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

Baldwin RL, Wu S, Li W, Li C, Bequette BJ, Li RW - Gene Regul Syst Bio (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, Bovine Functional Genomics Laboratory, Beltsville, MD, USA.

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.

No MeSH data available.


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.
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f3-grsb-6-2012-067: 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.

Mentions: The majority of our knowledge on regulatory impact of butyrate on global gene expression is derived from in vitro studies and observations.6,31,32 However, caution in interpretation of in vitro data and application of knowledge gained in vitro to in vivo models is warranted. SCFAs, such as butyrate, are known to promote rumen development and stimulate the proliferation of rumen epithelial cells in vivo.33 However, butyrate inhibits the proliferation of epithelial cells of the large intestine, rumen and kidney by down- regulating genes controlling cell proliferation in vitro.2,32 Butyrate also induces apoptosis and differentiation of tumor cells.34,35 Moreover, an in vitro study of epithelial cells of different origin (rat small intestine vs. human colon) has demonstrated that the cell type affects butyrate uptake characteristics.36 In fact, opposite effects of butyrate are observed for many cellular processes, such as cell proliferation and division, between in vitro and in vivo models and are clearly reflected in transcriptome characteristics.33,37 A number of genes related to cell proliferation and cell cycle progress were significantly down-regulated by a 24-h 10 mM butyrate incubation of established bovine rumen epithelial cells in long-term culturing (Wu et al 2012, personal communication). In contrast, expression of these same genes in the rumen epithelium in the present data set was not altered despite a 2-fold increase from 19.5 mM to 38.5 mM in intraruminal butyrate concentration (Fig. 1). Interestingly, the abundance of a butyrate transporter, SLC5A8, in the rumen epithelial transcriptome was significantly reduced concomitant with intra-ruminal butyrate concentration increases. However, SLC5A8 expression at the mRNA level was significantly increased ~21 fold by butyrate exposure in vitro (Fig. 3). This apparent opposite effect of butyrate on the expression of its transporter is suggestive of alternative regulatory mechanisms relating to butyrate uptake control and transport by the intact rumen epithelium and cells in culture. Moreover, cellular butyrate metabolism may be different between in vivo and in vitro models due to changes in the rate of removal of end product as well as changes between cell-cell interactions and micro-environments present in vivo, but disrupted in vitro. To this point, epithelial metabolism of butyrate, especially the pathways leading to ketogenesis, helps to maintain a butyrate concentration gradient in vivo, which in turn facilitates butyrate uptake and affects butyrate intracellular concentrations.30 Rate-limiting enzymes in the ruminal ketogenic process, such as acetyl-CoA acetyl transferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA synthases (HMGCS) 1 (cytoplasmic) and 2 (mitochondrial), play an important role in regulating butyrate metabolism at the substrate level. As depicted in Table 4, key enzymes in butyrate metabolic pathways exhibited a different expression pattern between the cell line and the rumen epithelium. As expected, HMGCS2 of mitochondrial origin was significantly up-regulated by butyrate in vitro, in response to increased butyrate concentration. However, expression of HMGCS2 remained unchanged in vivo.


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

Baldwin RL, Wu S, Li W, Li C, Bequette BJ, Li RW - Gene Regul Syst Bio (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362330&req=5

f3-grsb-6-2012-067: 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.
Mentions: The majority of our knowledge on regulatory impact of butyrate on global gene expression is derived from in vitro studies and observations.6,31,32 However, caution in interpretation of in vitro data and application of knowledge gained in vitro to in vivo models is warranted. SCFAs, such as butyrate, are known to promote rumen development and stimulate the proliferation of rumen epithelial cells in vivo.33 However, butyrate inhibits the proliferation of epithelial cells of the large intestine, rumen and kidney by down- regulating genes controlling cell proliferation in vitro.2,32 Butyrate also induces apoptosis and differentiation of tumor cells.34,35 Moreover, an in vitro study of epithelial cells of different origin (rat small intestine vs. human colon) has demonstrated that the cell type affects butyrate uptake characteristics.36 In fact, opposite effects of butyrate are observed for many cellular processes, such as cell proliferation and division, between in vitro and in vivo models and are clearly reflected in transcriptome characteristics.33,37 A number of genes related to cell proliferation and cell cycle progress were significantly down-regulated by a 24-h 10 mM butyrate incubation of established bovine rumen epithelial cells in long-term culturing (Wu et al 2012, personal communication). In contrast, expression of these same genes in the rumen epithelium in the present data set was not altered despite a 2-fold increase from 19.5 mM to 38.5 mM in intraruminal butyrate concentration (Fig. 1). Interestingly, the abundance of a butyrate transporter, SLC5A8, in the rumen epithelial transcriptome was significantly reduced concomitant with intra-ruminal butyrate concentration increases. However, SLC5A8 expression at the mRNA level was significantly increased ~21 fold by butyrate exposure in vitro (Fig. 3). This apparent opposite effect of butyrate on the expression of its transporter is suggestive of alternative regulatory mechanisms relating to butyrate uptake control and transport by the intact rumen epithelium and cells in culture. Moreover, cellular butyrate metabolism may be different between in vivo and in vitro models due to changes in the rate of removal of end product as well as changes between cell-cell interactions and micro-environments present in vivo, but disrupted in vitro. To this point, epithelial metabolism of butyrate, especially the pathways leading to ketogenesis, helps to maintain a butyrate concentration gradient in vivo, which in turn facilitates butyrate uptake and affects butyrate intracellular concentrations.30 Rate-limiting enzymes in the ruminal ketogenic process, such as acetyl-CoA acetyl transferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA synthases (HMGCS) 1 (cytoplasmic) and 2 (mitochondrial), play an important role in regulating butyrate metabolism at the substrate level. As depicted in Table 4, key enzymes in butyrate metabolic pathways exhibited a different expression pattern between the cell line and the rumen epithelium. As expected, HMGCS2 of mitochondrial origin was significantly up-regulated by butyrate in vitro, in response to increased butyrate concentration. However, expression of HMGCS2 remained unchanged in vivo.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, Bovine Functional Genomics Laboratory, Beltsville, MD, USA.

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.

No MeSH data available.