Publications by authors named "Jörg R Aschenbach"

69 Publications

Expression of proposed methionine transporters along the gastrointestinal tract of pigs and their regulation by dietary methionine sources.

Genes Nutr 2021 Sep 6;16(1):14. Epub 2021 Sep 6.

Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.

Background: Given the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs.

Methods: A total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of BAT1, ATB, rBAT, ASCT2, IMINO, LAT4, yLAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of BAT1, ASCT2, LAT2, and LAT4.

Results: Expression of all investigated transcripts differed significantly along the GIT. BAT1, rBAT, yLAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (yLAT1) intestine. Protein expression of BAT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met (small intestine) and L-Met (small intestine, oral mucosa, and stomach).

Conclusion: A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12263-021-00694-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8422629PMC
September 2021

The Combined Influence of Magnesium and Insulin on Central Metabolic Functions and Expression of Genes Involved in Magnesium Homeostasis of Cultured Bovine Adipocytes.

Int J Mol Sci 2021 May 31;22(11). Epub 2021 May 31.

Institute of Veterinary Physiology, Freie Universität Berlin, 14163 Berlin, Germany.

At the onset of lactation, dairy cows suffer from insulin resistance, insulin deficiency or both, similar to human diabetes, resulting in lipolysis, ketosis and fatty liver. This work explored the combined effects of different levels of magnesium (0.1, 0.3, 1 and 3 mM) and insulin (25, 250 and 25,000 pM) on metabolic pathways and the expression of magnesium-responsive genes in a bovine adipocyte model. Magnesium starvation (0.1 mM) and low insulin (25 pM) independently decreased or tended to decrease the accumulation of non-polar lipids and uptake of the glucose analog 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG). Activity of glycerol 3-phosphate dehydrogenase (GPDH) was highest at 25 pM insulin and 3 mM magnesium. Expression of and was reduced at 0.1 mM magnesium either across insulin concentrations () or at 250 pM insulin (). expression was reduced at 3 mM magnesium. expression was reduced at 3 mM and 0.1 mM magnesium at 25 and 250 pM insulin, respectively. Expression of , , and was not affected. We conclude that magnesium promotes lipogenesis in adipocytes and inversely regulates the transcription of genes that increase vs. decrease cytosolic magnesium concentration. The induction of GAPDH activity by surplus magnesium at low insulin concentration can counteract excessive lipomobilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22115897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199494PMC
May 2021

Changes in the Relationship between Ionized and Total Calcium in Clinically Healthy Dairy Cows in the Period around Calving.

Animals (Basel) 2021 Apr 6;11(4). Epub 2021 Apr 6.

Institute of Veterinary Physiology, Faculty of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany.

We aimed to establish a model for prediction of iCa from tCa, using multivariable regressions with diverse blood constituents. Blood was taken from 14 cows at days -2, 0, 2, 4, 7, and 14 relative to parturition. Cows were clinically healthy, and no hypocalcaemia prophylaxis and treatment were applied. Total calcium and further parameters were determined from frozen serum. Ionized calcium, blood gases, and electrolytes were determined from heparin-stabilized blood samples. Linear regression between iCa and tCa was estimated. Precision improved only slightly using a multivariable model. Best precision was achieved when estimating the iCa:tCa ratio from other blood constituents. To identify the reason behind the poorly predictive value of tCa for iCa, the relative changes of iCa and tCa around calving were calibrated to the respective values of day -2 (=100%) for each cow. An increase in the iCa:tCa ratio was observed from 0.43 at day -2 to 0.48 at day 0, followed by a gradual decrease towards 0.43 at day 7. We conclude that routine measurement of iCa should be implemented in the diagnosis of hypocalcaemia. An optimized estimate of iCa from tCa with non-esterified fatty acids (NEFA), beta-hydroxybutyric acid, cholesterol, and phosphorous as co-predictors is still poorly satisfying.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ani11041036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067466PMC
April 2021

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

PLoS One 2021 5;16(3):e0248165. Epub 2021 Mar 5.

Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine, Vienna, Austria.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935255PMC
March 2021

Functional Changes of the Community of Microbes With Ni-Dependent Enzyme Genes Accompany Adaptation of the Ruminal Microbiome to Urea-Supplemented Diets.

Front Microbiol 2020 22;11:596681. Epub 2020 Dec 22.

Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany.

