Response to Incremental Replacement of Palm Oil with Fish Oil in Starter Diet on Growth Performance, Plasma Metabolites, Ruminal Fermentation, and Behavior of Dairy Calves.

The objective of this study was to evaluate the effects of the incremental levels of n-3 fatty acids (FA) in starter feed (SF) on growth and metabolic performance of milk-fed calves. From day 3 of age, 30 female calves (39.4 ± 3.1 kg of body weight) were randomly assigned to one of three dietary treatments: (1) SF supplemented with 3.3% palm fatty acids (PO), (2) SF supplemented with 1.7% of PO and 1.9% fish oil (PFO), or (3) SF supplemented with 3.9% fish oil (FO). Chopped straw (7.5% of DM) was included in the SF of all treatments as total mixed ration (TMR). Diets had similar energy and protein contents. Total n-3 FA (% of total FA) and n-6/n-3 of PO, PFO, and FO were 1.90, 6.80, and 11.8 and 15.5, 4.50, and 2.70, respectively. The BW was greater for calves receiving FO (60.2 ± 0.3 kg) compared with PFO (58.7 ± 0.3 kg; p = 0.007) and tended to be greater for calves receiving FO vs. PO (59.0 ± 0.3 kg; p = 0.050). Because there was no interaction effect between diet × week of experiment, the greater BW of FO could not be attributed to the dietary treatment. Accordingly, average daily gain, total dry matter intake (DMI), starter DMI, and gain to intake ratio (G:FI) did not differ among dietary treatments during the entire period of the study (p > 0.05). Dietary treatments did not impact body size parameters such as body length, body girth, withers height, heart girth, hip height, and width (p > 0.05). Neither ruminal fermentation parameters nor blood variables were influenced by supplementing the types of oil at different time points. Calves' behavioral parameters, such as standing, lying, eating, and ruminating, were not influenced by different dietary treatments (p > 0.05). The number of days with abnormal fecal score was not different among dietary groups (p > 0.05). Overall, our findings suggest that changing the n-6/n-3 ratio in starter feed by incremental replacement of palm fatty acid with fish oil at a moderate supplemental level of ~3% of DM may not affect the growth and metabolic performance of young calves under non-challenged conditions.

Acid tolerance of lactate-utilizing bacteria of the order Bacteroidales contributes to prevention of ruminal acidosis in goats adapted to a high-concentrate diet.

The rapid accumulation of organic acids, particularly lactate, has been suggested as the main cause of ruminal acidosis (RA) for ruminants fed high-concentrate diets. Previous research has shown that a gradual shift from low-to high-concentrate diets within 4 to 5 weeks effectively reduces the risk for RA. However, the mechanisms remain unknown. In this study, 20 goats were randomly allocated into four groups (n = 5) and fed with a diet containing a weekly increasing concentrate portion of 20%, 40%, 60%, and 80% over 28 d. At d 7, 14, 21, and 28, one group (named C20, C40, C60, and C80 according to the last concentrate level that they received) was killed and the ruminal microbiome was collected. Ruminal acidosis was not detected in any of the goats during the experiment. Nonetheless, ruminal pH dropped sharply from 6.2 to 5.7 (P < 0.05) when dietary concentrate increased from 40% to 60%. A combined metagenome and metatranscriptome sequencing approach identified that this was linked to a sharp decrease in the abundance and expression of genes encoding nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (nLDH), catalyzing the enzymatic conversion of pyruvate to lactate (P < 0.01), whereas the expression of two genes encoding NAD-independent lactate dehydrogenase (iLDH), catalyzing lactate oxidation to pyruvate, showed no significant concomitant change. Abundance and expression alterations for nLDH- and iLDH-encoding genes were attributable to bacteria from Clostridiales and Bacteroidales, respectively. By analyzing the gene profiles of 9 metagenome bins (MAG) with nLDH-encoding genes and 5 MAG with iLDH-encoding genes, we identified primary and secondary active transporters as being the major types of sugar transporter for lactate-producing bacteria (LPB) and lactate-utilizing bacteria (LUB), respectively. Furthermore, more adenosine triphosphate was required for the phosphorylation of sugars to initiate their catabolic pathways in LPB compared to LUB. Thus, the low dependence of sugar transport systems and catabolic pathways on primary energy sources supports the acid tolerance of LUB from Bacteroidales. It favors ruminal lactate utilization during the adaptation of goats to a high-concentrate diet. This finding has valuable implications for the development of measures to prevent RA.

