. Silencing of FYN and HDAC7 was associated with impaired myotube formation, which for HDAC7 reduced muscle glucose uptake.Conclusions: The data provides evidence of impaired muscle development predisposing LBW individuals to T2D is linked to and potentially caused by distinct DNA methylation and transcriptional changes including down regulation of HDAC7 and FYN in their immature myoblast stem cells.

Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood.

Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.

Abnormal epigenetic changes during differentiation of human skeletal muscle stem cells from obese subjects.

BACKGROUND: Human skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects. METHODS: We used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model. RESULTS: We observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects. CONCLUSIONS: Our study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.

Adipose tissue transcriptomics and epigenomics in low birthweight men and controls: role of high-fat overfeeding.

AIMS/HYPOTHESIS: Individuals who had a low birthweight (LBW) are at an increased risk of insulin resistance and type 2 diabetes when exposed to high-fat overfeeding (HFO). We studied genome-wide mRNA expression and DNA methylation in subcutaneous adipose tissue (SAT) after 5 days of HFO and after a control diet in 40 young men, of whom 16 had LBW. METHODS: mRNA expression was analysed using Affymetrix Human Gene 1.0 ST arrays and DNA methylation using Illumina 450K BeadChip arrays. RESULTS: We found differential DNA methylation at 53 sites in SAT from LBW vs normal birthweight (NBW) men (false discovery rate <5%), including sites in the FADS2 and CPLX1 genes previously associated with type 2 diabetes. When we used reference-free cell mixture adjustments to potentially adjust for cell composition, 4,323 sites had differential methylation in LBW vs NBW men. However, no differences in SAT gene expression levels were identified between LBW and NBW men. In the combined group of all 40 participants, 3,276 genes (16.5%) were differentially expressed in SAT after HFO (false discovery rate <5%) and there was no difference between LBW men and controls. The most strongly upregulated genes were ELOVL6, FADS2 and NNAT; in contrast, INSR, IRS2 and the SLC27A2 fatty acid transporter showed decreased expression after HFO. Interestingly, SLC27A2 expression correlated negatively with diabetes- and obesity-related traits in a replication cohort of 142 individuals. DNA methylation at 652 CpG sites (including in CDK5, IGFBP5 and SLC2A4) was altered in SAT after overfeeding in this and in another cohort. CONCLUSIONS/INTERPRETATION: Young men who had a LBW exhibit epigenetic alterations in their adipose tissue that potentially influence insulin resistance and risk of type 2 diabetes. Short-term overfeeding influences gene transcription and, to some extent, DNA methylation in adipose tissue; there was no major difference in this response between LBW and control participants.

Epigenetic programming of adipose-derived stem cells in low birthweight individuals.

AIMS/HYPOTHESIS: Low birthweight (LBW) is associated with dysfunctions of adipose tissue and metabolic disease in adult life. We hypothesised that altered epigenetic and transcriptional regulation of adipose-derived stem cells (ADSCs) could play a role in programming adipose tissue dysfunction in LBW individuals. METHODS: ADSCs were isolated from the subcutaneous adipose tissue of 13 normal birthweight (NBW) and 13 LBW adult men. The adipocytes were cultured in vitro, and genome-wide differences in RNA expression and DNA methylation profiles were analysed in ADSCs and differentiated adipocytes. RESULTS: We demonstrated that ADSCs from LBW individuals exhibit multiple expression changes as well as genome-wide alterations in methylation pattern. Reduced expression of the transcription factor cyclin T2 encoded by CCNT2 may play a key role in orchestrating several of the gene expression changes in ADSCs from LBW individuals. Indeed, silencing of CCNT2 in human adipocytes decreased leptin secretion as well as the mRNA expression of several genes involved in adipogenesis, including MGLL, LIPE, PPARG, LEP and ADIPOQ. Only subtle genome-wide mRNA expression and DNA methylation changes were seen in mature cultured adipocytes from LBW individuals. CONCLUSIONS/INTERPRETATION: Epigenetic and transcriptional changes in LBW individuals are most pronounced in immature ADSCs that in turn may programme physiological characteristics of the mature adipocytes that influence the risk of metabolic diseases. Reduced expression of CCNT2 may play a key role in the developmental programming of adipose tissue.

Genome-wide analysis of DNA methylation in subjects with type 1 diabetes identifies epigenetic modifications associated with proliferative diabetic retinopathy.