Urea is an inexpensive non-protein nitrogen source commonly supplemented to the diets of ruminants. It is cleaved to ammonia by bacterial ureases, which require Ni as a catalyst for ureolysis. The key event in the changes of the ruminal microbiome after urea supplementation remains unknown. We have therefore investigated changes in the ruminal microbiome and its community with Ni-dependent enzyme genes following urea supplementation and analyzed the associations of rumen environmental factors, including fermentation variables and Ni concentrations, with the compositional and functional changes of these communities. We found that urea supplementation increased urease activity and the concentrations of ammonia and Ni, and tended to increase concentrations of short chain fatty acids and acetate, whereas it decreased rumen pH and the L-/D-lactate ratio. With standards for genome completeness >60% and strain heterogeneity <10%, 20 bacterial species containing five Ni-dependent enzyme genes were detected in the metagenome sequences. For the five Ni-dependent enzyme genes, urea supplementation increased the relative abundances of genes of urease and acetyl-CoA synthase, whereas it decreased the relative abundances of genes of glyoxalase I, [NiFe]-hydrogenase, and lactate racemase. For the 20 microbes with Ni-dependent enzyme genes, urea supplementation increased the relative abundances of five bacteria exhibiting high capacities for the utilization of hemicellulose and pectin for butyrate and fatty acid biosynthesis. For the ruminal microbiome, urea supplementation increased the metagenomic capacities for hemicellulose and pectin degradation, butyrate generation, fatty acid biosynthesis, and carbon fixation, whereas it decreased the metagenomic capacities for starch degradation, propionate generation, and sulfur and nitrogen metabolism. Constrained correspondence analysis identified rumen ammonia and Ni concentrations as likely driving factors in the reshaping of the ruminal microbiome and, together with pH, of the community of microbes with Ni-dependent enzyme genes. Thus, the functional change of the latter community is probably an important event in the adaptation of the ruminal microbiome to urea-supplemented diets. This result provides a new perspective for the understanding of the effects of urea supplementation on rumen fermentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.596681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782429PMC
December 2020

Dietary Mg Intake and the Na/Mg Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes.

Int J Mol Sci 2020 Nov 3;21(21). Epub 2020 Nov 3.

Biomedical Center Martin, Jessenius Faculty of Medicine, Comenius University in Bratislava, Mala Hora 4D, 036 01 Martin, Slovakia.

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na/Mg exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI-CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21218221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663288PMC
November 2020

Erratum to "The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic in vitro" [Vet. Anim. Sci. 3C (2017) 10-17].

Vet Anim Sci 2020 Jun 10;9:100093. Epub 2020 Jan 10.

Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vas.2020.100093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386718PMC
June 2020

Dietary Supplementation of dl-Methionine Potently Induces Sodium-Dependent l-Methionine Absorption in Porcine Jejunum Ex Vivo.

J Nutr 2020 07;150(7):1782-1789

Institute of Veterinary Physiology, Freie Universtität Berlin, Berlin, Germany.

Background: Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock.

Objectives: The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets.

Methods: A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met + cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 μM and a high concentration of 5 mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA.

Results: Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P < 0.05). Pigs supplemented with dl-Met had greater (P < 0.085) absorptive fluxes at 50 μM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 μM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P < 0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P < 0.05).

Conclusions: Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jn/nxaa115DOI Listing
July 2020

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in induction.

Adipocyte 2020 12;9(1):35-50

Institute of Veterinary-Physiology, Freie Universität Berlin, Berlin, Germany.

Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for ). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of and mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in induction, but this is not linearly related to the primarily BSL-driven lipid accumulation.: AcA: acetic acid; AsA: ascorbic acid; ASC: adipose-derived stem cells; BSL: bovine serum lipids; DAPI: 4´,6-diamidino-2-phenylindole; DLK: delta like non-canonical notch ligand; DMEM: Dulbecco's modified Eagle's medium; DPBS: Dulbecco's phosphate-buffered saline; ENG: endoglin; FABP: fatty acid binding protein; FAS: fatty acid synthase; GLUT4: glucose transporter type 4; IBMX: 3-isobutyl-1-methylxanthine; LPL: lipoprotein lipase; MSC: mesenchymal stem cells; α-MEM: α minimum essential medium; NT5E: ecto-5'-nucleotidase; PDGFRα: platelet derived growth factor receptor α; PPAR: peroxisome proliferator activated receptor γ; RPS19: ribosomal protein S19; SEM: standard error of the mean; THY1: Thy-1 cell surface antigen; TRT: treatment; TRT-Con: treatment negative control; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21623945.2020.1720480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999845PMC
December 2020

Effects of dietary menthol-rich bioactive lipid compounds on zootechnical traits, blood variables and gastrointestinal function in growing sheep.

J Anim Sci Biotechnol 2019 2;10:86. Epub 2019 Dec 2.

1Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

Background: The present study aimed at investigating the influence of 90% menthol-containing plant bioactive lipid compounds (PBLC, essential oils) on growth performance, blood haematological and biochemical profile, and nutrient absorption in sheep. Twenty-four growing Suffolk sheep were allotted into three dietary treatments: Control (without PBLC), lower dose of PBLC (PBLC-L; 80 mg/d) and higher dose of PBLC (PBLC-H; 160 mg/d). Sheep in all groups were fed meadow hay ad libitum plus 600 g/d of concentrate pellets for 28 d.

Results: Average daily gain was not affected by treatment. Feeding of PBLC increased hay and total feed intake per kg body weight ( < 0.05). Counts of total leucocytes, lymphocytes and monocytes were not different among treatments. However, neutrophil count decreased ( < 0.05) in PBLC-H with a similar trend in PBLC-L ( < 0.10). Concentrations of glucose, bilirubin, triglycerides, cholesterol, urea and magnesium in serum were not different among sheep fed different doses of PBLC. However, serum calcium concentration tended to increase in PBLC-H ( < 0.10) and serum concentrations of aspartate & asparagine ( < 0.01) and glutamate & glutamine ( < 0.05) increased linearly with increasing PBLC dose. In ruminal epithelia isolated from the rumen after killing, baseline conductance ( ;  < 0.05) and short-circuit current ( ;  < 0.01) increased in both PBLC groups. Ruminal uptakes of glucose and methionine in the presence of Na were not affected by the dietary PBLC supplementation. In the absence of Na, however, glucose and methionine uptakes increased ( < 0.05) in PBLC-H. In the jejunum, tended to increase in PBLC-H ( < 0.10), but baseline was not affected. Intestinal uptakes of glucose and methionine were not influenced by PBLC in the presence or absence of Na.

