Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 ± 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 ± 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

The Impact of Gastric Bypass on Telomere Length and Shelterin Complex Gene Expression: 6 Months Prospective Study.

BACKGROUND: Telomeres are structures located at the ends of chromosomes associated with a protein complex, known as the shelterin complex. In individuals with obesity, excess adipose tissue plays a key role in inducing a chronic and systemic inflammatory state, which can cause TL shortening. In this context, bariatric surgery is one of the most effective treatment modalities in improving metabolic control. AIM: Therefore, the present study aimed to evaluate how a short postoperative period of gastric bypass affects TL and expression of POT1, TRF1 and TRF2 genes. METHODS: Forty-eight women submitted to RYGB were evaluated before and after 6 months of the surgical procedure. Anthropometric measures of body weight and height (BMI), abdominal circumference (AC), body composition, food intake and blood collection for biochemical evaluation, TL analysis (DNA), and gene expression (RNA) were collected at each moment. RESULTS: There was a reduction of weight, BMI, AC, FM and FFM as well as of glycemia, total cholesterol, LDL-cholesterol, and triglycerides after gastric bypass. No difference in energy intake and macronutrients consumption was observed. There was no significant change in TL, but there was a significant increase of POT1 and TRF1 gene expression after surgery, while TRF2 expression did not change. CONCLUSIONS: Despite bariatric surgery is not capable of increasing telomere length in a short-term period, no reduction is observed; additionally, we found a correlation between serum triglycerides concentration and TL. The increase of POT1 and TRF1 gene expression may explain the maintenance of the TL after 6 months postoperative period.

Impact of green tea epigallocatechin-3-gallate on HIF1-α and mTORC2 expression in obese women: anti-cancer and anti-obesity effects?

INTRODUCTION: Introduction: epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea (Camellia sinensis) and has been associated with anti-obesity and anti-cancer effects, but the exact molecular mechanisms remain elusive. In this context, this study was designed to improve the understanding of the EGCG anti-obesity and anti-cancer action. Objectives: this study was designed to examine the effects of EGCG on the expression of genes involved in obesity and cancer pathways in the peripheral blood mononuclear cells of obese women. Material and methods: this longitudinal interventional study enrolled eleven women with severe obesity that were submitted to eight weeks of green tea (decaffeinated green tea capsules with 450.7 mg of EGCG, two capsules/day) supplementation (intervention group) and ten eutrophic women as a control group. Weight (kg), body mass index (BMI, kg/m2), fat mass (kg) and gene expression (qPCR method) were assessed before and after supplementation. HIF1-alpha (HIF1-α), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and rapamycin-insensitive companion of mTOR (RICTOR) were selected as potential targets. Results: after supplementation, body weight (114.9 ± 14.3 versus 115 ± 13.8 kg), body mass index (44.1 ± 3.7 versus 44.1 ± 3.9 kg/m2) and fat mass (47.6 ± 3.3 versus 47.3 ± 3.4 kg) did not change. EGCG upregulated the RICTOR and HIF1-α expression, however, did not modify PI3K expression. Conclusion: this study demonstrated that EGCG has a potential role to obesity and cancer related to obesity control and can be used not only for the purpose of weight loss, but also for the improvement of obesity-related comorbidities.

