Modulation of the porcine intestinal microbiota in the course of Ascaris suum infection.

BACKGROUND: The porcine roundworm Ascaris suum impairs feed conversion and weight gain, but its effects on intestinal microbiota remain largely unexplored. METHODS: Modulation of the intestinal microbiota was assessed in pigs that were infected once with 10,000 A. suum eggs and pigs that received a trickle infection (1000 eggs/day over 10 days), compared with a non-infected control group. Six pigs each were sacrificed per group at days 21, 35 and 49 post-infection (p.i.). Faecal samples taken weekly until slaughter and ingesta samples from different intestinal compartments were subjected to next-generation sequencing of the bacterial 16S rRNA gene. RESULTS: The results revealed marked differences between the single- and the trickle-infected group. Single infection caused a remarkable but transient decrease in microbial diversity in the caecum, which was not observed in the trickle-infected group. However, an increase in short-chain fatty acid-producing genera in the caecum on day 21 p.i., which shifted to a decrease on day 35 p.i., was common to both groups, possibly related to changes in excretory-secretory products following the parasite's final moult. Faecal microbial interaction networks were more similar between the single-infected and control group than the trickle-infected group. In addition, a lower degree of similarity over time indicated that A. suum trickle infection prevented microbiota stabilization. CONCLUSIONS: These different patterns may have important implications regarding the comparability of experimental infections with natural scenarios characterized by continuous exposure, and should be confirmed by further studies.

Ascaris suum Nutrient Uptake and Metabolic Release, and Modulation of Host Intestinal Nutrient Transport by Excretory-Secretory and Cuticle Antigens In Vitro.

Ascaris suum, the most important pig parasite, also infects humans as a zoonotic pathogen. Malabsorption upon infection probably results from impaired nutrient transport, presumably mediated by the parasite's excretory-secretory (ES) or cuticle somatic (CSO) antigens. The present study investigated the electrogenic transport (ΔIsc) of glucose, alanine and the dipeptide glycyl-l-glutamine (glygln), as well as glucose net flux rates in pig jejunal tissue after in vitro exposure to adult A. suum total ES or CSO antigens in Ussing chambers. ΔIsc of glucose, alanine and glucose net flux rate were significantly decreased after one hour of exposure to total ES antigen. In contrast, CSO antigens increased the transport of glygln. Additionally, nutrient uptake and ES antigen pattern were compared in culture medium from untreated adult worms and those with sealed mouth and anal openings. Untreated worms completely absorbed glucose, while cuticular absorption in sealed worms led to 90% reduction. Amino acid absorption was 30% less effective in sealed worms, and ammonia excretion decreased by 20%. Overall, the results show that A. suum total ES antigen rapidly impairs nutrient transport in vitro. Future studies confirming the results in vivo, narrowing down the ES components responsible and investigating underlying molecular mechanisms are needed.

Changes in porcine nutrient transport physiology in response to Ascaris suum infection.

BACKGROUND: The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to decreased nutrient absorption. This study aims at characterizing the effects of A. suum infection on intestinal nutrient transport processes and potential molecular mechanisms. METHODS: Three groups of six piglets each were infected orally (10,000 embryonated A. suum eggs) in a single dose ("single infection"). Another three groups were infected orally (1000 embryonated eggs) for 10 consecutive days ("trickle infection"). Animals were necropsied 21, 35 and 49 days post-infection (dpi). Three groups served as respective controls. The Ussing chamber technique was applied for the functional characterization of small intestinal tissues [short-circuit currents (Isc) as induced by glucose, alanine and peptides; 3H-glucose net flux rates; tissue conductance (Gt)]. Transcription and expression levels of relevant cytokines and nutrient transporters were evaluated (qPCR/western blot). RESULTS: Peptide- and alanine-induced changes in Isc were significantly decreased in the jejunum and ileum of the trickle-infected group at 49 dpi and in the ileum of the single-infected group at 49 dpi. No significant differences regarding glucose transport were observed between the Ascaris-infected groups and the control group in Ussing chamber experiments. Transcription levels of the glucose and peptide transporters as well as of selected transcription factors (transcription of signal transducer and activator of transcription 6 [STAT6] and hypoxia-inducible factor 1-alpha [Hif-1α]) were significantly increased in response to both infection types after some periods. The transcription of interleukins 4 and 13 varied between decrease and increase regarding the respective time points, as did the protein expression of glucose transporters. The expression of the peptide transporter PepT1 was significantly decreased in the ileal single-infected group at 35 dpi. Hif-1α was significantly increased in the ileal tissue from the single-infected group at 21 dpi and in the trickle-infected group at 35 dpi. The expression levels of Na+/K+-ATPase and ASCT1 remained unaffected. CONCLUSIONS: In contrast to the current hypothesis, these results indicate that the nutrient deprivation induced by A. suum cannot be explained by transcriptional or expression changes alone and requires further studies.

Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet.

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

Segment-specific effects of resveratrol on porcine small intestinal dipeptide absorption depend on the mucosal pH and are due to different mechanisms: potential roles of different transport proteins and protein kinases.

Numerous beneficial features of the polyphenol resveratrol (RSV) have been demonstrated in several tissues and cell culture models. There is also evidence, that RSV impairs intestinal nutrient transport but the underlying mechanisms are not understood. The aim of the present study was to evaluate whether RSV has also an impact on the H+-coupled transport of peptides via the peptide transporter 1 (PepT1) and to characterize RSV mediated changes in the apical abundance of nutrients transport proteins and protein kinases that may be involved. RSV decreased the H+-coupled transport of peptides in the porcine small intestines in a pH and location specific manner (jejunum vs ileum) as measured in Ussing chamber experiments. The comparison of the effects of RSV with the effects of the cAMP/PKA-activating agent forskolin indicates that different mechanisms may be responsible in the intestinal segments. Additionally, it seems that the transport of peptides and glucose in the jejunum are inhibited via the same mechanism while there might be two mechanisms involved in the ileum. Functional data and protein expression data indicate, that, besides PepT1, the activity of the Na+/H+-exchanger 3 (NHE3) may be involved. Protein kinase A (PKA) and AMP-activated kinase (AMPK) are both activated by RSV while the extracellular regulated kinase (ERK) and the serum and glucocorticoid induced kinase (SGK) are widely unaffected. Although PKA and AMPK are activated, AMPK seems not to be related to the effects of RSV. Additionally, both the functional data and the protein expression data reveal some interesting pH- and segment-specific differences.

Calcium-induced chloride secretion is decreased by Resveratrol in ileal porcine tissue.

OBJECTIVE: Chloride (Cl-) secretion is crucial for intestinal fluid secretion. Therefore, effects of the polyphenol Resveratrol (RSV) on Cl- secretion have been investigated. In a previous study, we observed effects of RSV on forskolin-induced Cl- secretion in the porcine jejunum but not the ileum although RSV itself induced a transepithelial ion current that may represent Cl- secretion in the ileum. The aim of this study was to gain further insights regarding the effects of RSV on characteristics of Cl- secretion in the porcine ileum using the Ussing chamber technique (recording of short circuit currents (Isc) as a measure for epithelial net ion transfer). RESULTS: RSV increased the Isc in the porcine ileum but not in the porcine jejunum as is already known. This increase was absent in a Cl--free buffer system, indicating that RSV indeed induces Cl- secretion. However, the carbachol-induced Isc was significantly inhibited by RSV indicating an inhibition of Ca2+-induced Cl- secretion. The cellular basis for these contradictory, segment specific results of RSV on Cl- secretion has to be subjected to further studies. The results also underline, that is difficult to generalize effects of RSV between different intestinal locations, organs, cell culture models or species.

Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na⁺/K⁺-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes.