Effects of Oil Supplements on Growth Performance, Eating Behavior, Ruminal Fermentation, and Ruminal Morphology in Lambs during Transition from a Low- to a High-Grain Diet.

The objectives of this study were to investigate the effect of a maximum recommended oil supplementation on growth performance, eating behavior, ruminal fermentation, and ruminal morphological characteristics in growing lambs during transition from a low- to a high-grain diet. A total of 21 Afshari male lambs with an initial body weight (BW) of 41.4 ± 9.1 kg (mean ± SD) and at 5−6 months of age were randomly assigned to one of three dietary treatments (n = 7 per group), including (1) a grain-based diet with no fat supplement (CON), (2) CON plus 80 g/d of prilled palm oil (PALM), and (3) CON plus 80 g/d soybean oil (SOY); oils were equivalent to 50 g/kg of dry matter based on initial dry matter intake (DMI). All lambs were adapted to the high-grain diet for 21 d. In the adaptation period, lambs were gradually transferred to a dietary forage-to-concentrate ratio of 20:80 by replacing 100 g/kg of the preceding diet every 3 d. Thereafter, lambs were fed experimental diets for another 22 days. Fat-supplemented lambs had greater DMI, body weight (BW), and average daily gain (ADG), with a lower feed to gain ratio (p < 0.05), compared to CON lambs. The highest differences of DMI between fat-supplemented and CON-lambs were observed in week 3 of the adaptation period (p = 0.010). PALM- or SOY-supplementation lowered DM and NDF digestibility compared with CON (p < 0.05), and SOY caused the lowest organic matter (OM) digestibility compared with CON and PALM lambs (62.0 vs. 67.6 and 66.9; p < 0.05). Ruminal pH was higher for PALM and SOY compared with CON (p = 0.018). Lambs in SOY tended to have the highest ammonia-N concentrations (p = 0.075), together with a trend for higher concentrations of propionic acid, at the expense of acetic acid in ruminal fluid, on the last day of the adaptation period (diet × time, p = 0.079). Fat-supplemented lambs had lower isovaleric and valeric acid concentrations compared with CON on d 40 (diet × time, p < 0.05). PALM and SOY-fed lambs had a longer eating time (min/d and min/kg of DMI), chewing activity (min/d), meal frequency (n), and duration of eating the first and second meals after morning feeding (p < 0.05), and the largest meal size (p < 0.001). Fat supplemented lambs had greater ruminal papillary length (p < 0.05) and width (p < 0.01), and thicker submucosal, epithelial, and muscle layers, compared with the CON (p < 0.01). Blood metabolites were not influenced by dietary treatments (p > 0.05). The results from this study suggest that fat supplementation to high-grain diets may improve the development of ruminal epithelia and modify ruminal fermentation via optimized eating behavior or the direct effect of oils on the ruminal environment, resulting in better growth performance in growing lambs.

Growth performance, nutrient digestibility, and ruminal fermentation of dairy calves fed starter diets with alfalfa hay versus corn silage as forage and soybean oil versus palm fatty acids as fat source.

