Different effects of bariatric surgery on epigenetic plasticity in skeletal muscle of individuals with and without type 2 diabetes.

AIM: Bariatric surgery is highly effective for the treatment of obesity in individuals without (OB1) and in those with type 2 diabetes (T2D2). However, whether bariatric surgery triggers similar or distinct molecular changes in OB and T2D remains unknown. Given that individuals with type 2 diabetes often exhibit more severe metabolic deterioration, we hypothesized that bariatric surgery induces distinct molecular adaptations in skeletal muscle, the major site of glucose uptake, of OB and T2D after surgery-induced weight loss. METHODS: All participants (OB, n=13; T2D, n=13) underwent detailed anthropometry before and one year after the surgery. Skeletal muscle biopsies were isolated at both time points and subjected to transcriptome and methylome analyses using a comprehensive bioinformatic pipeline. RESULTS: Before surgery, T2D had higher fasting glucose and insulin levels but lower whole-body insulin sensitivity, only glycemia remained higher in T2D than in OB after surgery. Surgery-mediated weight loss affected different subsets of genes with 2,013 differentially expressed in OB and 959 in T2D. In OB differentially expressed genes were involved in insulin, PPAR signaling and oxidative phosphorylation pathways, whereas ribosome and splicesome in T2D. LASSO regression analysis revealed distinct candidate genes correlated with improvement of phenotypic traits in OB and T2D. Compared to OB, DNA methylation was less affected in T2D in response to bariatric surgery. This may be due to increased global hydroxymethylation accompanied by decreased expression of one of the type 2 diabetes risk gene, TET2, encoding a demethylation enzyme in T2D. CONCLUSION: OB and T2D exhibit differential skeletal muscle transcriptome responses to bariatric surgery, presumably resulting from perturbed epigenetic flexibility.

Novel markers and networks related to restored skeletal muscle transcriptome after bariatric surgery.

OBJECTIVE: The aim of this study was to discover novel markers underlying the improvement of skeletal muscle metabolism after bariatric surgery. METHODS: Skeletal muscle transcriptome data of lean people and people with obesity, before and 1 year after bariatric surgery, were subjected to weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression. Results of LASSO were confirmed in a replication cohort. RESULTS: The expression levels of 440 genes differing between individuals with and without obesity were no longer different 1 year after surgery, indicating restoration. WGCNA clustered 116 genes with normalized expression in one major module, particularly correlating to weight loss and decreased plasma free fatty acids (FFA), 44 of which showed an obesity-related phenotype upon deletion in mice. Among the genes of the major module, 105 represented prominent markers for reduced FFA concentration, including 55 marker genes for decreased BMI in both the discovery and replication cohorts. CONCLUSIONS: Previously unknown gene networks and marker genes underlined the important role of FFA in restoring muscle gene expression after bariatric surgery and further suggest novel therapeutic targets for obesity.

Meta-analysis of genome-wide DNA methylation and integrative omics of age in human skeletal muscle.

BACKGROUND: Knowledge of age-related DNA methylation changes in skeletal muscle is limited, yet this tissue is severely affected by ageing in humans. METHODS: We conducted a large-scale epigenome-wide association study meta-analysis of age in human skeletal muscle from 10 studies (total n = 908 muscle methylomes from men and women aged 18-89 years old). We explored the genomic context of age-related DNA methylation changes in chromatin states, CpG islands, and transcription factor binding sites and performed gene set enrichment analysis. We then integrated the DNA methylation data with known transcriptomic and proteomic age-related changes in skeletal muscle. Finally, we updated our recently developed muscle epigenetic clock (https://bioconductor.org/packages/release/bioc/html/MEAT.html). RESULTS: We identified 6710 differentially methylated regions at a stringent false discovery rate <0.005, spanning 6367 unique genes, many of which related to skeletal muscle structure and development. We found a strong increase in DNA methylation at Polycomb target genes and bivalent chromatin domains and a concomitant decrease in DNA methylation at enhancers. Most differentially methylated genes were not altered at the mRNA or protein level, but they were nonetheless strongly enriched for genes showing age-related differential mRNA and protein expression. After adding a substantial number of samples from five datasets (+371), the updated version of the muscle clock (MEAT 2.0, total n = 1053 samples) performed similarly to the original version of the muscle clock (median of 4.4 vs. 4.6 years in age prediction error), suggesting that the original version of the muscle clock was very accurate. CONCLUSIONS: We provide here the most comprehensive picture of DNA methylation ageing in human skeletal muscle and reveal widespread alterations of genes involved in skeletal muscle structure, development, and differentiation. We have made our results available as an open-access, user-friendly, web-based tool called MetaMeth (https://sarah-voisin.shinyapps.io/MetaMeth/).

