Effects of a proinflammatory response on metabolic function of cultured, primary ruminal epithelial cells.

Inflammation of ruminal epithelium may occur during ruminal acidosis as a result of translocation and interaction of ruminal epithelial cells (REC) with molecules such as lipopolysaccharide (LPS). Such inflammation has been reported to alter cellular processes such as nutrient absorption, metabolic regulation, and energy substrate utilization in other cell types but has not been investigated for REC. The objectives of this study were to investigate the effects of LPS on metabolism of short-chain fatty acids by primary REC, as well as investigating the effects of media containing short-chain fatty acids on the proinflammatory response. Ruminal papillae from 9 yearling Speckle Park beef heifers were used to isolate and culture primary REC. Cells were grown in minimum essential medium (MEM) for 12 d before use and then reseeded in 24-well culture plates. The study was conducted as a 2 × 2 factorial, where cells were grown in unaltered MEM (REG) or medium containing 2 mM butyrate and 5 mM propionate (SCFA) with (50,000 EU/mL; +LPS) or without LPS (-LPS) for 24 h. Supernatant samples were collected for analysis of glucose and SCFA consumption. Cells were collected to determine the expression of mRNA for genes associated with inflammation (TNF, IL1B, CXCL2, CXCL8, PTGS2, and TLR4), purinergic signaling (P2RX7, ADORAB2, and CD73), nutrient transport [SLC16A1 (MCT1), SLC16A3 (MCT4), SLC5A8, and MCU], and cell metabolism [ACAT1, SLC2A1 (GLUT1), IGFBP3, and IGFBP5]. Protein expression of TLR4 and ketogenic enzymes (BDH1 and HMGCS1) were also analyzed using flow cytometry. Statistical analysis was conducted with the MIXED model of SAS version 9.4 (SAS Institute Inc., Cary, NC) with medium, LPS exposure, and medium × LPS interaction as fixed effects and animal within plate as a random effect. Cells tended to consume more glucose when exposed to LPS as opposed to no LPS exposure (31.8 vs. 28.7 ± 2.7), but consumption of propionate and butyrate was not influenced by LPS. Expression of TNF and IL1B was upregulated when exposed to LPS, and expression of CXCL2 and CXCL8 increased following LPS exposure with SCFA (medium × LPS). For cells exposed to LPS, we found a downregulation of ACAT1 and IGFBP5 and an upregulation of SLC2A1, SLC16A3, MCU, and IGFBP3. Medium with SCFA led to greater expression of MCU. SLC16A1 was upregulated in cells incubated with SCFA and without LPS compared with the other groups. Protein expression of ketogenic enzymes was not affected; however, BDH1 mean fluorescence intensity (MFI) expression tended to be less in cells exposed to LPS. These data are interpreted to indicate that when REC are exposed to LPS, they may increase glucose metabolism. Moreover, transport of solutes was affected by SCFA in the medium and by exposure to LPS. Overall, the results suggest that metabolic function of REC in vitro is altered by a proinflammatory response, which may lead to a greater glucose requirement.

Ruminal epithelial insulin-like growth factor-binding proteins 2, 3, and 6 are associated with epithelial cell proliferation.

The aim of this study was to identify factors that regulate ruminal epithelial insulin-like growth factor-binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short-chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin-like growth factor-I (IGF-I), -II (IGF-II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation rate of BREC was analyzed using a WST-1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d-Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF-I grew more rapidly than vehicle control-treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF-I-induced proliferation. IGF-II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF-I.

Short-chain fatty acids inhibit bovine rumen epithelial cells proliferation via upregulation of cyclin-dependent kinase inhibitors 1A, but not mediated by G protein-coupled receptor 41.