INTRODUCCIÓN: Introducción: la obesidad se asocia con altos niveles de estrés oxidativo (EO) e inflamación. Existe mucha evidencia de que algunos polifenoles, como el té verde, tienen un impacto positivo en el estado del sistema operativo y consecutivamente en la inflamación. Objetivos: los propósitos de este estudio fueron: a) acceso a biomarcadores de EO en mujeres obesas y de peso normal; y b) evaluar si la suplementación con té verde tiene impacto en los biomarcadores de citoquinas inflamatorias y de EO de mujeres obesas. Métodos: evaluamos mujeres obesas (índice de masa corporal - IMC ≥ 40 kg/m²) y peso normal (IMC entre 18,5 y 24,9 kg/m²). Se utilizaron muestras de sangre para acceder al malondialdehído (MDA), la capacidad antioxidante equivalente de Trolox (TEAC) y las citoquinas inflamatorias. Elegimos al azar pacientes obesos (18 individuos) y luego les dimos suplementos de té verde durante 8 semanas. El análisis estadístico incluyó las pruebas de Shapiro-Wilk, Wilcoxon, t pareadas e independientes, p < 0,05 se consideraron significativas. Resultados: se reclutaron 42 mujeres obesas (IMC: 48,2 ± 9,3 kg/m2) y 21 de peso normal (IMC: 22,5 ± 2 kg/m2) con una edad promedio de 36,2 ± 9,1 años. Los niveles séricos de MDA fueron más altos en las personas obesas (2,52 ± 0,31 µmol/L) que en las mujeres eutróficas (2,13 ± 0,26 µmol/L; p = 0.000). Por otro lado, se observaron valores de TEAC más bajos en obesos (0,75 ± 0,06 mM) que en el grupo eutrófico (0,78 ± 0,04 mM; p = 0,009). Después de la intervención del té verde, la MDA disminuyó 4,7% y el TEAC aumentó 10%. Los niveles séricos de interleucina-6 (IL-6) disminuyeron 12,7% después del tratamiento (p = 0,03). Conclusiones: el grupo obeso tenía menor capacidad antioxidante que el eutrófico. La suplementación con té verde mejoró TEAC y MDA y redujo los niveles séricos de IL-6 en mujeres obesas.

A new resting metabolic rate equation for women with class III obesity.

OBJECTIVE: Resting metabolic rate (RMR) is an important parameter to guide the nutritional therapy of class III obese patients. The aims of the present study were to develop a predictive equation for RMR estimation in class III obese women using anthropometric indicators and to compare indirect calorimetry with other predictive equations. METHODS: This was a cross-sectional study on women with class III obesity (body mass index >40 kg/m2). Weight, height, fat-free mass, fat mass, and RMR of all individuals were measured. Multiple linear regression was used to determine the new RMR equation and the Bland-Altman plot was used to analyze the agreement between indirect calorimetry and the results of predictive equations. RESULTS: We evaluated 101 women with obesity class III and a mean age of 36.3 ± 10 y. The anthropometric and body composition variables used in the new equation had a coefficient of determination of 0.80, and a significant influence on RMR (P = 0.01). Harris-Benedict and World Health Organization equations showed similar bias and limits (181.6, +2 SD = 765.5, -2 SD = -402.2; 156.4, +2 SD = 799.4, -2 SD = -486.6, respectively). The Mifflin-St Jeor and Owen equations showed large clinical bias (mean, 239.2 and 463.9, respectively), and a tendency to overestimate RMR. CONCLUSION: The prediction equations tested in the study had low accuracy in estimating RMR of women with class III obesity. However, our equation was developed specifically for this population, using variables known to influence their energy expenditure.

Mammalian target of rapamycin complex 2 signaling in obese women changes after bariatric surgery.

OBJECTIVES: After bariatric surgery, modifications to signaling pathway networks including those of the metabolic regulator called mammalian or mechanistic target of rapamycin (mTOR) may lead to molecular alterations related to energy source availability, systemic nutrients, and catabolic and anabolic cellular processes. This study aimed to identify gene expression changes with regard to the mTOR complex 2 subunit signaling pathway in obese patients before and after bariatric surgery. METHODS: The experimental group included 13 obese women who were examined before (preoperative) and 6 mo after (postoperative) Roux-en-Y gastric bypass (RYGB) surgery. The control group included nine apparently eutrophic women matched by age and without any other metabolic diseases (i.e., no diabetes and no liver or kidney diseases). Peripheral blood mononuclear cell samples were collected for RNA extraction and subsequent microarray analysis. RESULTS: After this methodological procedure, we identified 47 000 differentially expressed genes. A subsequent bioinformatic analysis showed that three diferentially expressed genes (rapamycin-insensitive companion of mTOR [RICTOR], phosphoinositide-3-kinase regulatory subunit 1 [PIK3 R1], and hypoxia inducible factor 1 alpha subunit 1A [HIF1 A]) participated in the mTOR signaling pathway. Real-time quantitative polymerase chain reaction revealed that RICTOR, PIK3 R1, and HIF1 A were upregulated 6 mo after RYGB surgery (P <0.05). In addition, patients in the experimental group lost weight significantly and presented significant improvement in biochemical/metabolic variables. CONCLUSIONS: The weight loss that was induced by RYGB surgery alters the mTOR signaling pathway and specifically the mTOR complex 2 subunit. The increased expression of genes that act in this pathway such as RICTOR, PIK3 R1, and HIF1 A reflects the induced weight loss and improved metabolic indicators (e.g., insulin resistance and lipolysis) that are evidenced in this study.