Conclusion: The results suggest that menthol-rich PBLC increase feed intake, and passive ion and nutrient transport, the latter specifically in the rumen. They also increased serum concentrations of urea precursor amino acids and tended to increase serum calcium concentrations. Future studies will have to show whether some of these findings might be commonly linked to a stimulation of transient receptor potential (TRP) channels in the gastrointestinal tract.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40104-019-0398-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886202PMC
December 2019

Inflammatory Responses of Porcine MoDC and Intestinal Epithelial Cells in a Direct-Contact Co-culture System Following a Bacterial Challenge.

Inflammation 2020 Apr;43(2):552-567

Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.

Intestinal epithelial cells (IEC) and immune cells, such as dendritic cells (DC), jointly control the immune response towards luminal pathogens in the intestinal mucosa. Crosstalk between IEC and DC is crucial for coordinating immune responses and occurs via soluble factors and direct cell-cell contacts. The present study aimed at establishing a direct-contact co-culture model of porcine IEC and DC to mimic these interactions. The effects of (1) co-cultivation of the two cell types and (2) bacterial infection on the inflammatory response patterns of each of the cell types were determined with a special focus on the canonical and non-canonical inflammasome signaling pathways. In infection experiments, in vitro cultures were exposed to either the probiotic Enterococcus (E.) faecium NCIMB 10415 or enterotoxigenic Escherichia coli (ETEC). In porcine IEC (IPEC-J2), co-cultivation with porcine monocyte-derived DC (MoDC) resulted in reduced basal NLRP3 (nucleotide oligomerization domain [NOD]-like receptor [NLR] family, pyrin domain containing 3) inflammasome mRNA levels in unstimulated conditions. In porcine MoDC, the presence of IPEC-J2 cells evoked a noticeable decrease of interleukin (IL)-8 and transforming growth factor-β (TGF-β) mRNA and protein expression. ETEC, in contrast to E. faecium, modulated the inflammasome pathway in IPEC-J2 cells and porcine MoDC. Co-cultured IPEC-J2 cells showed an augmented inflammasome response to ETEC infection. By contrast, MoDC revealed a weakened ETEC response under such co-culture conditions as indicated by a reduction of inflammasome-related IL-1β protein release. Our data indicate that the close contact between IEC and resident immune cells has a major effect on their immunological behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10753-019-01137-4DOI Listing
April 2020

Transcriptomic analyses suggest a dominant role of insulin in the coordinated control of energy metabolism and ureagenesis in goat liver.

BMC Genomics 2019 Nov 14;20(1):854. Epub 2019 Nov 14.

Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany.

Background: The ureagenesis plays a central role in the homeostatic control of nitrogen metabolism. This process occurs in the liver, the key metabolic organ in the maintenance of energy homeostasis in the body. To date, the understanding of the influencing factors and regulators of ureagenesis in ruminants is still poor. The aim of this study was to investigate the relationship between energy metabolism and ureagenesis and detect the direct regulators of ureagenesis in the liver by using RNA-seq technology.

Results: Eighteen four-month-old male goats were divided into two groups randomly and received a diet containing 10% (LNFC group, n = 9) or 30% non-fiber carbohydrate (MNFC group, n = 9), respectively, for four weeks. The global gene expression analysis of liver samples showed that, compared with a LNFC diet, the MNFC diet promoted the expression of genes required for synthesis of fatty acid and glycerol, whereas it suppressed those related to fatty acid oxidation, gluconeogenesis from amino acids and ureagenesis. Additionally, gene expression for rate-limiting enzymes of ureagenesis were highly correlated to the gene expression of key enzymes of both fatty acid synthesis and glycerol synthesis (Spearman correlation coefficient > 0.8 and p < 0.05). In the differentially expressed signaling pathways related to the endocrine system, the MNFC diet activated the insulin and PPAR signaling pathway, whereas it suppressed the leptin-JAK/STAT signaling pathway, compared with the LNFC diet. Reverse transcription quantitative PCR analyses of 40 differentially expressed genes confirmed the RNA-seq results (R = 0.78).

Conclusion: Our study indicated that a dietary NFC-induced increase of energy supply promoted lipid anabolism and decreased ureagenesis in the caprine liver. By combining our results with previously published reports, insulin signaling can be suggested to play the dominant role in the coordinated control of hepatic energy metabolism and ureagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-019-6233-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854773PMC
November 2019

Dietary supplementation of menthol-rich bioactive lipid compounds alters circadian eating behaviour of sheep.

BMC Vet Res 2019 Oct 21;15(1):352. Epub 2019 Oct 21.

Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg19b, Berlin, Germany.