BACKGROUND: Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). METHODS: DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. RESULTS: We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). CONCLUSIONS: Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR.

Predicting type 2 diabetes via machine learning integration of multiple omics from human pancreatic islets.

Type 2 diabetes (T2D) is the fastest growing non-infectious disease worldwide. Impaired insulin secretion from pancreatic beta-cells is a hallmark of T2D, but the mechanisms behind this defect are insufficiently characterized. Integrating multiple layers of biomedical information, such as different Omics, may allow more accurate understanding of complex diseases such as T2D. Our aim was to explore and use Machine Learning to integrate multiple sources of biological/molecular information (multiOmics), in our case RNA-sequening, DNA methylation, SNP and phenotypic data from islet donors with T2D and non-diabetic controls. We exploited Machine Learning to perform multiOmics integration of DNA methylation, expression, SNPs, and phenotypes from pancreatic islets of 110 individuals, with ~ 30% being T2D cases. DNA methylation was analyzed using Infinium MethylationEPIC array, expression was analyzed using RNA-sequencing, and SNPs were analyzed using HumanOmniExpress arrays. Supervised linear multiOmics integration via DIABLO based on Partial Least Squares (PLS) achieved an accuracy of 91 ± 15% of T2D prediction with an area under the curve of 0.96 ± 0.08 on the test dataset after cross-validation. Biomarkers identified by this multiOmics integration, including SACS and TXNIP DNA methylation, OPRD1 and RHOT1 expression and a SNP annotated to ANO1, provide novel insights into the interplay between different biological mechanisms contributing to T2D. This Machine Learning approach of multiOmics cross-sectional data from human pancreatic islets achieved a promising accuracy of T2D prediction, which may potentially find broad applications in clinical diagnostics. In addition, it delivered novel candidate biomarkers for T2D and links between them across the different Omics.

DNA methylation partially mediates antidiabetic effects of metformin on HbA1c levels in individuals with type 2 diabetes.

AIMS: Despite metformin being used as first-line pharmacological therapy for type 2 diabetes, its underlying mechanisms remain unclear. We aimed to determine whether metformin altered DNA methylation in newly-diagnosed individuals with type 2 diabetes. METHODS AND RESULTS: We found that metformin therapy is associated with altered methylation of 26 sites in blood from Scandinavian discovery and replication cohorts (FDR < 0.05), using MethylationEPIC arrays. The majority (88%) of these 26 sites were hypermethylated in patients taking metformin for âˆ¼ 3 months compared to controls, who had diabetes but had not taken any diabetes medication. Two of these blood-based methylation markers mirrored the epigenetic pattern in muscle and adipose tissue (FDR < 0.05). Four type 2 diabetes-associated SNPs were annotated to genes with differential methylation between metformin cases and controls, e.g., GRB10, RPTOR, SLC22A18AS and TH2LCRR. Methylation correlated with expression in human islets for two of these genes. Three metformin-associated methylation sites (PKNOX2, WDTC1 and MICB) partially mediate effects of metformin on follow-up HbA1c levels. When combining methylation of these three sites into a score, which was used in a causal mediation analysis, methylation was suggested to mediate up to 32% of metformin's effects on HbA1c. CONCLUSION: Metformin-associated alterations in DNA methylation partially mediates metformin's antidiabetic effects on HbA1c in newly-diagnosed individuals with type 2 diabetes.

Liver saturated fat content associates with hepatic DNA methylation in obese individuals.