Epigenetic Changes in Islets of Langerhans Preceding the Onset of Diabetes.

The identification of individuals with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention. Here, we used a translational approach and prediction criteria to identify changes in DNA methylation visible before the development of T2D. Islets of Langerhans were isolated from genetically identical 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and in liver fat content. The application of a semiexplorative approach identified 497 differentially expressed and methylated genes (P = 6.42e-09, hypergeometric test) enriched in pathways linked to insulin secretion and extracellular matrix-receptor interaction. The comparison of mouse data with DNA methylation levels of incident T2D cases from the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort, revealed 105 genes with altered DNA methylation at 605 cytosine-phosphate-guanine (CpG) sites, which were associated with future T2D. AKAP13, TENM2, CTDSPL, PTPRN2, and PTPRS showed the strongest predictive potential (area under the receiver operating characteristic curve values 0.62-0.73). Among the new candidates identified in blood cells, 655 CpG sites, located in 99 genes, were differentially methylated in islets of humans with T2D. Using correction for multiple testing detected 236 genes with an altered DNA methylation in blood cells and 201 genes in diabetic islets. Thus, the introduced translational approach identified novel putative biomarkers for early pancreatic islet aberrations preceding T2D.

Overexpression of Gjb4 impairs cell proliferation and insulin secretion in primary islet cells.

OBJECTIVE: Altered gene expression contributes to the development of type 2 diabetes (T2D); thus, the analysis of differentially expressed genes between diabetes-susceptible and diabetes-resistant mouse models is an important tool for the determination of candidate genes that participate in the pathology. Based on RNA-seq and array data comparing pancreatic gene expression of diabetes-prone New Zealand Obese (NZO) mice and diabetes-resistant B6.V-ob/ob (B6-ob/ob) mice, the gap junction protein beta 4 (Gjb4) was identified as a putative novel T2D candidate gene. METHODS: Gjb4 was overexpressed in primary islet cells derived from C57BL/6 (B6) mice and INS-1 cells via adenoviral-mediated infection. The proliferation rate of cells was assessed by BrdU incorporation, and insulin secretion was measured under low (2.8 mM) and high (20 mM) glucose concentration. INS-1 cell apoptosis rate was determined by Western blotting assessing cleaved caspase 3 levels. RESULTS: Overexpression of Gjb4 in primary islet cells significantly inhibited the proliferation by 47%, reduced insulin secretion of primary islets (46%) and INS-1 cells (51%), and enhanced the rate of apoptosis by 63% in INS-1 cells. Moreover, an altered expression of the miR-341-3p contributes to the Gjb4 expression difference between diabetes-prone and diabetes-resistant mice. CONCLUSIONS: The gap junction protein Gjb4 is highly expressed in islets of diabetes-prone NZO mice and may play a role in the development of T2D by altering islet cell function, inducing apoptosis and inhibiting proliferation.

Epigenetic contribution to obesity.

Obesity is a worldwide epidemic and contributes to global morbidity and mortality mediated via the development of nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), cardiovascular (CVD) and other diseases. It is a consequence of an elevated caloric intake, a sedentary lifestyle and a genetic as well as an epigenetic predisposition. This review summarizes changes in DNA methylation and microRNAs identified in blood cells and different tissues in obese human and rodent models. It includes information on epigenetic alterations which occur in response to fat-enriched diets, exercise and metabolic surgery and discusses the potential of interventions to reverse epigenetic modifications.