Background: Plant bioactive lipid compounds (PBLC), commonly known as essential oils, are increasingly evaluated as feed additives in ruminants due to beneficial effects on animal performance and health; however, there is no study evaluating circadian eating behaviour in ruminants. Altered eating behaviour may be implicated in changes of feed intake in ruminants. Therefore, the present study investigated the influence of menthol-rich PBLC on circadian eating behaviour in 24 growing sheep that were equally divided into three treatments, control (without PBLC), a lower dose (80 mg/d) or a higher dose (160 mg/d) of PBLC. Daily doses of PBLC were supplied with 600 g/d of concentrates fed in three equal portions at 07:00, 11:00 and 15:00 h for 4 weeks, whereas, meadow hay was fed ad libitum.

Results: The eating behaviour recorded by an automatic transponder-operated feeding system revealed that daily eating time and feeder visits increased with increasing doses of PBLC. The circadian distribution of eating time and feeder visits (with 1-h resolution) was influenced by the treatment. Eating time during concentrate-offering hours and between concentrate-offering hours increased or tended to increase linearly with greater concentrations of PBLC. Feeder visits did not change significantly during concentrate-offering hours, but were greater in the PBLC groups compared with the control between concentrate-feeding hours. Average length of the longest meals (5th percentile) decreased due to PBLC feeding. Daily feed intake was greater in the PBLC groups than the control.

Conclusions: Menthol-rich PBLC in the applied dose range stimulate circadian eating behaviour, which cannot only be attributed to their presence during concentrate feeding hours, but persist during post-concentrate feeding hours.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12917-019-2109-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805686PMC
October 2019

Dietary Bioactive Lipid Compounds Rich in Menthol Alter Interactions Among Members of Ruminal Microbiota in Sheep.

Front Microbiol 2019 4;10:2038. Epub 2019 Sep 4.

Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany.

This study aimed to investigate the effects of two practically relevant doses of menthol-rich plant bioactive lipid compounds (PBLC) on fermentation, microbial community composition, and their interactions in sheep rumen. Twenty-four growing Suffolk sheep were divided into three treatments and were fed hay plus 600 g/d of concentrate containing no PBLC (Control) or PBLC at low dose (80 mg/d; PBLC-L) or high dose (160 mg/d; PBLC-H). After 4 weeks on the diets, samples of ruminal digesta were collected and analyzed for short-chain fatty acid (SCFA), ammonia, and microbiota; microbiota being analyzed in the solid and the liquid digesta fractions separately. Ruminal SCFA and ammonia concentrations were not affected by the PBLC treatments. The microbiota in the solid fraction was more diverse than that in the liquid fraction, and the relative abundance of most taxa differed between these two fractions. In the solid fraction, phylogenetic diversity increased linearly with increased PBLC doses, whereas evenness (lowest in PBLC-L) and Simpson diversity index (greatest in PBLC-H) changed quadratically. In the liquid fraction, however, the PBLC supplementation did not affect any of the microbial diversity measurements. Among phyla, (highest in PBLC-L) and unclassified_bacteria (lowest in PBLC-L) were altered quadratically by PBLC. , (increased linearly), (increased in PBLC-L), (decreased in PBLC treatments), and (increased in PBLC treatments) were affected at the family level. Among genera, increased linearly in the solid fraction, increased linearly in the liquid fraction, whereas increased and increased linearly with increasing doses of PBLC in both fractions. The PBLC treatments also lowered methanogens within the classes and . Correlation network analysis revealed positive and negative correlations among many microbial taxa. Differential network analysis showed that PBLC supplementation changed the correlation between some microbial taxa and SCFA. The majority of the predicted functional features were different between the solid and the liquid digesta fractions, whereas the PBLC treatments altered few of the predicted functional gene categories. Overall, dietary PBLC treatments had little influence on the ruminal fermentation and microbiota but affected the associations among some microbial taxa and SCFA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.02038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738200PMC
September 2019

Effects of glucagon-like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep.

J Anim Physiol Anim Nutr (Berl) 2019 Nov 9;103(6):1727-1738. Epub 2019 Sep 9.

Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany.

Epidermal growth factor (EGF) and glucagon-like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP-1, GLP-2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP-1 or GLP-2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short-circuit current (I ) was affected by time and treatment and their interactions. Only 250 nM of either GLP-1 or GLP-2 decreased I in certain periods compared with 25 nM GLP-1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (J ) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in J between the first and last hour in epithelia incubated with 25 nM GLP-1 or GLP-2 and in epithelia incubated with EGF. After 7 hr incubation, claudin-7 mRNA expression was downregulated in all treatments. Claudin-1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin-4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP-2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP-1, GLP-2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin-7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jpn.13200DOI Listing
November 2019

Cholera toxin perturbs the paracellular barrier in the small intestinal epithelium of rats by affecting claudin-2 and tricellulin.

Pflugers Arch 2019 09 20;471(9):1183-1189. Epub 2019 Jun 20.

Institute of Veterinary Physiology, Freie Universität Berlin, 14163, Berlin, Germany.

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 μg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 μg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-019-02294-zDOI Listing
September 2019

The Inflammatory Response to Enterotoxigenic and Probiotic in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells.

Mediators Inflamm 2018 20;2018:9368295. Epub 2018 Dec 20.

Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor- (TGF-) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic (ETEC) and probiotic () NCIMB 10415. As potential mediators of IEC/DC interactions, TGF- and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF- was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/9368295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317115PMC
June 2019

Symposium review: The importance of the ruminal epithelial barrier for a healthy and productive cow.

J Dairy Sci 2019 Feb 20;102(2):1866-1882. Epub 2018 Dec 20.

Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8.

The stratified squamous ruminal epithelium is the main site for absorption of key nutrients (e.g., short-chain fatty acids; SCFA) and electrolytes (e.g., sodium and magnesium). The absorptive function has to be highly selective to prevent simultaneous entry of microbes and toxins from the rumen into the blood. As such, epithelial absorption is primarily transcellular, whereas the paracellular pathway appears rather tightly sealed. A network of tight junction (claudin-1, claudin-4, and occludin) and tight junction-associated proteins (e.g., zonula occludens) accomplishes the latter. When microbial fermentation activity is high such as with highly fermentable diets, rumen epithelial functions are often challenged by acidity, high osmolarity, toxins (e.g., endotoxin and histamine), and immune mediators (inflammatory mediators and cytokines) released during local and systemic inflammation. Epithelial damage by low pH in combination with high luminal SCFA concentrations is not immediately reversible and may initially aggravate upon return to physiological pH. In contrast, barrier opening upon hyperosmolarity is acutely transient. The initial insults set by luminal acidity and SCFA and the increasing concentrations of microbial-associated molecular patterns such as lipopolysaccharides are key factors that trigger inflammation not only in the rumen but also in the hindgut (cecum and colon), which reach out to the liver and other organs, causing systemic inflammation. Low feed intake during parturition, transportation, heat stress, or disease is the second most relevant challenge for the ruminal epithelial barrier. The barrier opening is usually only transient and quickly restored upon refeeding. Due to a rapid, dose-dependent, and prolonged decrease in absorption capacity for SCFA, however, any feed restriction increases the odds for postrestriction subacute ruminal acidosis. Inflammation due to acidosis can be alleviated by supplemental thiamine, yeasts, and plant bioactive (phytogenic) compounds. Butyrate is used in weaning calves to support ruminal barrier development; however, excess butyrate may promote hyperkeratosis, parakeratosis, and epithelial injury in the fully developed rumen of adult cows. Further research is needed to enhance the understanding of the various factors that counteract barrier impairment and help barrier restoration during acidogenic feeding, especially when concurring with unavoidable periods of feed restriction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3168/jds.2018-15243DOI Listing
February 2019

Normal Values of Thyroid Uptake of 99mTechnetium Pertechnetate SPECT in Mice with Respect to Age, Sex, and Circadian Rhythm.

Nuklearmedizin 2018 Sep 28;57(5):181-189. Epub 2018 Sep 28.

Aim: The aim of this study was to establish normal values for thyroid uptake of 99mtechnetium pertechnetate (TcO) as a function of age, sex and circadian rhythm in mice.

Methods: In 12 female (F) and 12 male (M) C57BL/6N mice, nine consecutive SPECT images of 10 min duration each were acquired as dynamic acquisitions beginning 5 min after intravenous injection of 80 MBq TcO. Each mouse was imaged in follow-up studies up to 24 months (A: 1 month; B: 3 months; C: 6 months; D: 12 months; E: 24 months). In order to assess for physiologic changes related to circadian rhythm, animals were imaged during light (sleeping phase, SP) as well as during night conditions (awake phase, AP). The percentage tracer uptake of the injected activity is expressed as median %ID.

Results: Female mice showed significantly higher uptake than males (F 1.6, M 1.1; p < 0.001). This effect was observed up to the age of 12 months: A (F 1.6, M 1.1; p < 0.001), B (F 1.7, M 1.1; p < 0.001), C (F 1.8, M 1.2; p < 0.001), D (F 1.6, M 1.2; p < 0.001), E (F 1.1, M 1.1; p = 0.79). Impact of age on uptake could be observed in females (p = 0.056) and was not present in males (p = 0.27). A significant effect of circadian rhythm could not be observed in females (SP 1.6, AP 1.7; p = 0.65) but in males (SP 1.2, AP 1.1; p = 0.02).

Conclusion: This study showed a significant influence of sex on thyroid TcO uptake in mice. Sex was also a significant factor affecting age-related changes in uptake in female mice but not in males. In contrast, circadian rhythm had no relevant impact on TcO uptake. Therefore, design of thyroid uptake studies in mice using TcO should consider animal sex, and in females, age as significant factors affecting uptake. ZIEL:: Ziel dieser Studie war die Erhebung von Normwerten für den Schilddrüsen Uptake von Technetium-Pertechnetat (TcO) in Abhängigkeit von Alter, Geschlecht und circadianem Rhythmus bei Mäusen.

Methoden: Bei 12 weiblichen (F) und 12 männlichen (M) C57BL/6N Mäusen wurden neun konsekutive SPECT Akquisitionen von jeweils 10 min Dauer 5 min nach intravenöser Injektion von 80 MBq TcO durchgeführt. Jede Maus wurde mehrmals bis zu 24 Monaten untersucht (A: 1 Mo; B: 3 Mo; C: 6 Mo; D: 12 Mo; E: 24 Mo). Um Veränderungen auf Grund des circadianen Rhythmus zu erfassen wurden die Tiere während der Hellphase (Schlafphase, SP) sowie während der Dunkelphase (Wachphase, AP) untersucht. Der prozentuale Tracer Uptake ist als Median %ID der injizierten Aktivität aufgeführt.