BACKGROUND: Accumulation of saturated fatty acids (SFAs) in the liver is known to induce hepatic steatosis and inflammation causing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although SFAs have been shown to affect the epigenome in whole blood, pancreatic islets, and adipose tissue in humans, and genome-wide DNA methylation studies have linked epigenetic changes to NAFLD and NASH, studies focusing on the association of SFAs and DNA methylation in human liver are missing. We, therefore, investigated whether human liver SFA content associates with DNA methylation and tested if SFA-linked alterations in DNA methylation associate with NAFLD-related clinical phenotypes in obese individuals. RESULTS: We identified DNA methylation (Infinium HumanMethylation450 BeadChip) of 3169 CpGs to be associated with liver total SFA content (q-value < 0.05) measured using proton NMR spectroscopy in participants of the Kuopio Obesity Surgery Study (n = 51; mean ± SD:49.3 ± 8.5 years old; BMI:43.7 ± 6.2 kg/m2). Of these 3169 sites, 797 overlapped with previously published NASH-associated CpGs (NASH-SFA), while 2372 CpGs were exclusively associated with SFA (Only-SFA). The corresponding annotated genes of these only-SFA CpGs were found to be enriched in pathways linked to satiety and hunger. Among the 54 genes mapping to these enriched pathways, DNA methylation of CpGs mapping to PRKCA and TSPO correlated with their own mRNA expression (HumanHT-12 Expression BeadChip). In addition, DNA methylation of another ten of these CpGs correlated with the mRNA expression of their neighboring genes (p value < 0.05). The proportion of CpGs demonstrating a correlation of DNA methylation with plasma glucose was higher in NASH-SFA and only-SFA groups, while the proportion of significant correlations with plasma insulin was higher in only-NASH and NASH-SFA groups as compared to all CpGs on the Illumina 450 K array (Illumina, San Diego, CA, USA). CONCLUSIONS: Our results suggest that one of the mechanisms how SFA could contribute to metabolic dysregulation in NAFLD is at the level of DNA methylation. We further propose that liver SFA-related DNA methylation profile may contribute more to hyperglycemia, while insulin-related methylation profile is more linked to NAFLD or NASH. Further research is needed to elucidate the molecular mechanisms behind these observations.

Genes with epigenetic alterations in human pancreatic islets impact mitochondrial function, insulin secretion, and type 2 diabetes.

Epigenetic dysregulation may influence disease progression. Here we explore whether epigenetic alterations in human pancreatic islets impact insulin secretion and type 2 diabetes (T2D). In islets, 5,584 DNA methylation sites exhibit alterations in T2D cases versus controls and are associated with HbA1c in individuals not diagnosed with T2D. T2D-associated methylation changes are found in enhancers and regions bound by ß-cell-specific transcription factors and associated with reduced expression of e.g. CABLES1, FOXP1, GABRA2, GLR1A, RHOT1, and TBC1D4. We find RHOT1 (MIRO1) to be a key regulator of insulin secretion in human islets. Rhot1-deficiency in ß-cells leads to reduced insulin secretion, ATP/ADP ratio, mitochondrial mass, Ca2+, and respiration. Regulators of mitochondrial dynamics and metabolites, including L-proline, glycine, GABA, and carnitines, are altered in Rhot1-deficient ß-cells. Islets from diabetic GK rats present Rhot1-deficiency. Finally, RHOT1methylation in blood is associated with future T2D. Together, individuals with T2D exhibit epigenetic alterations linked to mitochondrial dysfunction in pancreatic islets.

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

Glucocorticoids and glucolipotoxicity alter the DNA methylome and function of human EndoC-ßH1 cells.

AIMS: Synthetic glucocorticoids, including dexamethasone (DEX), are clinically prescribed due to their immunoregulatory properties. In excess they can perturb glucose homeostasis, with individuals predisposed to glucose intolerance more sensitive to these negative effects. While DEX is known to negatively impact ß-cell function, it is unclear how. Hence, our aim was to investigate the effect of DEX on ß-cell function, both alone and in combination with a diabetogenic milieu in the form of elevated glucose and palmitate. MAIN METHODS: Human pancreatic EndoC-ßH1 cells were cultured in the presence of high glucose and palmitate (glucolipotoxicity) and/or a pharmacological concentration of DEX, before functional and molecular analyses. KEY FINDINGS: Either treatment alone resulted in reduced insulin content and secretion, while the combination of DEX and glucolipotoxicity promoted a strong synergistic effect. These effects were associated with reduced insulin biosynthesis, likely due to downregulation of PDX1, MAFA, and the proinsulin converting enzymes, as well as reduced ATP response upon glucose stimulation. Genome-wide DNA methylation analysis found changes on PDE4D, MBNL1 and TMEM178B, all implicated in ß-cell function, after all three treatments. DEX alone caused very strong demethylation of the glucocorticoid-regulated gene ZBTB16, also known to influence the ß-cell, while the combined treatment caused altered methylation of many known ß-cell regulators and diabetes candidate genes. SIGNIFICANCE: DEX treatment and glucolipotoxic conditions separately alter the ß-cell epigenome and function. The combination of both treatments exacerbates these changes, showing that caution is needed when prescribing potent glucocorticoids in patients with dysregulated metabolism.