The present study was intended to evaluate the effect of forage source (alfalfa hay; ALF vs. corn silage; CS) along with a supplemental fat source (soybean oil; SO vs. rumen-inert palm fatty acids; PF) on growth performance, nutrient digestibility, and ruminal fermentation in dairy calves. Forty-eight new-born Holstein female calves (3 d old) were assigned to one of 4 treatments: (1) alfalfa hay with soybean oil (ALF-SO); (2) alfalfa hay with palm fatty acids (ALF-PF); (3) corn silage with soybean oil (CS-SO); (4) corn silage with palm fatty acids (CS-PF). Starter diets had equal amounts of forage (100 g/kg dry matter; DM) and fat source (30 g/kg DM). Calves were fed a constant amount of milk (d 1 to 63) and had ad libitum access to water and starters (d 1 to 83). The lowest and greatest starter intakes during the preweaning period occurred in ALF-SO and CS-PF, respectively. This coincided with forage × fat source interaction for average daily gain (ADG) during preweaning. The forage source affected total DM intake and ADG over the entire period, body weight (BW) at weaning, and final BW with greater values in calves that received CS compared with ALF. The concentrations of total short-chain fatty acids and butyrate were increased, whereas concentration of acetate and acetate:propionate ratio were decreased in the rumen of calves fed CS compared with ALF. Feeding CS increased urinary excretion of allantoin and, as a trend, total purine derivatives (PD) and estimated microbial protein synthesis in comparison with ALF. The fat source affected starter intake, ADG, and BW postweaning with the highest values in PF. The digestibility of neutral detergent fiber, crude protein and, as a trend, organic matter were higher in calves fed PF compared with SO. Calves fed PF had lower ruminal ammonia-N concentration and urinary N excretion and greater urinary excretion of allantoin and total PD. Calves receiving SO had a lower ruminal protozoa population. In conclusion, supplementing starter diets with CS and PF is superior to ALF and SO. Interaction of the positive effects of CS and PF on performance underlines that concurrent supplementation of CS with PF is especially recommendable in young calves before weaning.

Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources.

BACKGROUND: Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated. RESULTS: Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs. CONCLUSION: In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Effects of dietary oil sources (sunflower and fish) on fermentation characteristics, epithelial gene expression and microbial community in the rumen of lambs fed a high-concentrate diet.

The feeding of high-concentrate diets commonly results in lowered pH and ruminal dysbiosis which cause shifts in uptake dynamics of short-chain fatty acids (SCFA) and altered epithelial function. Therefore, the current study evaluated the effect of dietary polyunsaturated fatty acids (PUFA) on ruminal fermentation products, gene expression in the ruminal epithelium and the associated changes in ruminal microorganisms in lambs fed a high-concentrate diet. Twenty-six Afshari lambs adapted to a high-concentrate diet during a completely randomised design were fed with a basal diet supplemented with 100 g oil supplement (OS; 60 g sunflower oil and 40 g fish oil) for 10 (OS10), 20 (OS20) and 30 (OS30) d, respectively (n = 6). Lambs with no oil supplementation (OS0, n = 8) were considered as control and slaughtered at d 0 of the experiment, and the remaining lambs were slaughtered at 10, 20 and 30 d on feed. After slaughter, ruminal digesta was collected for evaluating fermentation and microbial community. Ruminal papillae were taken for assessment of epithelial gene expression. Compared with OS0 lambs, supplemental PUFA in OS30 lambs tended to decrease total SCFA concentration with decreased acetic and increased propionic acid concentrations. Acetate:propionate ratios were decreased and ruminal pH was increased in OS20 and OS30 lambs compared to OS0. All groups with included OS had decreased concentrations of iso-valeric and valeric acids compared to OS0. Relative mRNA abundance of monocarboxylate transporter isoforms 1 and 4, insulin-like growth factor binding protein 3, sterol regulatory element-binding proteins 1 and 2 decreased with increasing OS duration. The relative abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 1 mRNA transcript was higher for OS10 and OS20 lambs relative to OS0 lambs. OS20 and OS30 showed a decrease of lipopolysaccharide binding protein mRNA expression compared with OS0. Feeding supplemental PUFA decreased Ciliate protozoa and increased Butyrivibrio fibrisolvens in OS20 and OS30 lambs, whereas Megasphaera elsdenii was increased in OS30 lambs. In conclusion, combined supplementation of sunflower and fish oil to a high-concentrate diet affects the ruminal microbial community with prominent decreases in ruminal ciliate protozoa and increases in B. fibrisolvens and M. elsdenii. These results lead to a more stabilised ruminal pH and a fermentation shift towards more propionate generation. Consideration of nutrients digestion will help to fully understand the benefits of feeding PUFA with a high-concentrate diet.

