DNA methylation screening after roux-en Y gastric bypass reveals the epigenetic signature stems from genes related to the surgery per se

[Abstract] Background/objectives: Obesity has been associated with gene methylation regulation. Recent studies have shown that epigenetic signature plays a role in metabolic homeostasis after Roux-en Y gastric bypass (RYGB). To conduct a genome-wide epigenetic analysis in peripheral blood to investigate whether epigenetic changes following RYGB stem from weight loss or the surgical procedure per se. Subjects/methods: By means of the Infinium Human Methylation 450 BeadChip array, global methylation was analyzed in blood of 24 severely obese women before and 6 months after RYGB and in 24 normal-weight women (controls). Results: In blood cells, nine DMCpG sites showed low methylation levels before surgery, methylation levels increased after RYGB and neared the levels measured in the controls. Additionally, 44 CpG sites associated with the Wnt and p53 signaling pathways were always differently methylated in the severely obese patients as compared to the controls and were not influenced by RYGB. Finally, 1638 CpG sites related to inflammation, angiogenesis, and apoptosis presented distinct methylation in the post-surgery patients as compared to the controls. Conclusion: Bariatric surgery per se acts on CpGs related to inflammation, angiogenesis, and endothelin-signaling. However, the gene cluster associated with obesity remains unchanged, suggesting that weight loss 6 months after RYGB surgery cannot promote this effect.This study’s data collection, DNA and statistical analysis was supported by São Paulo Research Foundation (FAPESP) (grants #2017/07220–7, #2016/05638–1 and #2015/18669–0). Also, statistical and bioinformatics analysis was supported by “Centro de Investigacion Biomedica En Red” (CIBERobn) and grants (PI17/01287) from the “Instituto de Salud Carlos III” (ISCIII), Spain, co-financed by the European Regional Development Fund (FEDER), MINECO grants MTM2014–52876-R and MTM2017–82724-R, and by the Xunta de Galicia (Grupos de Referencia Competitiva ED431C-2016-015 and Centro Singular de Investigación de Galicia ED431G/01), all of them through the ERDF. A Diaz-Lagares is funded by a research contract “Juan Rodés” (JR17/00016) and Ana B Crujeiras is funded by a research contract “Miguel Servet” (CP17/00088) from the ISCIII, co-financed by the European Regional Development Fund (FEDER)Brasil. Fundação de Amparo à Pesquisa do Estado de São Paulo; #2017/07220–7Brasil. Fundação de Amparo à Pesquisa do Estado de São Paulo; #2016/05638–1Brasil. Fundação de Amparo à Pesquisa do Estado de São Paulo; #2015/18669–0Xunta de Galicia; ED431C-2016-015Xunta de Galicia; ED431G/0

Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women

[Abstract] DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m2) and 24 obese women (BMI: 43.3 ± 5.7 kg/m2) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip (cutoff of p ≤ 0.05 and fold change ≥2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.São Paulo Research Foundation; 2017/07220-7São Paulo Research Foundation; #2016/05638-1São Paulo Research Foundation; #2013/12819-4São Paulo Research Foundation; #2015/18669-0Instituto de Salud Carlos III; PI17/01287Ministerio de Economía y Empresa; MTM2014-52876-RMinisterio de Economía y Empresa; MTM2017-82724-RXunta de Galicia; ED431C-2016-015Xunta de Galicia; ED431G/0

Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women

DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 +/- 1.6 kg/m(2)) and 24 obese women (BMI: 43.3 +/- 5.7 kg/m(2)) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q /=2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated

Exercise training and DNA methylation profile in post-bariatric women: Results from an exploratory study

[Abstract]: Exercise training and bariatric surgery have been shown to independently modulate DNA methylation profile in clusters of genes related to metabolic and inflammatory pathways. This study aimed to investigate the effects of a 6-month exercise training program on DNA methylation profile in women who underwent bariatric surgery. In this exploratory, quasi-experimental study, we analyzed DNA methylation levels by array technology in eleven women who underwent Roux-en-Y Gastric Bypass and a 6-month, three-times-a-week, supervised exercise training program. Epigenome Wide Association Analysis showed 722 CpG sites with different methylation level equal to or greater than 5% (P < 0.01) after exercise training. Some of these CpGs sites were related to pathophysiological mechanisms of inflammation, specially Th17 cell differentiation (FDR value < 0.05 and P < 0.001). Our data showed epigenetic modification in specific CpG sites related to Th17 cell differentiation pathway in post-bariatric women following a 6-months exercise training program.This study was supported by São Paulo Research Foundation (FAPESP) (grants #2015/18669–0, #2016/05638–1, #2017/13552–2) and Coordenação de Aperfeiçoamento de Pesquisa de Pessoal de Nível Superior - CAPES (grant #88887.473556/2020–00)