BACKGROUND: Beneficial effects of Resveratrol (RSV) have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. METHODS: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min) in Ussing chambers (functional studies) and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs)). RESULTS: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc) and influenced forskolin-induced ΔIsc. The phosphorylation of sodium-glucose-linked transporter 1 (SGLT1), AMP-activated protein kinase (AMPK), protein kinase A substrates (PKA-S) and liver kinase B1 (LKB1) increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1) and (phosphorylated) Na⁺/H⁺-exchanger 3 (NHE3) did not change. CONCLUSION: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP) levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.

Degree of SGLT1 phosphorylation is associated with but does not determine segment-specific glucose transport features in the porcine small intestines.

Glucose-induced electrogenic ion transport is higher in the porcine ileum compared with the jejunum despite equal apical abundance of SGLT1. The objective of this study was a detailed determination of SGLT1 and GLUT2 expressions at mRNA and protein levels along the porcine small intestinal axis. Phosphorylation of SGLT1 at serine 418 was assessed as a potential modulator of activity. Porcine intestinal tissues taken along the intestinal axis 1 h or 3 h after feeding were analyzed for relative mRNA (RT-PCR) and protein levels (immunoblot) of SGLT1, pSGLT1, GLUT2, (p)AMPK, ß2 -receptor, and PKA substrates. Functional studies on electrogenic glucose transport were done (Ussing chambers: short circuit currents (Isc )). Additionally, effects of epinephrine (Epi) administration on segment-specific glucose transport and pSGLT1 content were examined. SGLT1 and GLUT2 expression was similar throughout the small intestines but lower in the duodenum and distal ileum. pSGLT1 abundance was significantly lower in the ileum compared with the jejunum associated with significantly higher glucose-induced Isc . SGLT1 phosphorylation was not inducible by Epi. Epi treatment decreased glucose-induced Isc and glucose flux rates in the jejunum but increased basal Isc in the ileum. Epi-induced PKA activation was detectable in jejunal tissue. These results may indicate that SGLT1 phosphorylation at Ser418 represents a structural change to compensate for certain conditions that may decrease glucose transport (unfavorable driving forces/changed apical membrane potential) rather than being the cause for the overall differences in glucose transport characteristics between the jejunum and ileum.

Gastrointestinal transport of calcium and glucose in lactating ewes.

During lactation, mineral and nutrient requirements increase dramatically, particularly those for Ca and glucose. In contrast to monogastric species, in ruminants, it is rather unclear to which extend this physiological change due to increased demand for milk production is accompanied by functional adaptations of the gastrointestinal tract (GIT). Therefore, we investigated potential modulations of Ca and glucose transport mechanisms in the GIT of lactating and dried-off sheep. Ussing-chamber technique was applied to determine the ruminal and jejunal Ca flux rates. In the jejunum, electrophysiological properties in response to glucose were recorded. Jejunal brush-border membrane vesicles (BBMV) served to characterize glucose uptake via sodium-linked glucose transporter 1 (SGLT1), and RNA and protein expression levels of Ca and glucose transporting systems were determined. Ruminal Ca flux rate data showed a trend for higher absorption in lactating sheep. In the jejunum, small Ca absorption could only be observed in lactating ewes. From the results, it may be assumed that lactating ewes compensate for the Ca loss by increasing bone mobilization rather than by increasing supply through absorption from the GIT Presence of SGLT1 in the jejunum of both groups was shown by RNA and protein identification, but glucose uptake into BBMV could only be detected in lactating sheep. This, however, could not be attributed to electrogenic glucose absorption in lactating sheep under Ussing-chamber conditions, providing evidence that changes in jejunal glucose uptake may include additional factors, that is, posttranslational modifications such as phosphorylation.

Effects of dietary starch source on electrophysiological intestinal epithelial properties and intestinal glucose uptake in growing goats.