Novel Subgroups of Type 2 Diabetes Display Different Epigenetic Patterns That Associate With Future Diabetic Complications.

OBJECTIVE: Type 2 diabetes (T2D) was recently reclassified into severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD), mild obesity-related diabetes (MOD), and mild age-related diabetes (MARD), which have different risk of complications. We explored whether DNA methylation differs between these subgroups and whether subgroup-unique methylation risk scores (MRSs) predict diabetic complications. RESEARCH DESIGN AND METHODS: Genome-wide DNA methylation was analyzed in blood from subjects with newly diagnosed T2D in discovery and replication cohorts. Subgroup-unique MRSs were built, including top subgroup-unique DNA methylation sites. Regression models examined whether MRSs associated with subgroups and future complications. RESULTS: We found epigenetic differences between the T2D subgroups. Subgroup-unique MRSs were significantly different in those patients allocated to each respective subgroup compared with the combined group of all other subgroups. These associations were validated in an independent replication cohort, showing that subgroup-unique MRSs associate with individual subgroups (odds ratios 1.6-6.1 per 1-SD increase, P < 0.01). Subgroup-unique MRSs were also associated with future complications. Higher MOD-MRS was associated with lower risk of cardiovascular (hazard ratio [HR] 0.65, P = 0.001) and renal (HR 0.50, P < 0.001) disease, whereas higher SIRD-MRS and MARD-MRS were associated with an increased risk of these complications (HR 1.4-1.9 per 1-SD increase, P < 0.01). Of 95 methylation sites included in subgroup-unique MRSs, 39 were annotated to genes previously linked to diabetes-related traits, including TXNIP and ELOVL2. Methylation in the blood of 18 subgroup-unique sites mirrors epigenetic patterns in tissues relevant for T2D, muscle and adipose tissue. CONCLUSIONS: We identified differential epigenetic patterns between T2D subgroups that associated with future diabetic complications. These data support a reclassification of diabetes and the need for precision medicine in T2D subgroups.

Statin therapy is associated with epigenetic modifications in individuals with Type 2 diabetes.

Aim: Statins lower cholesterol and reduce the risk of cardiovascular disease. However, the exact mechanisms of statins remain unknown. We investigated whether statin therapy associates with epigenetics in Type 2 diabetes (T2D) patients. Materials & methods: DNA methylation was analyzed in blood from newly diagnosed T2D patients in All New Diabetics in Scania (ANDIS) and a replication cohort All New Diabetics in Uppsala County (ANDiU). Results: Seventy-nine sites were differentially methylated between cases on statins and controls (false discovery rate <5%) in ANDIS. These include previously statin-associated methylation sites annotated to DHCR24 (cg17901584), ABCG1 (cg27243685) and SC4MOL (cg05119988). Differential methylation of two sites related to cholesterol biosynthesis and immune response, cg17901584 (DHCR24) and cg23011663 (ARIH2), were replicated in ANDiU. Conclusion: Statin therapy associates with epigenetic modifications in T2D patients.

Lifestyle Intervention in Pregnant Women With Obesity Impacts Cord Blood DNA Methylation, Which Associates With Body Composition in the Offspring.

Maternal obesity may lead to epigenetic alterations in the offspring and might thereby contribute to disease later in life. We investigated whether a lifestyle intervention in pregnant women with obesity is associated with epigenetic variation in cord blood and body composition in the offspring. Genome-wide DNA methylation was analyzed in cord blood from 208 offspring from the Treatment of Obese Pregnant women (TOP)-study, which includes pregnant women with obesity randomized to lifestyle interventions comprised of physical activity with or without dietary advice versus control subjects (standard of care). DNA methylation was altered at 379 sites, annotated to 370 genes, in cord blood from offspring of mothers following a lifestyle intervention versus control subjects (false discovery rate [FDR] <5%) when using the Houseman reference-free method to correct for cell composition, and three of these sites were significant based on Bonferroni correction. These 370 genes are overrepresented in gene ontology terms, including response to fatty acids and adipose tissue development. Offspring of mothers included in a lifestyle intervention were born with more lean mass compared with control subjects. Methylation at 17 sites, annotated to, for example, DISC1, GBX2, HERC2, and HUWE1, partially mediates the effect of the lifestyle intervention on lean mass in the offspring (FDR <5%). Moreover, 22 methylation sites were associated with offspring BMI z scores during the first 3 years of life (P < 0.05). Overall, lifestyle interventions in pregnant women with obesity are associated with epigenetic changes in offspring, potentially influencing the offspring's lean mass and early growth.

