Reduction in inflammatory gene expression in skeletal muscle from Roux-en-Y gastric bypass patients randomized to omentectomy

Abstract

Objectives: To examine the effects of Roux-en-Y gastric bypass (RYGB) surgery with and without laparoscopic removal of omental fat (omentectomy) on the temporal gene expression profiles of skeletal muscle.

Design: Previously reported were the whole-body metabolic effects of a randomized, single-blinded study in patients receiving RYGB surgery stratified to receive or not receive omentectomy. In this follow up study we report on changes in skeletal muscle gene expression in a subset of 21 patients, for whom biopsies were collected preoperatively and at either 6 months or 12 months postoperatively.

Methodology/principal findings: RNA isolated from skeletal muscle biopsies of 21 subjects (8 without omentectomy and 13 with omentectomy) taken before RYGB or at 6 and 12 months postoperatively were subjected to gene expression profiling via Exon 1.0 S/T Array and Taqman Low Density Array. Robust Multichip Analysis and gene enrichment data analysis revealed 84 genes with at least a 4-fold expression difference after surgery. At 6 and 12 months the RYGB with omentectomy group displayed a greater reduction in the expression of genes associated with skeletal muscle inflammation (ANKRD1, CDR1, CH25H, CXCL2, CX3CR1, IL8, LBP, NFIL3, SELE, SOCS3, TNFAIP3, and ZFP36) relative to the RYGB non-omentectomy group. Expressions of IL6 and CCL2 were decreased at all postoperative time points. There was differential expression of genes driving protein turnover (IGFN1, FBXW10) in both groups over time and increased expression of PAAF1 in the non-omentectomy group at 12 months. Evidence for the activation of skeletal muscle satellite cells was inferred from the up-regulation of HOXC10. The elevated post-operative expression of 22 small nucleolar RNAs and the decreased expression of the transcription factors JUNB, FOS, FOSB, ATF3 MYC, EGR1 as well as the orphan nuclear receptors NR4A1, NR4A2, NR4A3 suggest dramatic reorganizations at both the cellular and genetic levels.

Conclusions/significance: These data indicate that RYGB reduces skeletal muscle inflammation, and removal of omental fat further amplifies this response.

Trial registration: ClinicalTrials.gov NCT00212160.

Figure 1. CONSORT Diagram.

Figure 2. Box plot depicting the difference among microarray expression datasets (chips) before and after normalization by Robust Microarray Averaging (RMA).

Boxes show the 25th and 75th percentiles in the distribution of log-transformed (log base 2) intensities. The median is the horizontal bar in the middle of the box. The whiskers (dotted lines extending from the boxes) illustrate the maximum value or 1.5 times the interquartile range of data (IQR), whichever is smaller. The circles display any points beyond these whiskers.

Figure 3. Heat map displaying hierarchical clustering results from microarray expression profile data derived from skeletal muscle RNA pools.

Samples include 0, 6 and 12 months “No” omentectomy and 0, 6 and 12 months “Yes” omentectomy. The expression of 2,697 genes is represented as a dendrogram after applying filtering criteria for selecting genes with a log₂ ratio intensity greater than 1 (ratio>2). Gene expression ratios are displayed by applying progressively brighter shades of red (down-regulated) or green (up-regulated) to log₂ ratios that increasingly deviate from zero.

Figure 4. Bar graph illustrating the fold change of individual genes at 6 vs. 0 months without omentectomy as determined by microarray (grey) and TLDA profiling (black).

Figure 5. Bar graph illustrating the fold change of individual genes at 12 vs. 0 months without omentectomy as determined by microarray (grey) and TLDA profiling (black).

Figure 6. Bar graph illustrating the fold change of individual genes at 6 vs. 0 months with omentectomy as determined by microarray (grey) and TLDA profiling (black).

Figure 7. Bar graph illustrating the fold change of individual genes at 12 vs. 0 months with omentectomy as determined by microarray (grey) and TLDA profiling (black).

Figure 8. Correlation in fold change (log₂) of gene expression as determined by Affymetrix Exon microarray and TLDA profiling.

A) 6 vs. 0 months, no omentectomy, Pearson correlation coefficient rho = 0.559, p<0.0001; B) 12 vs. 0 months, no omentectomy, Pearson correlation coefficient rho = 0.646, p = 0.0002; C) 6 vs. 0 months, yes omentectomy, Pearson correlation coefficient rho = 0.649, p = 0.0002; D) 12 vs. 0 months, yes omentectomy, Pearson correlation coefficient rho = 0.640, p = 0.0002.