Ergebnisse: Weibchen zeigten einen signifikant höheren Uptake als Männchen (F 1,6, M 1,1; p < 0,001). Dieser Effekt wurde bis zu einem Alter von 12 Mo beobachtet: A (F 1,16, M 1,1; p < 0,001), B (F 1,7, M 1,1; p < 0,001), C (F 1,8, M 1,2; p < 0,001), D (F 1,6, M 1,2; p < 0,001), E (F 1,1, M 1,1; p = 0,79). Ein altersabhängiger Einfluss zeigte sich bei Weibchen (p = 0,056), jedoch nicht bei Männchen (p = 0,27). Ein signifikanter Effekt des circadianen Rhythmus konnte bei Männchen (SP 1,2, AP 1,1; p = 0,02) nicht aber bei Weibchen (SP 1,6, AP 1,7; p = 0,65) beobachtet werden.

Schlussfolgerung: Diese Studie zeigt einen signifikant geschlechtsabhängigen Einfluss auf den Schilddrüsen Uptake von TcO bei Mäusen. Im Gegensatz zu Männchen treten bei Weibchen altersabhängige Veränderungen im Uptake auf. Dagegen hat der circadiane Rhythmus keinen relevanten Einfluss. Folglich sollte bei dem Design von Schilddrüsenuntersuchungen mit TcO bei der Maus Geschlecht und bei weiblichen Tieren auch Alter als signifikante Faktoren berücksichtigt werden.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3413/Nukmed-0978-18-05DOI Listing
September 2018

Effects of a pathogenic ETEC strain and a probiotic Enterococcus faecium strain on the inflammasome response in porcine dendritic cells.

Vet Immunol Immunopathol 2018 Sep 15;203:78-87. Epub 2018 Aug 15.

Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany. Electronic address:

Dendritic cells (DC) are crucial for maintaining intestinal homeostasis and generating proper immune responses to bacteria occurring in the gut. Microbial stimuli can be recognized by intracellular receptors called inflammasomes, e.g., nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3). The aim of the present study was to unravel the inflammasome response of porcine monocyte-derived DC (MoDC). We investigated the capacity of probiotic Enterococcus faecium NCIMB 10415 (E. faecium) and enterotoxigenic Escherichia coli (ETEC) to elicit inflammasome activation. Since inflammasome activation normally requires a two-step process, MoDC were initially incubated with lipopolysaccharide (LPS) in order to prime cells. Primed and unprimed cells were then stimulated with the aforementioned bacterial strains. We also assessed whether preincubation with the probiotic prior to ETEC infection modified the immune response via the inflammasome pathway. Phenotypical analysis by flow cytometry showed that monocytes and MoDC expressed the surface markers CD14, CD16, and CD1 continuously, whereas swine leucocyte antigen (SLA) II was upregulated during differentiation. Following LPS priming, NLRP3, interleukin (IL)-1β and IL-18 mRNA expression, and IL-1β protein release increased. In unprimed cells, ETEC upregulated the expression of inflammasome components at later time points than in LPS-primed MoDC. Preincubation with the probiotic did not influence NLRP3 inflammasome activation in comparison with cells infected with ETEC alone. We conclude that ETEC, but not E. faecium, was able to stimulate inflammasome components in porcine MoDC. The present experimental conditions revealed no NLRP3 inflammasome-dependent protective effects of E. faecium during a pathogenic ETEC challenge.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetimm.2018.08.004DOI Listing
September 2018

Overexpression of the mitochondrial Mg channel MRS2 increases total cellular Mg concentration and influences sensitivity to apoptosis.

Metallomics 2018 07;10(7):917-928

Transfusion Medicine Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, 42123, Italy.

The mechanism of action of the mitochondrial Mg channel MRS2 and its involvement in cell viability remain unclear. Deletion of MRS2 has been reported to abolish Mg influx into mitochondria, to induce functional defects in mitochondrial organelles, and to result in cell death. We evaluated whether MRS2 expression had an impact on total Mg cellular content by inducing the overexpression of MRS2 in HEK-293 cells. We observed a remarkable increase of total intracellular Mg concentration in cells overexpressing MRS2 compared with control cells. In order to investigate whether and in what manner the detected Mg increment was involved in the MRS2 influence on cell viability, we treated MRS2-overexpressing cells with two known apoptotic inducers. We found that cells overexpressing the MRS2 channel became less responsive to these pharmacological insults. Our experimental evidence indicates that the MRS2 channel controls overall intracellular Mg levels, the alteration of which might have a role in the molecular signaling leading to apoptotic cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8mt00050fDOI Listing
July 2018

Effects of a combination of plant bioactive lipid compounds and biotin compared with monensin on body condition, energy metabolism and milk performance in transition dairy cows.

PLoS One 2018 27;13(3):e0193685. Epub 2018 Mar 27.

Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Oertzenweg, Germany.