Dynamic changes of muscle insulin sensitivity after metabolic surgery.

The mechanisms underlying improved insulin sensitivity after surgically-induced weight loss are still unclear. We monitored skeletal muscle metabolism in obese individuals before and over 52 weeks after metabolic surgery. Initial weight loss occurs in parallel with a decrease in muscle oxidative capacity and respiratory control ratio. Persistent elevation of intramyocellular lipid intermediates, likely resulting from unrestrained adipose tissue lipolysis, accompanies the lack of rapid changes in insulin sensitivity. Simultaneously, alterations in skeletal muscle expression of genes involved in calcium/lipid metabolism and mitochondrial function associate with subsequent distinct DNA methylation patterns at 52 weeks after surgery. Thus, initial unfavorable metabolic changes including insulin resistance of adipose tissue and skeletal muscle precede epigenetic modifications of genes involved in muscle energy metabolism and the long-term improvement of insulin sensitivity.

Epigenetic regulation of hepatic Dpp4 expression in response to dietary protein.

Dipeptidyl peptidase 4 (DPP4) is known to be elevated in metabolic disturbances such as obesity, type 2 diabetes and fatty liver disease. Lowering DPP4 concentration by pharmacological inhibition improves glucose homeostasis and exhibits beneficial effects to reduce hepatic fat content. As factors regulating the endogenous expression of Dpp4 are unknown, the aim of this study was to examine whether the Dpp4 expression is epigenetically regulated in response to dietary components. Primary hepatocytes were treated with different macronutrients, and Dpp4 mRNA levels and DPP4 activity were evaluated. Moreover, dietary low-protein intervention was conducted in New Zealand obese (NZO) mice, and subsequently, effects on Dpp4 expression, methylation as well as plasma concentration and activity were determined. Our results indicate that Dpp4 mRNA expression is mediated by DNA methylation in several tissues. We therefore consider the Dpp4 southern shore as tissue differentially methylated region. Amino acids increased Dpp4 expression in primary hepatocytes, whereas glucose and fatty acids were without effect. Dietary protein restriction in NZO mice increased Dpp4 DNA methylation in liver leading to diminished Dpp4 expression and consequently to lowered plasma DPP4 activity. We conclude that protein restriction in the adolescent and adult states is a sufficient strategy to reduce DPP4 which in turn contributes to improve glucose homeostasis.

The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth.

BACKGROUND: Even if genetics play an important role, individual variation in stature remains unexplained at the molecular level. Indeed, genome-wide association study (GWAS) have revealed hundreds of variants that contribute to the variability of height but could explain only a limited part of it, and no single variant accounts for more than 0.3% of height variance. At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits, an emerging challenge in humans, has not been attempted for height. Since insulin-like growth factor 1 (IGF1) controls postnatal growth, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene is a potential epigenetic contributor to the individual variation in circulating IGF1 and stature in growing children. RESULTS: Child height was closely correlated with serum IGF1. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. The highest association was for CG-137 methylation, which contributed 13% to the variance of height and 10% to serum IGF1. CG methylation (studied in children undergoing surgery) was approximately 50% lower in liver and growth plates, indicating that the IGF1 promoters are tissue-differentially methylated regions (t-DMR). CG methylation was inversely correlated with the transcriptional activity of the P2 promoter in mononuclear blood cells and in transfection experiments, suggesting that the observed association of methylation with the studied traits reflects true biological causality. CONCLUSIONS: Our observations introduce epigenetics among the individual determinants of child growth and serum IGF1. The P2 promoter of the IGF1 gene is the first epigenetic quantitative trait locus (QTL(epi)) reported in humans. The CG methylation of the P2 promoter takes place among the multifactorial factors explaining the variation in human stature.