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

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.

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction.

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 NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). 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 FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 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 D3 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 FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation.Abbreviations: 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; PPARγ: 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.

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

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.

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

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.

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

The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC-induced decrease in transepithelial resistance (Rt) (p ≤ 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 (p ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein (p ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells (p ≤ 0.05). These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC.

The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions.

Epithelial cell layers are interconnected by a meshwork of tight junction (TJ) protein strands, which are localized within apicolateral membranes. The proteins that form TJs are regarded to provide a static barrier, determining epithelial properties. However, recent findings in the field of barriology suggest that TJs contribute to more physiological aspects than indicated by the sum of the qualities of the single TJ proteins. Generally, TJs exhibit four major functions: (i) a "gate function," defining transepithelial permeability (i.e., barrier) properties, (ii) a "fence function" determining epithelial cell polarity, (iii) a "signaling function," affecting regulatory pathways, and (iv) a "stabilizing function," maintaining the integrity of the epithelium. This review presents a critical view on how the efficacy of physiological processes in epithelia and thus organ function might be improved by changes in the expression of claudins, the latter representing the largest and most variable family of TJ proteins. Major focus is set on (i) the coordinated regulation of transport and barrier in the intestine, (ii) the role of TJs in defining the route for antigen uptake and presentation in intestinal Peyer's patches, and (iii) the TJ function in mammary glands in response to milk accumulation, which represent impressive examples to highlight the amplification of epithelial functions by TJ proteins. © 2017 IUBMB Life, 69(5):290-296, 2017.

Gastrointestinal methionine shuttle: Priority handling of precious goods.

Methionine (Met) is a neutral, sulfur-containing, essential amino acid with biological functions in the initiation and prolongation step of protein synthesis, transmethylation reactions, the synthesis of cysteine and cystine, and as a component of antioxidant systems. Its key importance is reflected by the fact that it is usually absorbed from the diet with highest efficiency among all proteinogenic amino acids but may yet not optimally support metabolism and health. As such, crystalline Met supplements are partly used in man and heavily used in production of animal species (poultry, fish, shrimps, pigs and cattle) to provide improved health and performance. The main intention of this review is to analyze the current knowledge on transport proteins with proven or hypothetical relevance for Met absorption in the gastrointestinal tract, especially the small intestine. These transporters include Na+ -dependent B0 AT1 and ATB0,+ and the Na+ -independent exchanger b0,+ /rBAT in the apical membrane, which may be supported by the Na+ -dependent systems ASCT2 and IMINO. The basolateral exit of Met appears to be largely limited to a single uniporter protein, LAT4. Insufficient or overtaxed efflux via LAT4 may lead to significant intracellular accumulation and metabolism of Met in the absorptive state. The latter can release large amounts of homocysteine into the blood, which favors atherosclerosis and other cardiovascular, as well as neurological, diseases. When LAT4 is defective, basolateral Met exit may be compensated to a certain degree by the Met exchange proteins 4F2hc/LAT2 or 4F2hc/LAT1; while carriers 4F2hc/y+ LAT1, 4F2hc/y+ LAT2, SNAT1 and SNAT2, may serve primarily for basolateral Met import. Expression of SNAT2 is increased when amino acid supply from the lumen ceases, suggesting a key role for Met supply of enterocytes in interdigestive periods. Enterocytes themselves have a huge requirement for Met to synthesize mucins and glutathione. © 2016 IUBMB Life, 68(12):924-934, 2016.

