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.
• Here, we investigated whether RSV has also an impact on the H+-coupled transport of peptides via the peptide transporter 1 (PepT1).
• We also characterized RSV mediated changes in the apical abundance of nutrients transport proteins and protein kinases.

Introduction

• Efforts to implement the multi capable, putatively health promoting, polyphenolic substance Resveratrol (RSV) necessitate studies on the effects that RSV may exert on the functionality of the gastrointestinal tract.
• One important cellular mechanisms of RSV is the inhibition of phosphodiesterase and the stimulation of adenylate cyclase, leading to elevated intracellular cAMP and Ca2+ levels, respectively
• RSV might also influence the intestinal absorption of di- and tripeptides via the H+-coupled peptide transporter 1 (PepT1) and that the effects are different between the proximal and distal part of the small intestine.

Animals and tissue removal

• In total, tissues from 20 male weaned piglets (Sus scrofa domestica, German Landrace × Large White) were used in this study.
• Tissues from 10 animals were used for Ussing chamber studies, and tissues from another 10 animals was used for tissue incubation experiments
• The animals were kept on a plant-based conventional diet (deuka primo pro, 17.4 MJ ME kg−1) for at least 10 days (feeding twice daily) with free access to drinking water.

Ussing chamber experiments

• Electrophysiological studies were carried out using the third meter distal to the pylorus (jejunum) and parts of the first meter proximal to the ileocecal valve (ileum, first 30 cm discarded).
• Stripped mucosa was incubated in standard chambers with an exposed serosal area of 1.13 cm2 under short circuit conditions (application of a direct current setting the transepithelial potential difference (PDt) to zero using a computer controlled voltage clamp devise from Mussler Scientific instruments, Aachen, Germany)
• Continuous monitoring of tissue conductances (Gt) and short circuit currents (Isc) was done under these conditions.
• Changes in the Isc reflect changes in the transgenic net ion transfer (increase or decrease).

Tissue incubations and preparation of apical brush border membranes and cell lysates

• The general procedure for the incubation of tissue samples for western blot analysis was as described previously [9].
• Six tissue bathes were available per intestinal segment whereby two were used for each mucosal pH level from which one served as control (mucosal buffer with ethanol (20 μl/10 mL)) and one was treated with RSV in ethanol (final concentration 300 μM).
• Glycyl-glycin was used instead of glygln, which was used in the Ussing chamber experiments.
• Ethanol and RSV were also added to the mucosal buffer solutions directly before applying the buffer to the tissue samples.

Western blot SDS-PAGE and Western blot

• Immunoblotting was essentially performed as described previously using the Laemmli system, nitrocellulose membranes and a tank blot procedure [28].
• The primary and secondary antibodies that were used and detailed information about sample denaturing conditions, the amount of protein per lane, and details on the dilution factors used for primary & secondary antibodies are given in Table 2.
• Empty Cellμg/laneDenaturing Conditions
• PAK(overnight 4°C)SAK(90 min, RT)PEPT11
• 10 μg95°C5 min1:5005%BSA/TBST1:2000
• Non-phosphorylated proteins and their phosphorylated forms were detected on the same membrane with stripping and new blocking membranes [28]
• After detection of the membrane detection, washing protein was done with femalization, and detection of non-pH, non-deoxyribonucleic acid, and non-spondylinositol (NDE) was detected with the Femalization software [29]
• Phospho-(Ser/Thr) PKA Substrate Antibody

Ussing chamber experiments

• Total number of animals: n=6 for ileal tissues but n=10 for jejunal tissues
• The samples were assumed to be paired within one animal.
• Jejunum and ileum were analyzed separately using RM Two-way ANOVA for matched values (factors: pH and RSV- or forskolin-treatment).
• Tukey post-test was used for multiple comparison of the means

Western blot data

• Tissue preparations from one animal were blotted on one membrane and the intensity of the bands was analyzed.
• Specific band intensities were normalized to the Indian Ink signal (total protein).
• The band intensity for jejunal samples was set to 1 (ileal intensity/jejunal intensity) and for acidity, the band intensity was set in relation to pH.

RSV differently affects jejunal and ileal basal short circuit current at different pH

• RSV segment-specifically affects the basal Isc
• The decreasing action of RSV in the jejunum was enhanced with decreasing pH.
• These effects may be associated with the pH dependent differences in the basal isc (Fig. 3A).

Glygln-induced short circuit currents: Effects of RSV and forskolin depend on the extracellular pH and the intestinal localization

• Compared to control chambers, RSV significantly decreased the jejunal ΔIsc at all pH levels but to different extents (pH × RSV interaction).
• Forskolin preincubation and irradiation was also effective in reducing it compared to the control chambers.
• No inhibitory effect of FSK was observed at pH 5.4. The difference was not statistically significant.