Short-chain fatty acids (SCFAs) play a critical role in regulation of rumen epithelial growth. The mechanisms underlying the regulatory effects of SCFAs on the proliferation of bovine rumen epithelial cells (BRECs) remain unknown; however, SCFAs can bind to G protein-coupled receptor 41 (GPR41); hence, the regulatory effects of SCFAs on BRECs proliferation may be mediated by GPR41. Here, we investigated the molecular mechanisms underlying the effects of SCFAs and GPR41 on BRECs proliferation. We demonstrated that SCFAs activate the expression of GPR41 and inhibit (p < .05) BRECs proliferation, while the GPR41 knockdown (GPR41KD) BRECs exhibited (p < .05) slow proliferation compared with controls. The treatment of BRECs with 10 mM SCFAs significantly enhanced (p < .05) expression of cyclin-dependent kinase inhibitors 1A (CDKN1A), 2A (CDKN2A) and 2B (CDKN2B) and inhibited (p < .05) their transition from G1 to S phase of the cell cycle, compared with controls. Remarkably, the GPR41KD BRECs treated with SCFAs restored high level of CDKN1A, relative to GPR41KD BRECs, but did not affect (p > .05) the expression of CDKN2A and CDKN2B. The GPR41KD BRECs had significantly reduced (p < .05) cyclin-dependent kinase 4 (CDK4) and cyclin D2 mRNA abundance compared with controls. The GPR41KD BRECs treated with SCFAs significantly decreased (p < .05) CDK4, cyclin D2, CDKN2A and CDKN2B mRNA abundance compared with BRECs treated with SCFAs. Overall, our results demonstrated that downregulation of CDK4 and cyclin D2 likely mediates the inhibitory effects of GPR41KD on BRECs proliferation. Additionally, CDKN1A plays a vital role in mediating the inhibitory effect of SCFAs on the BRECs proliferation, and that these changes are not mediated by GPR41.

Form of calf diet and the rumen. I: Impact on growth and development.

Preweaning diet is known to affect rumen tissue appearance at the gross level. The objectives of this experiment were to investigate effects of different preweaning diets on the growth and development of the rumen epithelium and on putative rumen epithelial stem and progenitor cell measurements at the gene and cell levels. Neonatal Holstein bull calves (n = 11) were individually housed and randomly assigned to 1 of 2 diets. The diets were milk replacer only (MRO; n = 5) or milk replacer with starter (MRS; n = 6). Diets were isoenergetic (3.87 ± 0.06 Mcal of metabolizable energy per day) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Milk replacer was 22% crude protein, 21.5% fat (dry matter basis). The textured calf starter was 21.5% crude protein (dry matter basis). Water was available ad libitum and feed and water intake were recorded daily. Putative stem and progenitor cells were labeled by administering a thymidine analog (5-bromo-2'-deoxyuridine, BrdU; 5 mg/kg of body weight in sterile saline) for 5 consecutive days and allowed a 25-d washout period. Calves were killed at 43 ± 1 d after a 6 h exposure to a defined concentration of volatile fatty acids. We obtained rumen tissue from the ventral sac and used it for immunohistochemical analyses of BrdU (putative stem and progenitor cells) and Ki67 (cell proliferation), gene expression analysis, and morphological measurements via hematoxylin and eosin staining. Epithelial stem and progenitor cell gene markers of interest, analyzed by real-time quantitative PCR, were ß1-integrin, keratin-14, notch-1, tumor protein p63, and leucine-rich repeat-containing G protein-coupled receptor 5. Body growth did not differ by diet, but empty reticulorumens were heavier in MRS calves (MRS: 0.67 ± 0.04 kg; MRO: 0.39 ± 0.04 kg). The percentage of label-retaining BrdU basale cells was higher in MRO calves than in MRS calves (2.0 ± 0.3% vs. 0.3 ± 0.2%, respectively). We observed a higher percentage of basale cells undergoing proliferation in MRS calves than in MRO calves (18.4 ± 2.6% vs. 10.8 ± 2.8%, respectively). Rumen epithelial gene expression was not affected by diet, but the submucosa was thicker in MRO calves and the epithelium and corneum/keratin layers were thicker in MRS calves. Presumptive stem and progenitor cells in the rumen epithelium were identifiable by their ability to retain labeled DNA in the long term, changed proliferative status in response to diet, and likely contributed to observed treatment differences in rumen tissue thickness.

