Weight loss after Roux-En-Y gastric bypass surgery reveals skeletal muscle DNA methylation changes

Abstract

Background: The mechanisms of weight loss and metabolic improvements following bariatric surgery in skeletal muscle are not well known; however, epigenetic modifications are likely to contribute. The aim of our study was to investigate skeletal muscle DNA methylation after weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery. Muscle biopsies were obtained basally from seven insulin-resistant obese (BMI > 40 kg/m²) female subjects (45.1 ± 3.6 years) pre- and 3-month post-surgery with euglycemic hyperinsulinemic clamps to assess insulin sensitivity. Four lean (BMI < 25 kg/m²) females (38.5 ± 5.8 years) served as controls. We performed reduced representation bisulfite sequencing next generation methylation on DNA isolated from the vastus lateralis muscle biopsies.

Results: Global methylation was significantly higher in the pre- (32.97 ± 0.02%) and post-surgery (33.31 ± 0.02%) compared to the lean (30.46 ± 0.02%), P < 0.05. MethylSig analysis identified 117 differentially methylated cytosines (DMCs) that were significantly altered in the post- versus pre-surgery (Benjamini-Hochberg q < 0.05). In addition, 2978 DMCs were significantly altered in the pre-surgery obese versus the lean controls (Benjamini-Hochberg q < 0.05). For the post-surgery obese versus the lean controls, 2885 DMCs were altered (Benjamini-Hochberg q < 0.05). Seven post-surgery obese DMCs were normalized to levels similar to those observed in lean controls. Of these, 5 were within intergenic regions (chr11.68,968,018, chr16.73,100,688, chr5.174,115,531, chr5.1,831,958 and chr9.98,547,011) and the remaining two DMCs chr17.45,330,989 and chr14.105,353,824 were within in the integrin beta 3 (ITGB3) promoter and KIAA0284 exon, respectively. ITGB3 methylation was significantly decreased in the post-surgery (0.5 ± 0.5%) and lean controls (0 ± 0%) versus pre-surgery (13.6 ± 2.7%, P < 0.05). This decreased methylation post-surgery was associated with an increase in ITGB3 gene expression (fold change + 1.52, P = 0.0087). In addition, we showed that ITGB3 promoter methylation in vitro significantly suppressed transcriptional activity (P < 0.05). Transcription factor binding analysis for ITGB3 chr17.45,330,989 identified three putative transcription factor binding motifs; PAX-5, p53 and AP-2alphaA.

Conclusions: These results demonstrate that weight loss after RYGB alters the epigenome through DNA methylation. In particular, this study highlights ITGB3 as a novel gene that may contribute to the metabolic improvements observed post-surgery. Future additional studies are warranted to address the exact mechanism of ITGB3 in skeletal muscle.

Keywords: Bariatric surgery; DNA methylation; Next generation sequencing; Obesity; Skeletal muscle.

Fig. 1

Methylation sites captured were mapped in the context of gene regions (a) and CpG island features (b). The regions were defined using UCSC browser refGene and CpG island tracks. The promoter region was defined as 1000 bp (basepairs) upstream of the transcription start site (TSS); untranslated region (UTR); CpG island is 200–3000 bp stretch of DNA with a C + G content of 50% and observed CpG/expected CpG in excess of 0.6; North (N) and South (S) shores flank the CpG island by 0–2000 bp; the North (N) and South (S) shelf flank the shores by 2000 bp (2000–4000 bp from the island)

Fig. 2

Reduced representation bisulfite sequencing analysis of global DNA methylation of DNA extracted from muscle in lean participants and patients pre- versus post-surgery. Data are mean ± SEM. *P < 0.05 using Mann–Whitney U test. **P < 0.05 using Wilcoxon signed-rank test

Fig. 3

Workflow of methylation analysis across the lean, pre-surgery obese and post-surgery groups

Fig. 4

Differentially methylation cytosines that post-surgery were normalized to levels similar to those observed in the lean control subjects. Data are mean ± SEM. *Benjamini–Hochberg q < 0.05

Fig. 5

In vitro DNA methylation of the ITGB3 human promoter is associated with decreased gene expression. Data are mean ± SEM. The mean represents 3 independent experiments with 5 replicates per experiment