Genome-wide DNA methylation pattern in visceral adipose tissue differentiates insulin-resistant from insulin-sensitive obese subject

Elucidating the potential mechanisms involved in the detrimental effect of excess body weight on insulin action is an important priority in counteracting obesity-associated diseases. The present study aimed to disentangle the epigenetic basis of insulin resistance by performing a genome-wide epigenetic analysis in visceral adipose tissue (VAT) from morbidly obese patients depending on the insulin sensitivity evaluated by the clamp technique. The global human methylome screening performed in VAT from 7 insulin-resistant (IR) and 5 insulin-sensitive (IS) morbidly obese patients (discovery cohort) analyzed using the Infinium HumanMethylation450 BeadChip array identified 982 CpG sites able to perfectly separate the IR and IS samples. The identified sites represented 538 unique genes, 10% of which were diabetes-associated genes. The current work identified novel IR-related genes epigenetically regulated in VAT, such as COL9A1, COL11A2, CD44, MUC4, ADAM2, IGF2BP1, GATA4, TET1, ZNF714, ADCY9, TBX5, and HDACM. The gene with the largest methylation fold-change and mapped by 5 differentially methylated CpG sites located in island/shore and promoter region was ZNF714. This gene presented lower methylation levels in IR than in IS patients in association with increased transcription levels, as further reflected in a validation cohort (n = 24; 11 IR and 13 IS). This study reveals, for the first time, a potential epigenetic regulation involved in the dysregulation of VAT that could predispose patients to insulin resistance and future type 2 diabetes in morbid obesity, providing a potential therapeutic target and biomarkers for counteracting this process

Epigenetic Landscape in Blood Leukocytes Following Ketosis and Weight Loss Induced by a Very Low Calorie Ketogenic Diet (VLCKD) in Patients With Obesity

[Abstract] Background:The molecular mechanisms underlying the potential health benefits of a ketogenic diet areunknown and could be mediated by epigenetic mechanisms.Objective:To identify the changes in the obesity-related methylome that are mediated by the inducedweight loss or are dependent on ketosis in subjects with obesity underwent a very-low calorie ketogenicdiet (VLCKD).Methods:Twenty-one patients with obesity (n¼12 women, 47.9±1.02 yr, 33.0±0.2 kg/m2) after 6months on a VLCKD and 12 normal weight volunteers (n¼6 women, 50.3±6.2 yrs, 22.7±1.5 kg/m2)were studied. Data from the Infinium MethylationEPIC BeadChip methylomes of blood leukocytes wereobtained at time points of ketotic phases (basal, maximum ketosis, and out of ketosis) during VLCKD(n¼10) and at baseline in volunteers (n¼12). Results were further validated by pyrosequencing inrepresentative cohort of patients on a VLCKD (n¼18) and correlated with gene expression.Results:After weight reduction by VLCKD, differences were found at 988 CpG sites (786 unique genes).The VLCKD altered methylation levels in patients with obesity had high resemblance with those fromnormal weight volunteers and was concomitant with a downregulation of DNA methyltransferases(DNMT)1, 3a and 3b. Most of the encoded genes were involved in metabolic processes, protein meta-bolism, and muscle, organ, and skeletal system development. Novel genes representing the top scoringassociated events were identified, includingZNF331,FGFRL1(VLCKD-induced weight loss) andCBFA2T3,C3orf38,JSRP1, andLRFN4(VLCKD-induced ketosis). Interestingly,ZNF331andFGFRL1were validated inan independent cohort and inversely correlated with gene expression.Conclusions:The beneficial effects of VLCKD therapy on obesity involve a methylome more suggestive ofnormal weight that could be mainly mediated by the VLCKD-induced ketosis rather than weight loss.This work was supported by the PronoKal Group® and grants from the Fondo de Investigacion Sanitaria as well as PI17/01287, PI20/00628 and PI20/00650 research projects and CIBERobn from the Instituto de Salud Carlos III (ISCIII)-Subdireccion General de Evaluacion y Fomento de la Investigación; Fondo Europeo de Desarrollo Regional (FEDER) Ana B Crujeiras is funded by a research contract “Miguel Servet” (CP17/00088) from the ISCIII, co-financed by the European Regional Development Fund (FEDER) and Xunta de Galicia-GAIN (IN607B2020)Xunta de Galicia; IN607B202