Telocytes as a Novel Structural Component in the Muscle Layers of the Goat Rumen.

Telocytes (TCs) have been identified as a distinct type of interstitial cells, but have not yet been reported in the gastrointestinal tract (GIT) of ruminants. In this study, we used transmission electron microscopy (TEM) and double-labelling immunofluorescence (IF) (antibodies: CD34, vimentin and PGP9.5) to seek TCs and investigate their potential functions in the muscle layers of the goat rumen. TCs were distributed widely in the myenteric plexus (TC-MYs) between the circular and longitudinal muscle layers, within circular muscle layers (TC-CMs) as well as in longitudinal muscle layers (TC-LMs). Ultrastructurally, TCs displayed small cell bodies with several long prolongations-telopodes-harboring alternate thin segments (podomers) and dilated segments (podoms). The podoms contained mitochondria, rough endoplasmic reticulum, and caveolae. Telopodes frequently established close physical interactions with near telopodes, collagen fibers (CFs), nerve fibers (NFs), smooth muscle cells (SMCs), nerve tracts, and smooth muscle bundles, as well as with blood vessels (BVs). Furthermore, both homo- and heterotypic connections were observed. In addition, telopodes were capable of releasing extracellular vesicles (EVs). IF analyses proved that TCs were reliably labeled as CD34+/vimentin+ cells, displaying spindle- or triangle-shaped bodies with long prolongations, consistent with TEM results. Specifically, podoms were visible as obvious bright spots. These positive cells covered entire muscular layers, surrounding ganglions, intermuscular BVs as well as entire smooth muscle bundles, forming a network. TC-MYs were distributed as clusters in the external ganglion, encompassing the entire ganglion and spreading to the muscle layers where TC-CMs and TC-LMs seemingly surround whole smooth muscle bundles. TC-MYs were also scattered within the interior of the ganglion, surrounding each ganglionic neuron, following the glial cells layer. We speculate that TCs support the muscle layer structure of the goat rumen and facilitate intercellular signaling directly or indirectly via the TC network.

Transcriptome analysis of rumen epithelium and meta-transcriptome analysis of rumen epimural microbial community in young calves with feed induced acidosis.

Many common management practices used to raise dairy calves while on milk and during weaning can cause rumen acidosis. Ruminal pH has long been used to identify ruminal acidosis. However, few attempts were undertaken to understand the role of prolonged ruminal acidosis on rumen microbial community or host health in young calves long after weaning. Thus, the molecular changes associated with prolonged rumen acidosis in post weaning young calves are largely unknown. In this study, we induced ruminal acidosis by feeding a highly processed, starch-rich diet to calves starting from one week of age through 16 weeks. Rumen epithelial tissues were collected at necropsy at 17 weeks of age. Transcriptome analyses on the rumen epithelium and meta-transcriptome analysis of rumen epimural microbial communities were carried out. Calves with induced ruminal acidosis showed significantly less weight gain over the course of the experiment, in addition to substantially lower ruminal pH in comparison to the control group. For rumen epithelial transcriptome, a total of 672 genes (fold-change, FC ≥ 1.5; adjusted-p ≤ 0.05) showed significant differential expression in comparison to control. Biological pathways impacted by these differentially expressed genes included cell signaling and morphogenesis, indicating the impact of ruminal acidosis on rumen epithelium development. rRNA read-based microbial classification indicated significant increase in abundance of several genera in calves with induced acidosis. Our study provides insight into host rumen transcriptome changes associated with prolonged acidosis in post weaning calves. Shifts in microbial species abundance are promising for microbial species-based biomarker development and artificial manipulation. Such knowledge provides a foundation for future more precise diagnosis and preventative management of rumen acidosis in dairy calves.

Mechanism of peptide absorption in the isolated forestomach epithelial cells of dairy cows.