In ruminants, the potential benefit of by-pass starch to improve energy supply is under discussion. As efficient intestinal starch digestion and monosaccharide absorption are important prerequisites for an energetic benefit compared to ruminal fermentation, this study was conducted to characterise potential adaptations of intestinal tissues to different dietary starch sources qualitatively. The Ussing chamber technique was used to determine electrophysiological parameters of jejunal tissues and glucose flux rates. Kinetics of sodium-dependent glucose uptake into isolated brush-border membrane vesicles (BBMV) were calculated, and the expression level of sodium-dependent glucose transporter 1 (SGLT1) was determined. Samples were collected from goats that were assigned to three dietary treatments differing in starch content (hay/concentrate) and starch source (wheat/corn). Additionally, ingesta samples were analysed for starch and glucose contents. Jejunal tissues from hay-fed animals showed higher tissue conductances (G t) and numerically higher short-circuit currents (I sc). Unidirectional glucose flux rates were higher for hay-fed animals, whereas net flux rates were unaffected. The maximal glucose transport capacity into BBMV was increased for concentrate-fed animals, but the affinity and SGLT1 expression were not affected. Our results may indicate an adaptation of glucose uptake via SGLT1 to variations in dietary starch but it could not be excluded that intestinal uptake capacity was saturated under the given conditions or that the measured capacity was sufficient for absorption of available glucose.

trans-Resveratrol and ε-viniferin decrease glucose absorption in porcine jejunum and ileum in vitro.

trans-Resveratrol and ε-viniferin are used as dietary supplements. They are reported to be supportive in preventing arteriosclerosis and diabetes and a previous study could demonstrate an inhibitory potential on sodium-dependent glucose transport (SGLT1) in oocytes und mouse intestinal everted rings (Schulze et al., 2012, Genes Nutr. 6, S61). The in vitro effects of trans-resveratrol and ε-viniferin on intestinal glucose uptake in the porcine small intestines (Sus Scrofa) have not yet been evaluated. It was hypothesized that trans-resveratrol/ε-viniferin may have an adverse effect on porcine intestinal sodium-dependent glucose uptake. The effects on electrogenic small intestinal glucose absorption and sodium-dependent (3)H-glucose uptake in brush border membrane vesicles (BBMV) were evaluated. Pieces of mucosa were mounted into Ussing chambers and were incubated with either trans-resveratrol (0.3 mmol/L), ε-viniferin (0.3 mmol/L), or ethanol. Sodium-dependent glucose absorption into BBMV was measured. (3)H-glucose uptake studies were performed using the same concentrations of the respective substances. SGLT1-mediated glucose absorption was approximately 3-fold higher in ileum compared to jejunum. After preincubation with trans-resveratrol and ε-viniferin, glucose-induced increases of short-circuit currents were significantly decreased. BBMV-studies revealed comparable results and glucose uptake was also significantly decreased. As the glucose transport/uptake was decreased after preincubation with either trans-resveratrol or ε-viniferin this active transport mechanism was directly influenced by inhibiting the SGLT1 transport system.

Segmental diversity of electrogenic glucose transport characteristics in the small intestines of weaned pigs.

It has been shown in several species that the intestinal Na(+)-dependent glucose co-transporter 1 (SGLT1) is more abundant in the jejunum than in ileum. In contrast, the efficiency of intestinal glucose uptake rates in suckling piglets or weaned pigs is not clearly fitting with this segmental distribution. The aim of this study was to evaluate SGLT1 mediated glucose absorption in the jejunum and ileum of growing pigs (Sus scrofa) in more detail. In Ussing chambers, basal short-circuit currents were significantly more positive in the jejunum. It could be demonstrated that the electrogenic ileal glucose transport was significantly more pronounced in different breeds and occurred at 5 mmol∙L(-1) glucose 7 times faster in the ileum, although slightly higher jejunal expression of glycosylated SGLT1 was detected by Western blotting. This expression pattern was connected to significantly lower phlorizin sensitivity in the jejunum. As the more efficient ileal glucose absorption was also observable with glucose uptake studies into isolated brush-border membrane vesicles without differences in abundance and activity of the Na(+)/K(+)-ATPase in both segments, we conclude that the segmental differences in porcine glucose transport characteristics may be based on direct or indirect modulations of SGLT1 activity.