Influence of expression of UCP3, PLIN1 and PPARG2 on the oxidation of substrates after hypocaloric dietary intervention.

BACKGROUND & AIMS: In addition to environmental and psychosocial factors, it is known that genetic factors can also influence the regulation of energy metabolism, body composition and determination of excess weight. The objective of this study was to evaluate the influence of UCP3, PLIN1 and PPARG2 genes on the substrates oxidation in women with grade III obesity after hypocaloric dietary intervention. SUBJECTS/METHODS: This is a longitudinal study with 21 women, divided into two groups: Intervention Group (G1): 11 obese women (Body Mass Index (BMI) ≥40 kg/m2), and Control Group (G2): 10 eutrophic women (BMI between 18.5 kg/m2 and 24.9 kg/m2). Weight (kg), height (m), BMI (kg/m2), substrate oxidation (by Indirect Calorimetry) and abdominal subcutaneous adipose tissue were collected before and after the intervention. For the dietary intervention, the patients were hospitalized for 6 weeks receiving 1200 kcal/day. RESULTS: There was a significant weight loss (8.4 ± 4.3 kg - 5.2 ± 1.8%) and reduction of UCP3 expression after hypocaloric dietary intervention. There was a positive correlation between carbohydrate oxidation and UCP3 (r = 0.609; p = 0.04), PLIN1 (r = 0.882; p = 0.00) and PPARG2 (r = 0.791; p = 0.00) expression before dietary intervention and with UCP3 (r = 0.682; p = 0.02) and PLIN1 (r = 0.745; p = 0.00) genes after 6 weeks of intervention. There was a negative correlation between lipid oxidation and PLIN1 (r = -0.755; p = 0.00) and PPARG2 (r = 0.664; p = 0.02) expression before dietary intervention and negative correlation with PLIN1 (r = 0.730; p = 0.02) expression after 6 weeks of hypocaloric diet. CONCLUSION: Hypocaloric diet reduces UCP3 expression in individuals with obesity and the UCP3, PLIN1 and PPARG2 expression correlate positively with carbohydrate oxidation and negatively with lipid oxidation.

Pregnancy After Roux en Y Gastric Bypass: Nutritional and Biochemical Aspects.

INTRODUCTION: Although pregnancy after bariatric surgery is related to risk reduction, nutritional complications may occur. This study aimed to evaluate nutritional and biochemical indicators of women who became pregnant after Roux en Y gastric bypass (RYGB). MATERIALS AND METHODS: We carried out a retrospective study with women who became pregnant after RYGB. We evaluated anthropometric, biochemical, and dietary intake indicators in the preoperative period and before, during, and after pregnancy by analysis of medical records. Shapiro-Wilk test and ANOVA for repeated measures were performed (p < 0.05). RESULTS: The study included 25 patients (35.7 ± 3.8 years), who became pregnant 31.3 ± 21.7 months after RYGB. Weight loss until the beginning of pregnancy was 32.4%, and the gestational weight gain was 3.8 ± 12 kg. There was a higher frequency of patients with hypertension in the preoperative time when compared to that during the pregnancy period. Total cholesterol (180.9 ± 24.8 versus 148.5 ± 30.4 mg/dL), LDL-cholesterol (103.5 ± 19.2 versus 85.8 ± 23.1 mg/dL), HDL-cholesterol (56.4 ± 8 versus 46.9 ± 8.7 mg/dL), and latent iron-binding capacity (337.6 ± 95.8 versus 277.8 ± 65 µg/dL) were higher during the pregnancy compared to that before the pregnancy, while hemoglobin values (11.2 ± 1 versus 12.3 ± 1.2 g/dL) and sodium (138.8 ± 2.9 versus 141 ± 3 mmol/L) were lower. No differences of food intake were found among times. There is no difference on gestational weight gain between women who became pregnant before or after the first year. CONCLUSION: During pregnancy, there was an expected weight gain and maintenance of the lipid profile within the normal range; however, there was a reduction of hemoglobin levels. These findings show the need for individualized follow-up with adequate nutritional intervention in the event of deficiencies.