BACKGROUND: Peptide absorption from the forestomach plays a vital role in protein nutrition of dairy cows. This study was conducted to investigate the mechanism of dipeptide absorption in the forestomach of dairy cows using isolated omasal epithelial cells (OECs) and ruminal epithelial cells (RECs). RESULTS: Compared with RECs, the OECs formed a less tight monolayer, but had greater ability to transport glycylsarcosine (Gly-Sar) (P < 0.05). The OEC monolayers were immunopositive for the antibodies of anti-junction proteins. Gly-Sar transport was significantly greater at 37 °C than that at 4 °C, with an optimal pH of 6.0-6.5, and was decreased significantly by diethylpyrocarbonate and dipeptide Met-Gly (P < 0.05). The apical-to-basolateral transport was significantly greater than basolateral-to-apical transport (P < 0.05). Knockdown of peptide transporter 1 (PepT1) resulted in less Gly-Sar uptake in OECs, whereas overexpression of PepT1 in OECs resulted in higher Gly-Sar uptake (P < 0.05). Additionally, the expression of PepT1 was upregulated by the treatment with various dipeptides (P < 0.05). CONCLUSION: The OECs have a greater ability to transport Gly-Sar than RECs do. Both passive and active routes are involved in the process of Gly-Sar absorption in the isolated cultured forestomach epithelial cells from dairy cows. © 2018 Society of Chemical Industry.

Prenatal histomorphological development of the rumen in Dama dama.

This work studies the morphological changes taking place in the Dama dama rumen during prenatal development using histomorphometrics, surface microstructure and immunohistochemistry analysis as well as carrying out a comparative analysis of this species with other wild (red deer) and domestic-type ruminants. A total of 25 fallow deer embryos and fetuses were used, from the first stage of prenatal life until birth. The appearance of the rumen from the primitive gastric tube was observed at 51 days of prenatal life (CRL 3 cm, 21% gestation). By 57 days (CRL 4.3 cm, 24% gestation) the ruminal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Ruminal pillars were visible at 72 days (CRL 6 cm, 30% gestation), and by 85 days (CRL 7.2 cm, 35% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 80 days (CRL 7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Morphometric results showed accelerated growth of the epithelial layer and the tunica muscularis at 180 days (75% gestation). By contrast, the growth-rate of the lamina propria and submucosa declined from the early embryonic stages until birth. The serosa maintained a steady rate of growth until birth. Neuroendocrine cells (synaptophysin) were detected at 85 days (CRL 7.2 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein and vimentin) were found at 108 days (CRL 31 cm, 45% gestation) and 63 days (CRL 4.4 cm, 26% gestation) respectively. Neuropeptide Y and vasoactive intestinal polypeptide were detected immunohistochemically at 180 days (CRL 33 cm, 75% gestation) and 192 days (CRL 35 cm, 80% gestation) respectively. In comparison to other wild and domestic-type ruminants, histomorphogenesis of the rumen in Dama dama was similar to that reported in red deer and goats, but rather slower than that observed for sheep or cattle.

Modulation of ovine SBD-1 expression by Saccharomyces cerevisiae in ovine ruminal epithelial cells.

BACKGROUND: The ovine rumen is involved in host defense responses and acts as the immune interface with the environment. The ruminal mucosal epithelium plays an important role in innate immunity and secretes antimicrobial innate immune molecules that have bactericidal activity against a variety of pathogens. Defensins are cationic peptides that are produced by the mucosal epithelia and have broad-spectrum antimicrobial activity. Sheep ß-defensin-1 (SBD-1) is one of the most important antibacterial peptides in the rumen. The expression of SBD-1 is regulated by the probiotic, Saccharomyces cerevisiae (S.c); however, the regulatory mechanism has not yet been elucidated. In the current study, the effects of S.c on the expression and secretion of SBD-1 in ovine ruminal epithelial cells were investigated using quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). In addition, specific inhibitors were used to block the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), p38, JNK, and ERK1/2 signalling pathways separately or simultaneously, to determine the regulatory mechanism(s) governing S.c-induced SBD-1 upregulation. RESULTS: Incubation with S.c induced release of SBD-1 by ovine ruminal epithelial cells, with SBD-1 expression peaking after 12 h of incubation. The highest SBD-1 expression levels were achieved after treatment with 5.2 × 107 CFU∙mL- 1 S.c. Treatment with S.c resulted in significantly increased NF-κB, p38, JNK, ERK1/2, TLR2, and MyD88 mRNA expression. Whereas inhibition of mitogen-activated protein kinases (MAPKs) and NF-κB gene expression led to a decrease in SBD-1 expression. CONCLUSIONS: S.c was induced SBD-1 expression and the S.c-induced up-regulation of SBD-1 expression may be related to TLR2 and MyD88 in ovine ruminal epithelial cells. This is likely simultaneously regulated by the MAPKs and NF-κB pathways with the p38 axis of the MAPKs pathway acting as the primary regulator. Thus, the pathways regulating S.c-induced SBD-1 expression may be related to TLR2-MyD88-NF-κB/MAPKs, with the TLR2-MyD88-p38 component of the TLR2-MyD88-MAPKs signalling acting as the main pathway.