Solute carrier 41A3 encodes for a mitochondrial Mg(2+) efflux system.

The important role of magnesium (Mg(2+)) in normal cellular physiology requires flexible, yet tightly regulated, intracellular Mg(2+) homeostasis (IMH). However, only little is known about Mg(2+) transporters of subcellular compartments such as mitochondria, despite their obvious importance for the deposition and reposition of intracellular Mg(2+) pools. In particular, knowledge about mechanisms responsible for extrusion of Mg(2+) from mitochondria is lacking. Based on circumstantial evidence, two possible mechanisms of Mg(2+) release from mitochondria were predicted: (1) Mg(2+) efflux coupled to ATP translocation via the ATP-Mg/Pi carrier, and (2) Mg(2+) efflux via a H(+)/Mg(2+) exchanger. Regardless, the identity of the H(+)-coupled Mg(2+) efflux system is unknown. We demonstrate here that member A3 of solute carrier (SLC) family 41 is a mitochondrial Mg(2+) efflux system. Mitochondria of HEK293 cells overexpressing SLC41A3 exhibit a 60% increase in the extrusion of Mg(2+) compared with control cells. This efflux mechanism is Na(+)-dependent and temperature sensitive. Our data identify SLC41A3 as the first mammalian mitochondrial Mg(2+) efflux system, which greatly enhances our understanding of intracellular Mg(2+) homeostasis.

Human CNNM2 is not a Mg(2+) transporter per se.

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg(2+), and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg(2+) in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg(2+) depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg(2+) transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg(2+) homeostatic factor without being a Mg(2+) transporter per se.

Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na(+)/H(+) exchange (NHE) of the ruminal epithelium plays a key role in Na(+) absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na(+) transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na(+) transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na(+) by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na(+) transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na(+) transport in omasal epithelium; and (3) SCFA and acidic pH acutely activate Na(+) transport but suppress the expression of NHE2 and NHE3 in the longer term. By contrast, the expression of NHE1 is increased by SCFA and acidic pH, indicating a prominent role for NHE1 in the regulation of intracellular pH of omasal epithelium. Our results suggest a regulatable Na(+) absorption in ruminal and omasal epithelium. It is of benefit for intracellular pH homeostasis and highly relevant to dairy cows fed on high-concentrate diets.

Short-chain fatty acids and acidic pH upregulate UT-B, GPR41, and GPR4 in rumen epithelial cells of goats.

Currently, the mechanism(s) responsible for the regulation of urea transporter B (UT-B) expression levels in the epithelium of the rumen remain unclear. We hypothesized that rumen fermentation products affect ruminal UT-B expression. Therefore, the effects of short-chain fatty acids (SCFA), pH, ammonia, and urea on mRNA and protein levels of UT-B were assayed in primary rumen epithelial cell cultures and in rumen epithelium obtained from intact goats. In vitro, SCFA and acidic pH were found to synergetically stimulate both mRNA and protein expression of UT-B, whereas NH4Cl decreased mRNA and protein levels of UT-B at pH 6.8. Treatment with urea increased both levels at pH 7.4. When goats received a diet rich in nitrogen (N) and nonfiber carbohydrates (NFC), their rumen epithelium had higher levels of UT-B, and the rumen contained higher concentrations of SCFA and NH3-N with a lower pH. An increase in plasma urea-N concentration was also observed compared with the plasma of the goats that received a diet low in N and NFC. In a second feeding trial, goats that received a NFC-rich, but isonitrogenous, diet had higher mRNA and protein levels of UT-B, and higher levels of G protein-coupled receptor (GPR) 41 and GPR4, in their rumen epithelium. The ruminal concentrations of SCFA and NH3-N also increased, while a lower pH was detected. In contrast, the serum urea-N concentrations remained unchanged. These data indicate that ruminal SCFA and pH are key factors, via GPR4 and GPR41, in the dietary regulation of UT-B expression, and they have priority over changes in plasma urea.