The aim of this study was to test whether a combination of plant bioactive lipid compounds (also termed 'essential oils') and biotin (PBLC+B) could decrease the mobilization of body reserves and ketosis incidence in postpartum dairy cows. We compared non-supplemented control (CON) cows with cows receiving monensin (MON) as a controlled-release capsule at d -21, and with cows receiving PBLC+B from day (d) -21 before calving until calving (Phase 1) and further until d 37 after calving (Phase 2), followed by PBLC+B discontinuation from d 38 to d 58 (Phase 3). The PBLC+B cows had higher body weight and higher back fat thickness than CON cows and lesser body weight change than MON and CON cows in Phase 3. Body condition score was not different among groups. Milk protein concentration tended to be higher on the first herd test day in PBLC+B vs. CON cows. Milk fat concentration tended to be highest in PBLC+B cows throughout Phases 2 and 3, with significantly higher values in PBLC+B vs. MON cows on the second herd test day. Yields of energy-corrected milk were higher in PBLC+B vs. CON and MON cows in Phase 2 and higher in PBLC+B and MON cows vs. CON cows in Phase 3. The incidence of subclinical ketosis was 83%, 61% and 50% in CON, PBLC+B and MON cows, respectively, with lower mean β-hydroxybutyrate values in MON than in PBLC+B cows in Phase 1 prepartum. The serum triglyceride concentration was higher in PBLC+B vs. CON cows on d 37. No differences were observed in serum glucose, urea, non-esterified fatty acids, cholesterol and bilirubin concentrations. Aspartate transaminase and γ-glutamyltranspeptidase but not glutamate dehydrogenase activities tended to be highest in MON and lowest in PBLC+B in Phase 2. We conclude that PBLC+B prevent body weight loss after parturition and are associated with similar ketosis incidence and partly higher yields of energy-corrected milk compared to MON supplementation of dairy cows.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870966PMC
June 2018

Overexpression of Na/Mg exchanger SLC41A1 attenuates pro-survival signaling.

Oncotarget 2018 Jan 22;9(4):5084-5104. Epub 2017 Dec 22.

Institute of Veterinary-Physiology, Free University of Berlin, Berlin, Germany.

The Na/Mg exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg efflux system, and its overexpression decreases [Mg]. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg] impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg] in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr and/or Ser and of Erk1/2 on Thr/Tyr in the presence of 0 or 1 mM (physiological) Mg in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg] in the presence of [Mg] = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB - Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg] (and consequently also [Mg]) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.23598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797035PMC
January 2018

Effect of individual SCFA on the epithelial barrier of sheep rumen under physiological and acidotic luminal pH conditions.

J Anim Sci 2018 Feb;96(1):126-142

Institute of Veterinary Physiology, Freie Universität Berlin, Germany.

The objective of this study was to investigate whether individual short-chain fatty acids (SCFA) have a different potential to either regulate the formation of the ruminal epithelial barrier (REB) at physiological pH or to damage the REB at acidotic ruminal pH. Ruminal epithelia of sheep were incubated in Ussing chambers on their mucosal side in buffered solutions (pH 6.1 or 5.1) containing no SCFA (control), 30 mM of either acetate, propionate or butyrate, or 100 mM acetate. Epithelial conductance (Gt), short-circuit current (Isc), and fluorescein flux rates were measured over 7 h. Thereafter, mRNA and protein abundance, as well as localization of the tight junction proteins claudin (Cldn)-1, -4, -7, and occludin were analyzed. At pH 6.1, butyrate increased Gt and decreased Isc, with additional decreases in claudin-7 mRNA and protein abundance (each P < 0.05) and disappearance of Cldn-7 immunosignals from the stratum corneum. By contrast, the mRNA abundance of Cldn-1 and/or Cldn-4 were upregulated by 30 mM propionate, 30 mM butyrate, or 100 mM acetate (P < 0.05), however, without coordinated changes in protein abundance. At luminal pH 5.1, neither Gt, Isc, nor TJ protein abundance was altered in the absence of SCFA; only fluorescein flux rates were slightly increased (P < 0.05) and fluorescein signals were no longer restricted to the stratum corneum. The presence of acetate, propionate, or butyrate at pH 5.1 increased fluorescein flux rates and Gt, and decreased Isc (each P < 0.05). Protein abundance of Cldn-1 was decreased in all SCFA treatments but 30 mM butyrate; abundance of Cldn -4 and -7 was decreased in all SCFA treatments but 30 mM acetate; and abundance of occludin was decreased in all SCFA treatments but 30 mM propionate (each P < 0.05). Immunofluorescence staining of SCFA-treated tissues at pH 5.1 showed disappearance of Cldn-7, discontinuous pattern for Cldn-4 and blurring of occludin and Cldn-1 signals in tight junction complexes. The fluorescein dye appeared to freely diffuse into deeper cell layers. The strongest increase in Gt and consistent decreases in the abundance and immunosignals of tight junction proteins were observed with 100 mM acetate at pH 5.1. We conclude that SCFA may contribute differently to the REB formation at luminal pH 6.1 with possible detrimental effects of butyrate at 30 mM concentration. At luminal pH 5.1, all SCFA elicited REB damage with concentration appearing more critical than SCFA species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jas/skx017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140886PMC
February 2018

Characterization of Inflammasome Components in Pig Intestine and Analysis of the Influence of Probiotic Enterococcus Faecium during an Escherichia Coli Challenge.