Associations among dietary non-fiber carbohydrate, ruminal microbiota and epithelium G-protein-coupled receptor, and histone deacetylase regulations in goats.

BACKGROUND: Diet-derived short-chain fatty acids (SCFAs) in the rumen have broad effects on the health and growth of ruminants. The microbe-G-protein-coupled receptor (GPR) and microbe-histone deacetylase (HDAC) axes might be the major pathway mediating these effects. Here, an integrated approach of transcriptome sequencing and 16S rRNA gene sequencing was applied to investigate the synergetic responses of rumen epithelium and rumen microbiota to the increased intake of dietary non-fiber carbohydrate (NFC) from 15 to 30% in the goat model. In addition to the analysis of the microbial composition and identification of the genes and signaling pathways related to the differentially expressed GPRs and HDACs, the combined data including the expression of HDACs and GPRs, the relative abundance of the bacteria, and the molar proportions of the individual SCFAs were used to identify the significant co-variation of the SCFAs, clades, and transcripts. RESULTS: The major bacterial clades promoted by the 30% NFC diet were related to lactate metabolism and cellulose degradation in the rumen. The predominant functions of the GPR and HDAC regulation network, under the 30% NFC diet, were related to the maintenance of epithelium integrity and the promotion of animal growth. In addition, the molar proportion of butyrate was inversely correlated with the expression of HDAC1, and the relative abundance of the bacteria belonging to Clostridum_IV was positively correlated with the expression of GPR1. CONCLUSIONS: This study revealed that the effects of rumen microbiota-derived SCFA on epithelium growth and metabolism were mediated by the GPR and HDAC regulation network. An understanding of these mechanisms and their relationships to dietary components provides better insights into the modulation of ruminal fermentation and metabolism in the promotion of livestock production.

Histamine Induces Bovine Rumen Epithelial Cell Inflammatory Response via NF-κB Pathway.

BACKGROUND/AIMS: Subacute ruminal acidosis (SARA) is a common disease in high-producing lactating cows. Rumenitis is the initial insult of SARA and is associated with the high concentrations of histamine produced in the rumen of dairy cows during SARA. However, the exact mechanism remains unclear. The objective of the current study is to investigate whether histamine induces inflammation of rumen epithelial cells and the underlying mechanism of this process. METHODS: Bovine rumen epithelial cells were cultured and treated with different concentrations of histamine and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) cultured in different pH medium (pH 7.2 or 5.5). qRT-PCR, Western-blotting, ELISA and immunocytofluorescence were used to evaluate whether histamine activated the NF-κB pathway and inflammatory cytokines. RESULTS: The results showed that histamine significantly increased the activity of IKK ß and the phosphorylation levels of IκB α, as well as upregulated the mRNA and protein expression levels of NF-κB p65 in the rumen epithelial cells cultured in neutral (pH=7.2) and acidic (pH=5.5) medium. Furthermore, histamine treatment also significantly increased the transcriptional activity of NF-κB p65. High expression and transcriptional activity of NF-κB p65 significantly increased the mRNA expressions and concentrations of inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1ß), thereby inducing the inflammatory response in bovine rumen epithelial cells. However, inhibition of NF-κB p65 by PDTC significantly decreased the expressions and concentrations of the inflammatory cytokines induced by histamine in the rumen epithelial cells cultured in the neutral and acidic medium. CONCLUSION: The present data indicate that histamine induces the inflammatory response of bovine rumen epithelial cells through the NF-κB pathway.