Differential DNA Methylation and Expression of miRNAs in Adipose Tissue From Twin Pairs Discordant for Type 2 Diabetes.

The prevalence of type 2 diabetes (T2D) is increasing worldwide, but current treatments have limitations. miRNAs may play a key role in the development of T2D and can be targets for novel therapies. Here, we examined whether T2D is associated with altered expression and DNA methylation of miRNAs using adipose tissue from 14 monozygotic twin pairs discordant for T2D. Four members each of the miR-30 and let-7-families were downregulated in adipose tissue of subjects with T2D versus control subjects, which was confirmed in an independent T2D case-control cohort. Further, DNA methylation of five CpG sites annotated to gene promoters of differentially expressed miRNAs, including miR-30a and let-7a-3, was increased in T2D versus control subjects. Luciferase experiments showed that increased DNA methylation of the miR-30a promoter reduced its transcription in vitro. Silencing of miR-30 in adipocytes resulted in reduced glucose uptake and TBC1D4 phosphorylation; downregulation of genes involved in demethylation and carbohydrate/lipid/amino acid metabolism; and upregulation of immune system genes. In conclusion, T2D is associated with differential DNA methylation and expression of miRNAs in adipose tissue. Downregulation of the miR-30 family may lead to reduced glucose uptake and altered expression of key genes associated with T2D.

VPS39-deficiency observed in type 2 diabetes impairs muscle stem cell differentiation via altered autophagy and epigenetics.

Insulin resistance and lower muscle quality (strength divided by mass) are hallmarks of type 2 diabetes (T2D). Here, we explore whether alterations in muscle stem cells (myoblasts) from individuals with T2D contribute to these phenotypes. We identify VPS39 as an important regulator of myoblast differentiation and muscle glucose uptake, and VPS39 is downregulated in myoblasts and myotubes from individuals with T2D. We discover a pathway connecting VPS39-deficiency in human myoblasts to impaired autophagy, abnormal epigenetic reprogramming, dysregulation of myogenic regulators, and perturbed differentiation. VPS39 knockdown in human myoblasts has profound effects on autophagic flux, insulin signaling, epigenetic enzymes, DNA methylation and expression of myogenic regulators, and gene sets related to the cell cycle, muscle structure and apoptosis. These data mimic what is observed in myoblasts from individuals with T2D. Furthermore, the muscle of Vps39+/- mice display reduced glucose uptake and altered expression of genes regulating autophagy, epigenetic programming, and myogenesis. Overall, VPS39-deficiency contributes to impaired muscle differentiation and reduced glucose uptake. VPS39 thereby offers a therapeutic target for T2D.

Epigenetic markers associated with metformin response and intolerance in drug-naïve patients with type 2 diabetes.

Metformin is the first-line pharmacotherapy for managing type 2 diabetes (T2D). However, many patients with T2D do not respond to or tolerate metformin well. Currently, there are no phenotypes that successfully predict glycemic response to, or tolerance of, metformin. We explored whether blood-based epigenetic markers could discriminate metformin response and tolerance by analyzing genome-wide DNA methylation in drug-naïve patients with T2D at the time of their diagnosis. DNA methylation of 11 and 4 sites differed between glycemic responders/nonresponders and metformin-tolerant/intolerant patients, respectively, in discovery and replication cohorts. Greater methylation at these sites associated with a higher risk of not responding to or not tolerating metformin with odds ratios between 1.43 and 3.09 per 1-SD methylation increase. Methylation risk scores (MRSs) of the 11 identified sites differed between glycemic responders and nonresponders with areas under the curve (AUCs) of 0.80 to 0.98. MRSs of the 4 sites associated with future metformin intolerance generated AUCs of 0.85 to 0.93. Some of these blood-based methylation markers mirrored the epigenetic pattern in adipose tissue, a key tissue in diabetes pathogenesis, and genes to which these markers were annotated to had biological functions in hepatocytes that altered metformin-related phenotypes. Overall, we could discriminate between glycemic responders/nonresponders and participants tolerant/intolerant to metformin at diagnosis by measuring blood-based epigenetic markers in drug-naïve patients with T2D. This epigenetics-based tool may be further developed to help patients with T2D receive optimal therapy.