Immunol Invest 2017 Oct;46(7):742-757

a Department of Veterinary Medicine, Institute of Veterinary Physiology , Freie Universitaet Berlin , Berlin , Germany.

The aim of the present study was to investigate systematically the expression of inflammasome components in pig intestine and to analyze the influence of age and long-term supplementation with the probiotic Enterococcus faecium NCIMB 10415 (E. faecium). In order to examine probiotic effects on the inflammasomes during a challenge with pathogens, enterotoxigenic Escherichia coli (ETEC) and E. faecium were directly added to pig jejunum in Ussing chambers. The mRNA expression of inflammasome components generally decreased in an oral-aboral direction in intestinal tissues. In 29-day-old piglets, the expression levels of NLRP3 were significantly higher and ASC (apoptotic speck-like protein containing a caspase recruitment domain) expression were lower compared with those in the ileum of 70-day-old pigs (p ≤ 0.05). Long-term supplementation with E. faecium significantly increased ASC expression levels in the jejunum and ileum of 29-day-old piglets compared to control animals (p ≤ 0.05). Ex vivo addition of ETEC or E. faecium did not affect mRNA expression of inflammasome components significantly, whereas IL-1β protein release was significantly elevated in ETEC-incubated jejunum (p ≤ 0.05), providing evidence for the functional activation of the inflammasome, which was prevented by pre-incubation with E. faecium. We conclude that pre-incubation with E. faecium has a protective effect during ETEC challenge; this effect is probably not located at the inflammasome transcription level. The results of this study of the expression and regulation of inflammasome components in pigs are similar to those obtained in humans, reinforcing the use of pigs as a suitable model for translational inflammasome research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/08820139.2017.1360341DOI Listing
October 2017

Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic and Probiotic .

Mediators Inflamm 2017 14;2017:2748192. Epub 2017 May 14.

Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

The aim of the present study was to elucidate the effects of the probiotic feed additive NCIMB 10415 () on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic (ETEC). Cells were incubated with , ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with abolished the ETEC-induced decrease in transepithelial resistance () ( ≤ 0.05). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation ( ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-, IL-1, and IL-6 which could be prevented by coincubation for TNF- mRNA expression and IL-6 protein ( ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells ( ≤ 0.05). These findings indicate a protective effect of the probiotic on inflammatory responses during infection with ETEC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2017/2748192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457759PMC
March 2018

Key role of short-chain fatty acids in epithelial barrier failure during ruminal acidosis.

J Dairy Sci 2017 Aug 24;100(8):6662-6675. Epub 2017 May 24.

Institute of Veterinary Physiology, Freie Universität Berlin, D-14163 Berlwin, Germany. Electronic address:

Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (G), short-circuit current (I), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either G or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in G and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3168/jds.2016-12262DOI Listing
August 2017

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes.

Cells Tissues Organs 2017 12;204(1):13-24. Epub 2017 May 12.

Institute of Veterinary Physiology, Free University of Berlin, Berlin, Germany.

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000472708DOI Listing
March 2018

The regulon affects virulence gene expression and adhesion of porcine enteropathogenic .

Vet Anim Sci 2017 Jun 27;3:10-17. Epub 2017 Apr 27.

Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.

The ability of enteropathogenic (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated expression and downregulated mRNA synthesis of the bacterial adhesin . EPEC strains transformed with the gene produced 2.1-3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vas.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386710PMC
June 2017

Finishing pigs that are divergent in feed efficiency show small differences in intestinal functionality and structure.

PLoS One 2017 5;12(4):e0174917. Epub 2017 Apr 5.

Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.

Controversial information is available regarding the feed efficiency-related variation in intestinal size, structure and functionality in pigs. The present objective was therefore to investigate the differences in visceral organ size, intestinal morphology, mucosal enzyme activity, intestinal integrity and related gene expression in low and high RFI pigs which were reared at three different geographical locations (Austria, AT; Northern Ireland, NI; Republic of Ireland, ROI) using similar protocols. Pigs (n = 369) were ranked for their RFI between days 42 and 91 postweaning and low and high RFI pigs (n = 16 from AT, n = 24 from NI, and n = 60 from ROI) were selected. Pigs were sacrificed and sampled on ~day 110 of life. In general, RFI-related variation in intestinal size, structure and function was small. Some energy saving mechanisms and enhanced digestive and absorptive capacity were indicated in low versus high RFI pigs by shorter crypts, higher duodenal lactase and maltase activity and greater mucosal permeability (P < 0.05), but differences were mainly seen in pigs from AT and to a lesser degree in pigs from ROI. Additionally, low RFI pigs from AT had more goblet cells in duodenum but fewer in jejunum compared to high RFI pigs (P < 0.05). Together with the lower expression of TLR4 and TNFA in low versus high RFI pigs from AT and ROI (P < 0.05), these results might indicate differences in the innate immune response between low and high RFI pigs. Results demonstrated that the variation in the size of visceral organs and intestinal structure and functionality was greater between geographic location (local environmental factors) than between RFI ranks of pigs. In conclusion, present results support previous findings that the intestinal size, structure and functionality do not significantly contribute to variation in RFI of pigs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174917PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381887PMC
September 2017
-->