Effects of dietary physical or nutritional factors on morphology of rumen papillae and transcriptome changes in lactating dairy cows based on three different forage-based diets.

BACKGROUND: Rumen epithelial tissue plays an important role in nutrient absorption and rumen health. However, whether forage quality and particle size impact the rumen epithelial morphology is unclear. The current study was conducted to elucidate the effects of forage quality and forage particle size on rumen epithelial morphology and to identify potential underlying molecular mechanisms by analyzing the transcriptome of the rumen epithelium (RE). To achieve these objectives, 18 mid-lactation dairy cows were allocated to three groups (6 cows per group), and were fed with one of three different forage-based diets, alfalfa hay (AH), corn stover (CS), and rice straw (RS) for 14 weeks, respectively. Ruminal volatile fatty acids (VFAs) and epithelial thickness were determined, and RNA-sequencing was conducted to identify the transcriptomic changes of rumen epithelial under different forage-based diets. RESULTS: The RS diet exhibited greater particle size but low quality, the AH diet was high nutritional value but small particle size, and CS diet was low quality and small particle size. The ruminal total VFA concentration was greater in AH compared with those in CS or RS. The width of the rumen papillae was greater in RS-fed cows than in cows fed AH or CS. In total, 31, 40, and 28 differentially expressed (DE, fold change > 2, FDR < 0.05) genes were identified via pair-wise comparisons including AH vs. CS, AH vs. RS, and RS vs. CS, respectively. Functional classification analysis of DE genes revealed dynamic changes in ion binding (such as DSG1) between AH and CS, proliferation and apoptotic processes (such as BAG3, HLA-DQA1, and UGT2B17) and complement activation (such as C7) between AH or RS and CS. The expression of HLA-DQA1 was down-regulated in RS compared with AH and CS, and the expression of UGT2B17 was down-regulated in RS compared with CS, with positive (R = 0.94) and negative (R = -0.96) correlation with the width of rumen epithelial papillae (P < 0.05), respectively. CONCLUSION: Our results suggest that both nutrients (VFAs) and particle sizes can alter expression of genes involved in cell proliferation/apoptosis process and complement complex. Our results suggest that particle size may be more important in regulating rumen epithelial morphology when animals are fed with low-quality forage diets and the identified DE genes may affect the RE nutrient absorption or morphology of RE. Our findings provide insights into the effects of the dietary particle size in the future management of dairy cow feeding, that when cows were fed with low-quality forage (such as rice straw), smaller particle size may be beneficial for nutrients absorption and milk production.

Taxonomic Identification of Ruminal Epithelial Bacterial Diversity during Rumen Development in Goats.

Understanding of the colonization process of epithelial bacteria attached to the rumen tissue during rumen development is very limited. Ruminal epithelial bacterial colonization is of great significance for the relationship between the microbiota and the host and can influence the early development and health of the host. MiSeq sequencing of 16S rRNA genes and quantitative real-time PCR (qPCR) were applied to characterize ruminal epithelial bacterial diversity during rumen development in this study. Seventeen goat kids were selected to reflect the no-rumination (0 and 7 days), transition (28 and 42 days), and rumination (70 days) phases of animal development. Alpha diversity indices (operational taxonomic unit [OTU] numbers, Chao estimate, and Shannon index) increased (P < 0.01) with age, and principal coordinate analysis (PCoA) revealed that the samples clustered together according to age group. Phylogenetic analysis revealed that Proteobacteria, Firmicutes, and Bacteroidetes were detected as the dominant phyla regardless of the age group, and the abundance of Proteobacteria declined quadratically with age (P < 0.001), while the abundances of Bacteroidetes (P = 0.088) and Firmicutes (P = 0.009) increased with age. At the genus level, Escherichia (80.79%) dominated at day zero, while Prevotella, Butyrivibrio, and Campylobacter surged (linearly; P < 0.01) in abundance at 42 and 70 days. qPCR showed that the total copy number of epithelial bacteria increased linearly (P = 0.013) with age. In addition, the abundances of the genera Butyrivibrio, Campylobacter, and Desulfobulbus were positively correlated with rumen weight, rumen papilla length, ruminal ammonia and total volatile fatty acid concentrations, and activities of carboxymethylcellulase (CMCase) and xylanase. Taking the data together, colonization by ruminal epithelial bacteria is age related (achieved at 2 months) and might participate in the anatomic and functional development of the rumen.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a ß-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions.

