A porcine gluteus medius muscle genome-wide transcriptome analysis: dietary effects of omega-6 and omega-3 fatty acids on biological mechanisms

Abstract

Background: The level of omega-6 and omega-3 polyunsaturated fatty acids can affect many cellular systems and function via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate changes in the muscle transcriptome and the biological functions regulated by increased consumption of omega-3 and omega-6 fatty acids in the pig gluteus medius muscle.

Results: The transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database pointing differentially expressed genes (DEG). Comparative expression analyses identified 749 genes significantly differing at least in the twofold of change between two groups of animals fed with divergent level of omega-3 and omega-6 fatty acids. The expression of 219 genes was upregulated, and the expression of 530 genes was downregulated in the group of pigs supplemented with omega-3 and omega-6 fatty acids in relation to control group pigs. Results of RNA-seq indicated a role of fatty acid in the regulation of the expression of genes which are essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including inflammatory response, signaling, lipid metabolism, and homeostasis.

Conclusions: Summarizing, obtained results provide strong evidence that omega-6 and omega-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.

Keywords: Gluteus medius muscle; Omega-3 fatty acid; Omega-6 fatty acid; Pig; Transcriptome.

Fig. 1

The validation of NGS results with real-time PCR—comparison of control and experimental group. Mean expression of ten genes: transthyretin (TTR), apolipoprotein C-III (APOC3), retinol-binding protein 4 (RBP4), ADAM metallopeptidase domain 19 (ADAM19), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), adenosine A1 receptor (ADORA1), calsequestrin 1 (CASQ1), mitogen-activated protein kinase 1 (MAPK1), Fas (TNFRSF6)-associated via death domain (FADD), and C-C motif chemokine ligand 4 (CCL4) determined using real-time PCR for 24 pigs from two diet treatments. Expression was normalized to the topoisomerase (DNA) II beta (TOP2B) gene as an endogenous control. Error bars represent standard error.*P value <0.05; **P value <0.01; ***P value <0.001, ns not significant

Fig. 2

The validation of NGS results with real-time PCR—comparison of two methods. Log2 fold changes of ten differentially expressed genes measured by NGS (blue) versus real-time PCR (orange): transthyretin (TTR), apolipoprotein C-III (APOC3), retinol-binding protein 4 (RBP4), ADAM metallopeptidase domain 19 (ADAM19), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), adenosine A1 receptor (ADORA1), calsequestrin 1 (CASQ1), mitogen-activated protein kinase 1 (MAPK1), Fas (TNFRSF6)-associated via death domain (FADD), and C-C motif chemokine ligand 4 (CCL4)

Fig. 3

Cytoscape-ClueGo analysis of differentially expressed genes (DEG). Gene networks of upregulated (red) and downregulated (blue) genes for the diet supplemented with n-6 and n-3 fatty acids versus the control diet. The edges connecting the nodes are based on the kappa statistic (kappa score of 0.9) that measures the overlap of shared genes between terms

Fig. 4

Cytoscape-ClueGo analysis of functional distribution of differentially expressed genes (DEG). Analysis of genes identified with differential expression in muscle of pigs fed with a diet supplemented with fatty acids versus pigs fed a control diet. Categories of genes were based on GO annotation. Significant terms (Kappa = 0.9) and distribution were determined according to percentage of number of gene association