UCP2 and PLIN1 Expression Affects the Resting Metabolic Rate and Weight Loss on Obese Patients.

BACKGROUND: Analysis of the expression of genes related to the energy metabolism can elucidate the understanding of physiological and genetic factors that contribute to obesity. This study aimed to evaluate the expression of genes and its influence on resting metabolic rate and weight loss in obese patients before and after bariatric surgery. METHODS: This study was conducted on 23 women, who were divided into two groups: bariatric surgery (preoperative and 6 months after surgery) and control. Abdominal subcutaneous adipose tissue samples were collected to analyze the gene expression, and the resting metabolic rate (RMR) was measured by indirect calorimetry. RESULTS: Significant differences were observed in weight reduction (22 %, p = 0.01), BMI (22.5 %, p = 0.01), and RMR values (10.5 %, p = 0.01) after the bariatric surgery, while the weight-adjusted RMR increased (15.8 %, p = 0.01). Increased UCP2 expression after 6 months of Roux-en-Y gastric bypass (RYGB) as compared to preoperative period (0.764 to 1.268, p = 0.01) was observed. Analysis with weight-adjusted RMR as dependent variable revealed that UCP2 (r 2 = 0.517, p = 0.01) and PLIN1 (r 2 = 0.420, p = 0.04) expression determine the RMR values in preoperative period. Moreover, UCP2 and PLIN1 expression in preoperative period influenced the percentage of weight loss, even when adjusted for age and BMI. CONCLUSIONS: We have demonstrated that after 6 months of bariatric surgery, there is significant increase in the UCP2 expression. Additionally, the expression of UCP2 and PLIN1 genes influences the resting metabolic rate in obese individuals and could predict the weight loss after bariatric surgery.

The Genetic Predisposition Score of Seven Obesity-Related Single Nucleotide Polymorphisms Is Associated with Better Metabolic Outcomes after Roux-en-Y Gastric Bypass.

BACKGROUND/AIMS: Genetic variants associated with obesity have cumulative effects on obesity risk and related phenotypes. This study aimed to estimate the contribution of a genetic predisposition score (GPS) calculated from 7 obesity-related polymorphisms to the improvement of biochemical parameters 1 year after Roux-en-Y gastric bypass (RYGB). METHODS: Obese patients (n = 150; aged 47.2 ± 10.5 years) were enrolled and weight, body mass index (BMI), and biochemical parameters (glycemia and lipid profile) were evaluated preoperatively and 1 year after RYGB. A GPS was calculated with the polymorphisms rs1801282 of PPARG2, rs4994 of ADRB3, rs1800592 of UCP1, rs659366 and rs669339 of UCP2, rs7121 of GNAS1, and rs5443 of GNB3. We observed that 66.3% of the patients has a GPS >5. RESULTS: During the preoperative period, the GPS showed a significant association with weight (ß = -0.163; p = 0.020), BMI (ß = -0.169; p = 0.038), and glucose concentrations (ß = -0.177; p = 0.036). After sex and age adjustment, a higher GPS was associated with a greater reduction in glycemia (ß = -0.158; p = 0.048), triglycerides (ß = -0.256; p = 0.002), and total cholesterol (ß = -0.172; p = 0.038) concentrations 1 year after surgery. CONCLUSION: Our data elucidated that a higher GPS provides a greater metabolic benefit of RYGB.

DNA Methylation and Hydroxymethylation Levels in Relation to Two Weight Loss Strategies: Energy-Restricted Diet or Bariatric Surgery.