Expression and cellular localization of monocarboxylate transporters (MCT2, MCT7, and MCT8) along the cattle gastrointestinal tract.

Fourteen members of the monocarboxylate transporter (MCT, SLC16) family have been identified, each having a different tissue distribution and substrate specificity. The expression of monocarboxylate transporters MCT1 and MCT4 have been studied in the gastrointestinal tract of ruminants; however, details of the expression of other MCT isoforms in the various parts of ruminant gastrointestinal tract are lacking. Reverse transcription with the polymerase chain reaction was used to study the regional distribution of MCT2, MCT3, and MCT5-MCT14 in the cattle gastrointestinal tract and verified the existence of MCT mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT9, MCT10, MCT13, and MCT14 in the ruminal and abomasal epithelia, mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT10, MCT13, and MCT14 in the jejunum, and mRNA transcripts for MCT2, MCT3, MCT4, MCT7, MCT8, MCT13, and MCT14 in the caecum of cattle. At the cellular level, immunohistochemical studies localized MCT2, MCT7, and MCT8 proteins in the cattle rumen, abomasum, jejunum, and caecum. This is the first study to detect the expression of various MCT isoforms in the gastrointestinal tract of a ruminant species. Our data suggest that these transporter proteins are involved in essential physiologic processes and are possible molecular targets for studying the regulation of the transport of short-chain monocarboxylates, aromatic amino acids, and thyroid hormones across the gastrointestinal tract of cattle.

Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet.

Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.

Role of metabolic and cellular proliferation genes in ruminal development in response to enhanced plane of nutrition in neonatal Holstein calves.

We evaluated expression of 50 genes encoding enzymes involved in metabolism, cellular growth, and various transporters in ruminal epithelium tissue when calves were fed conventional milk replacer (MR) and starter (control) or enhanced MR and enhanced starter. Male Holstein calves were fed reconstituted control MR [20% crude protein (CP), 20% fat; 0.57 kg of solids/calf] plus conventional starter (19.6% CP, dry matter basis) or a high-protein MR (ENH; 28.5% CP, 15% fat; at ∼2% of body weight) plus high-CP starter (25.5% CP, dry matter basis). Groups of calves in control and ENH were harvested after 43 d (wk 5) and 71 d (wk 10) of feeding. Ruminal epithelium from 5 calves (3 to 42 d age) in each group was used for transcript profiling using quantitative reverse transcription PCR. No differences were observed for plasma ß-hydroxybutyrate (BHBA) concentration but BHBA increased by wk 10 regardless of treatment. Reticulorumen mass postweaning was greater in calves consuming the ENH diet and corresponded with overall greater serum insulin. A marked upregulation of the ketogenic genes HMGCS2, HMGCL, and BDH1 was observed, concomitant with downregulation of expression of genes involved in fatty acid oxidation (CPT1A, ACADVL) at wk 10. Higher relative percentage mRNA abundance of HMGCS2 (∼40% of total genes assayed), the rate-controlling enzyme in hepatic ketogenesis, underscored its importance for ruminal cell energy metabolism. Higher PPARA expression and blood nonesterified fatty acids at wk 5 due to ENH were suggestive of more extensive long-chain fatty acid oxidation in ruminal epithelial cells during the milk-fed phase. In contrast, calves fed control consumed more starter during the milk-fed phase, which likely increased production of volatile fatty acids and accounted for higher expression of propionyl-CoA carboxylase (PCCA) and the Na(+)/H(+) exchanger 2 (SLC9A2) at wk 5. Expression of G-coupled protein receptors for short-chain fatty acids was undetectable. The expression of the urea transporter (SLC14A1) increased markedly with age and was correlated with the increase in blood urea N. Expression of genes involved in cell proliferation (INSR, FOXO1, AKT3) was greater for ENH primarily during the milk-fed period and corresponded with greater serum insulin. The greater reticuloruminal mass in calves fed ENH postweaning underscores the importance of feeding high-quality starter and indicates that fermentability of the diet, by providing metabolic fuel for ruminal epithelial cells, is a primary driver of ruminal development postweaning. From a mechanistic standpoint, the 7-fold increase in expression of the nuclear receptor PPARD (∼40-fold more abundant than PPARA) suggests a key role in controlling biological processes driving ruminal epithelial cell development. Elucidating ligands of PPARD may provide the means for nutritional regulation of rumen development.