Epigenetic Landscape in Blood Leukocytes Following Ketosis and Weight Loss Induced by a Very Low Calorie Ketogenic Diet (VLCKD) in Patients with Obesity

Background: The molecular mechanisms underlying the potential health benefits of a ketogenic diet are unknown and could be mediated by epigenetic mechanisms. Objective: To identify the changes in the obesity-related methylome that are mediated by the induced weight loss or are dependent on ketosis in subjects with obesity underwent a very-low calorie ketogenic diet (VLCKD). Methods: Twenty-one patients with obesity (n ¼ 12 women, 47.9 ± 1.02 yr, 33.0 ± 0.2 kg/m2 ) after 6 months on a VLCKD and 12 normal weight volunteers (n ¼ 6 women, 50.3 ± 6.2 yrs, 22.7 ± 1.5 kg/m2 ) were studied. Data from the Infinium MethylationEPIC BeadChip methylomes of blood leukocytes were obtained at time points of ketotic phases (basal, maximum ketosis, and out of ketosis) during VLCKD (n ¼ 10) and at baseline in volunteers (n ¼ 12). Results were further validated by pyrosequencing in representative cohort of patients on a VLCKD (n ¼ 18) and correlated with gene expression. Results: After weight reduction by VLCKD, differences were found at 988 CpG sites (786 unique genes). The VLCKD altered methylation levels in patients with obesity had high resemblance with those from normal weight volunteers and was concomitant with a downregulation of DNA methyltransferases (DNMT)1, 3a and 3b. Most of the encoded genes were involved in metabolic processes, protein metabolism, and muscle, organ, and skeletal system development. Novel genes representing the top scoring associated events were identified, including ZNF331, FGFRL1 (VLCKD-induced weight loss) and CBFA2T3, C3orf38, JSRP1, and LRFN4 (VLCKD-induced ketosis). Interestingly, ZNF331 and FGFRL1 were validated in an independent cohort and inversely correlated with gene expression. Conclusions: The beneficial effects of VLCKD therapy on obesity involve a methylome more suggestive of normal weight that could be mainly mediated by the VLCKD-induced ketosis rather than weight loss.This work was supported by the PronoKal Group® and grants from the Fondo de Investigacion Sanitaria as well as PI17/01287, PI20/00628 and PI20/00650 research projects and CIBERobn from the Instituto de Salud Carlos III (ISCIII)-Subdireccion General de Evaluacion y Fomento de la Investigación; Fondo Europeo de Desarrollo Regional (FEDER) Ana B Crujeiras is funded by a research contract “Miguel Servet” (CP17/00088) from the ISCIII, co-financed by the European Regional Development Fund (FEDER) and Xunta de Galicia-GAIN (IN607B2020). The funding source had no involvement in the study design or interpretation of the result

Effect of a Very-Low-Calorie Ketogenic Diet on Circulating Myokine Levels Compared with the Effect of Bariatric Surgery or a Low-Calorie Diet in Patients with Obesity

The preservation of muscle mass and muscle function after weight loss therapy is currently a considerable challenge in the fight against obesity. Muscle mass secretes proteins called myokines that have relevant functions in the regulation of metabolism and health. This study was aimed to evaluate whether a very low-calorie ketogenic (VLCK) diet may modulate myokine levels, in addition to changes in body composition, compared to a standard, balanced low-calorie (LC) diet or bariatric surgery in patients with obesity. Body composition, ketosis, insulin sensitivity and myokines were evaluated in 79 patients with overweight/obesity after a therapy to lose weight with a VLCK diet, a LC diet or bariatric surgery. The follow-up was 6 months. The weight loss therapies induced changes in myokine levels in association with changes in body composition and biochemical parameters. The effects on circulating myokine levels compared to those at baseline were stronger after the VLCK diet than LC diet or bariatric surgery. Differences reached statistical significance for IL-8, MMP2 and irisin. In conclusion, nutritional interventions or bariatric surgery to lose weight induces changes in circulating myokine levels, being this effect potentially most notable after following a VLCK diet