BACKGROUND: Weight loss can be influenced by genetic factors and epigenetic mechanisms that participate in the regulation of body weight. This study aimed to investigate whether the weight loss induced by two different obesity treatments (energy restriction or bariatric surgery) may affect global DNA methylation (LINE-1) and hydroxymethylation profile, as well as the methylation patterns in inflammatory genes. METHODS: This study encompassed women from three differents groups: 1. control group (n = 9), normal weight individuals; 2. energy restriction group (n = 22), obese patients following an energy-restricted Mediterranean-based dietary treatment (RESMENA); and 3. bariatric surgery group (n = 14), obese patients underwent a hypocaloric diet followed by bariatric surgery. Anthropometric measurements and 12-h fasting blood samples were collected before the interventions and after 6 months. Lipid and glucose biomarkers, global hydroxymethylation (by ELISA), LINE-1, SERPINE-1, and IL-6 (by MS-HRM) methylation levels were assessed in all participants. RESULTS: Baseline LINE-1 methylation was associated with serum glucose levels whereas baseline hydroxymethylation was associated with BMI, waist circumference, total cholesterol, and triglycerides. LINE-1 and SERPINE-1 methylation levels did not change after weight loss, whereas IL-6 methylation increased after energy restriction and decreased in the bariatric surgery group. An association between SERPINE-1 methylation and weight loss responses was found. CONCLUSIONS: Global DNA methylation and hydroxymethylation might be biomarkers for obesity and associated comorbidities. Depending on the obesity treatment (diet or surgery), the DNA methylation patterns behave differently. Baseline SERPINE-1 methylation may be a predictor of weight loss values after bariatric surgery.

Influence of excess weight loss and weight regain on biochemical indicators during a 4-year follow-up after Roux-en-Y gastric bypass.

BACKGROUND: Bariatric surgery produces a substantial weight loss and improves the comorbidities associated with obesity such as diabetes mellitus and dyslipidemia, although inability to lose weight or weight regain has been estimated to occur in 20 % of cases. The objective of the present study was to assess the influence of weight variations on biochemical indicators during a 4-year period after bariatric surgery. METHODS: A 4-year retrospective longitudinal study was conducted on 138 patients with grade III obesity submitted to Roux-en-Y gastric bypass, with the assessment of anthropometric measurements and biochemical indicators. The patients were divided into two groups according to percent excess weight loss (%EWL): %EWL > 50 % and %EWL < 50 %, and into two groups according to weight regain: <10 % and >10 %. The Student t test for independent samples was used to assess the differences in biochemical indicators between groups (p ≤ 0.05). RESULTS: Four years after surgery, there was a weight loss of 49.4 ± 21.8 kg and %EWL of 61 ± 21.2 %, with 73.2 % (n = 101) of the patients showing %EWL of 50 % or more. Significant weight regain occurred in 24.6 % of the sample. There was a difference in weight, BMI, total cholesterol, LDL-cholesterol, triglycerides, and albumin between patients with different %EWL. No difference in biochemical indicators was observed between subjects with and without regain. CONCLUSION: Four years after surgery, greater %EWL was associated with a better lipid profile. In addition, weight regain did not change the biochemical indicators of this patient series.

Red meat intolerance in patients submitted to gastric bypass: a 4-year follow-up study.

BACKGROUND: Bariatric surgery provides significant weight reduction; however, it may result in food intolerance followed by gastrointestinal complications that may lead to nutritional deficiencies. This study evaluated the influence of red meat intolerance on the dietary pattern, biochemical indicators, and clinical symptoms after Roux-en-Y gastric bypass (RYGB). METHODS: This retrospective study evaluated patients 4 years after RYGB. The patients were divided into 2 groups: patients with and without red meat intolerance, and data for the following were collected: food intake, anthropometric data, biochemical data, and presence of nausea, vomiting, weakness, weak nails, and hair loss. The difference between groups in the times postoperative was determined by ANOVA. RESULTS: Of the 72 patients included in the study, 63 were evaluated during the first postoperative year, 45 during the second, 56 during the third, and 41 during the fourth. Red meat intolerance was observed in 49.2%, 42.2%, 46.4%, and 39% of the patients after 1, 2, 3, and 4 years, respectively. After 1 year, the intolerant group showed lower calorie, carbohydrate, and iron intake. After 3 years, tolerant patients showed weight regain (2.9 ± 5.3 kg), while the intolerant ones remained stable. There was no difference in the presence of clinical symptoms or biochemical indicators between groups. CONCLUSION: Red meat intolerance is frequent after bariatric surgery and may alter energy, iron intake, and weight loss; however, it is not associated with the presence of clinical symptoms and biochemical profile.