Intracellular magnesium detection by fluorescent indicators.

Magnesium is essential for a wide variety of biochemical reactions and physiological functions, but its regulatory mechanisms (both at the cellular and at the systemic level) are still poorly characterized. Not least among the reasons for this gap are the technical difficulties in sensing minor changes occurring over a high background concentration. Specific fluorescent indicators are highly sensitive tools for dynamic evaluation of intracellular magnesium concentration. We herein discuss the main criteria to consider when choosing a magnesium-specific fluorescent indicator and provide examples among commercial as well as developmental sensors. We focus on spectrofluorimetric approaches to quantify Mg(2+) concentration in cell or mitochondria suspensions, and on imaging techniques to detect intracellular magnesium distribution and fluxes by live microscopy, reporting a detailed description of standard protocols for each method. The general guidelines we provide should be applicable to specific issues by any researcher in the field.

Na+ transport across rumen epithelium of hay-fed sheep is acutely stimulated by the peptide IGF-1 in vitro.

An energy-rich diet leads to enhanced ruminal Na(+) absorption, which is associated with elevated plasma insulin-like growth factor 1 (IGF-1) levels and an increased number of IGF-1 receptors in rumen papillae. This study examined the in vitro effect of IGF-1 on Na(+) transport across the rumen epithelium of hay-fed sheep, in which the IGF-1 concentration in plasma is lower than in concentrate-fed animals. At concentrations ranging from 20 to 100 µg l(-1), serosal LR3-IGF-1, a recombinant analogue of IGF-1, rapidly (within 30 min) stimulated the mucosal-to-serosal Na(+) flux (J(ms)Na) and consequently the net Na(+) flux (J(net)Na). Compared with controls, J(net)Na increased by about 60% (P < 0.05) following the serosal application of LR3-IGF-1 (20 µg l(-1)). The IGF-1-induced increment of J(ms)Na and J(net)Na was inhibited by mucosal amiloride (1 mmol l(-1)). Neither IGF-1 nor amiloride altered tissue conductance or the short-circuit current of the isolated rumen epithelium. These data support the assumption that the stimulating effect of serosally applied IGF-1 on Na(+) transport across the rumen epithelium is mediated by Na(+)-H(+) exchange (NHE). A further study was performed with cultured rumen epithelial cells and a fluorescent probe (BCECF) to estimate the rate of pH(i) recovery after acid loading. The pH(i) of isolated rumen epithelial cells was 6.43 ± 0.15 after butyrate loading and recovered by 0.26 ± 0.02 pH units (15 min)(-1). Application of LR3-IGF-1 (20 µg l(-1)) significantly increased the rate of pH(i) recovery to 0.33 ± 0.02 pH units (15 min)(-1). Amiloride administration reduced the recovery rate in both control and IGF-1-stimulated cells. These results show, for the first time, that an acute effect of IGF-1 on Na(+) absorption across rumen epithelium results from increased NHE activity. Insulin-like growth factor 1 is thus important for the fast functional adaptation of ruminal Na(+) transport via NHE.