Quantification of Transcriptome Responses of the Rumen Epithelium to Butyrate Infusion using RNA-seq Technology

Abstract

Short-chain fatty acids (SCFAs), such as butyrate, produced by gut microorganisms, play a critical role in energy metabolism and physiology of ruminants as well as in human health. In this study, the temporal effect of elevated butyrate concentrations on the transcriptome of the rumen epithelium was quantified via serial biopsy sampling using RNA-seq technology. The mean number of genes transcribed in the rumen epithelial transcriptome was 17,323.63 ± 277.20 (±SD; N = 24) while the core transcriptome consisted of 15,025 genes. Collectively, 80 genes were identified as being significantly impacted by butyrate infusion across all time points sampled. Maximal transcriptional effect of butyrate on the rumen epithelium was observed at the 72-h infusion when the abundance of 58 genes was altered. The initial reaction of the rumen epithelium to elevated exogenous butyrate may represent a stress response as Gene Ontology (GO) terms identified were predominantly related to responses to bacteria and biotic stimuli. An algorithm for the reconstruction of accurate cellular networks (ARACNE) inferred regulatory gene networks with 113,738 direct interactions in the butyrate-epithelium interactome using a combined cutoff of an error tolerance (ɛ = 0.10) and a stringent P-value threshold of mutual information (5.0 × 10(-11)). Several regulatory networks were controlled by transcription factors, such as CREBBP and TTF2, which were regulated by butyrate. Our findings provide insight into the regulation of butyrate transport and metabolism in the rumen epithelium, which will guide our future efforts in exploiting potential beneficial effect of butyrate in animal well-being and human health.

Keywords: RNA-seq; butyrate; epithelial; networks; ruminant; transcriptome.

Figure 1

Ruminal concentrations (mM) of short-chain fatty acids in response to butyrate infusion. Notes: 0 h = baseline, immediately prior to initiation of continuous infusion with butyrate. 24 h, 72 h, and 168 h = 24, 72, and 168 h after continuous infusion, respectively. Post 24 h and post 168 h = 24 h and 168 h after infusion withdrawal, respectively. Red: acetate; Blue: butyrate; Green: propionate. Error bars = standard deviation, N = 4.

Figure 2

A regulatory gene network controlled by CREB binding protein (CREBBP). Notes: The network was inferred using ARACNE at a combined stringent cutoff of an error tolerance ɛ = 0.10 and a P-value threshold of mutual information (MI) at 5.0 × 10⁻¹¹. CREBBP had 87 direct interactions (the first neighbors) and 4139 indirect interactions (the second neighbors, not shown). The expression of the CREBBP gene, a transcription factor, at the mRNA level was significantly regulated by butyrate. The color cycle with gene symbols represented genes significantly regulated by butyrate.

Figure 3

Relative expression of a butyrate transporter, solute carrier family 5 (iodide transporter), member 8 (SLC5A8). Notes: The number denotes the relative abundance of the transcript in both the bovine epithelial cell and in the bovine rumen epithelium. CT: control, cells treated with PBS; BT: cells treated with 10 mM butyrate for 24 h in vitro. *False discovery rate (FDR) < 0.05; ***FDR < 0.001.

Figure 4

A regulatory gene network controlled by FOS. Notes: The network was inferred using ARACNE at a combined stringent cutoff of an error tolerance ɛ = 0.10 and a P-value threshold of mutual information (MI) at 5.0 × 10⁻¹¹. FOS had 4 direct interactions (the first neighbors) and 32 indirect interactions. The expression of all genes in this network at the mRNA level was significantly regulated by butyrate. The green/yellow color with gene symbols represents transcription factors.