Diets high in heat-treated soybean meal reduce the histamine-induced epithelial response in the colon of weaned piglets and increase epithelial catabolism of histamine.

We examined the influence of dietary fermentable protein (fCP) and fermentable carbohydrates (fCHO) on the colonic epithelial response to histamine in pigs. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO, low fCP/high fCHO, high fCP/low fCHO and high fCP/high fCHO. After 21-23 days, the pigs were killed and tissue from the proximal colon was stimulated with carbachol, histamine, PGE2 or sodium hydrogen sulphide in Ussing chambers. Changes in short-circuit current and tissue conductance were measured. Diamine oxidase, histamine N-methyltransferase, stem cell growth factor receptor, Fc-epsilon receptor I and cystic fibrosis transmembrane conductance regulator gene expression was determined. Activities of diamine oxidase and histamine N-methyltransferase and numbers of colonic mast cells were measured. The change in the short-circuit current in response to histamine was lower (P = 0.002) and tended to be lower for PGE2 (P = 0.053) in high fCP groups compared to low fCP groups, irrespective of fCHO. Additionally, the change in tissue conductance after the application of histamine was lower (P = 0.005) in the high fCP groups. The expression of histamine N-methyltransferase mRNA (P = 0.033) and the activities of diamine oxidase (P = 0.001) and histamine N-methyltransferase (P = 0.006) were higher with high fCP in comparison with low fCP. The expression of mast cell markers, stem cell growth factor receptor (P = 0.005) and Fc-epsilon receptor I (P = 0.049) was higher with high fCP diets compared to diets low in fCP, whereas the mast cell count did not differ between groups. The expression of the cystic fibrosis transmembrane conductance regulator was reduced (P = 0.001) with high fCP diets compared to low fCP diets. The lower epithelial response to histamine and PGE2 and elevated epithelial histamine inactivation suggests an adaptation to high fCP diets.

Increased papillae growth and enhanced short-chain fatty acid absorption in the rumen of goats are associated with transient increases in cyclin D1 expression after ruminal butyrate infusion.

We tested the hypothesis that the proliferative effects of intraruminal butyrate infusions on the ruminal epithelium are linked to upregulation in cyclin D1 (CCND1), the cyclin-dependent kinase 4 (CDK4), and their possible association with enhanced absorption of short-chain fatty acids (SCFA). Goats (n=23) in 2 experiments (Exp.) were fed 200 g/d concentrate and hay ad libitum. In Exp. 1, goats received an intraruminal infusion of sodium butyrate at 0.3 (group B, n=8) or 0 (group C, n=7) g/kg of body weight (BW) per day before morning feeding for 28 d and were slaughtered 8 h after the butyrate infusion. In Exp. 2, goats (n=8) received butyrate infusion and feeding as in Exp. 1. On d 28, epithelial samples were biopsied from the antrium ruminis at 0, 3, and 7 h after the last butyrate infusion. In Exp. 1, the ruminal molar proportional concentration of butyrate increased in group B by about 110% after butyrate infusion and remained elevated for 1.5 h; thereafter, it gradually returned to the baseline (preinfusion) level. In group C, the molar proportional concentration of butyrate was unchanged over the time points. The length and width of papillae increased in B compared with C; this was associated with increased numbers of cells and cell layers in the epithelial strata and an increase in the surface area of 82%. The mRNA expression of CCND1 increased transiently at 3 h but returned to the preinfusion level at 7 h following butyrate infusion in Exp. 2. However, it did not differ between B and C in Exp. 1, in which the ruminal epithelium was sampled at 8 h after butyrate infusion. The mRNA expression of the monocarboxylate transporter MCT4, but not MCT1, was stably upregulated in B compared with C. The estimated absorption rate of total SCFA (%/h) increased in B compared with C. We conclude that transient increases in cyclin D1 transcription contribute to butyrate-induced papillae growth and subsequently to the increased absorption of SCFA in the ruminal epithelium of goats.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep.

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.