Glucose-induced short circuit currents are differently affected by RSV and forskolin depending on the extracellular pH

• The effects of RSV, FSK and pH on jejunal and ileal glucose induced ΔIsc and respective details on the statistical analysis are shown in Fig. 4B and 4C.
• RSV inhibited glucose- and glygln-induced changes in the intestinal segments at the different pH levels after RSV incubation (Fig. 4D).
• While the effects in the jejunum were correlated at all pH stages, the effects were only significantly correlated at pH 6.4 and 7.4.

Time courses of glygln-induced short circuit currents differ between jejunum and ileum and at different pH levels

• In order to quantify these different curve characteristics, the ΔIsc at different time points was analyzed (see also Fig. 5A).
• The time course of the glyglyn-induced Isc in control chambers (light blue line) was different between the intestinal segments and differed also within the intestinal segment between the different pH level.
• Briefly, the change of the Isc per second was dependent on the pH that differently affected the mean Δ Isc per second in the both intestinal segments
• Assuming that PepT1 is not regulated during the short period until t2 is reached, the plateau phases or decreases of Isc during t3 and t4 might be attributable to a stop or decrease in peptide transport due to differences in the activity of NHE3 or the activation for NHE2.

RSV and FSK affect the time courses of jejunal and ileal glygln-induced short circuit currents

• Jejunum: RSV decreased ΔIsc t2 at pH 6.4 (P<.0001) and less pronounced at pH 7.4.
• ileum: FSK was not able to significantly inhibit γIII in the jejunum, and was almost unaffected by RSV
• The effects of FSK and RSV differed between the intestinal segments and pH levels.

Effects of intestinal location, mucosal pH and RSV on the expression of transporters and protein kinases

• The effect of the intestinal segment at defined pH on the apical abundance of intestinal nutrient transporter and expression of protein kinase was calculated independently for both control and treated tissues.
• RSV treatment tended to reduce the mean band height in the jejunum while the ileal mean was reduced at pH 7.4 (data not shown).
• Apart from effects on the band intensity, there were differences in the band height under some conditions (e.g. Western Blotting).

The abundance of apical pNHE3 Ser522 was also affected by the intestinal location.

• In the ileum, there was an increase at pH 5.4 compared to pH 7.4 while in the jejunum there was a decrease at pH 6.4
• RSV affected the apical abundance of Ser522 in a similar way as for total NHE3
• There was no clear effect of pH

The amount of apical CFTR was lower in the ileum than in the jejunum at pH 6.4 and pH 7.4 than at pH 5.4.

• RSV treatment decreased the mean band height in western blot experiments (see example in Fig. 2), whereby both intestinal segments at all pH levels were significantly affected (data not shown).
• There was no significant effect of the mucosal pH on the expression of SGLT1.

pAMPK Thr172/AMPK

• The phosphorylation level of AMPK was significantly different between the intestinal segments at pH 7.4 and pH 6.4 but not at pH 5.4.
• RSV treatment had a strong increasing effect on AMPK phosphorylated at all pH levels in both intestinal segments (e.g., ileum/jejunum ratio increased for pSGK Thr256).
• There was no effect on SGK in control tissues (Fig. 7L) or RSV treated tissues (data not shown).

Correlation Analysis

• To work out whether the multiple effects on the expression of the different proteins caused by the intestinal segment, the mucosal pH or RSV treatment followed a similar pattern some correlation analysis were carried out.
• The ratios calculated for each protein were correlated with the ratios for the other proteins.

Correlation between the effects of the intestinal segment on protein expression

• The expression of almost all transport proteins except PepT1 and pNHE3 Ser605 (high variability) and some protein kinases was strongly dependent on the intestinal location at least at some pH levels.
• RSV treatment did not change the slope of the respective regression lines (data not shown).
• Furthermore, the segment effects for the transport proteins were more or less strongly correlated with each other.

Correlation between the effects of pH on protein expression

• In both intestinal segments, RSV treatment leads to significant correlations of pH effects of RSV on pPKA-S with the respective effects on many transport proteins (jejunum: pSGLT1, NHE3, pNHE3 Ser552, Ileum: PepT1), pAMPK/AMPK, ERK, and SGK.

Correlation between RSV and protein expression

• In the jejunum, the effects of RSV on the amount of pPKA-S were correlated with PepT1, SGLT1
• and tended to be correlated with pNHE3 Ser522 and total NHE3.
• in the ileum, RSV effect on pK-S was only significantly correlated with PSGLT 1 Ser418, although it was negatively correlated with other proteins.

Discussion

• The results of the Ussing chamber experiments reveal several differences between jejunal and ileal tissues with regard to their basal features, their response to mucosal nutrient supply at different pH levels, their reaction to a FSK-mediated elevation of intracellular cAMP concentrations, and in their reactivity to RSV
• There were segment-specific, pH-dependent differences in the RSV-evoked ΔIsc after the addition of RSV.
• RSV is able to inhibit the intestinal absorption of peptides in the jejunum compared to the ileum, which may indicate that either different mechanisms were initiated by RSV in the different segments that showed segment specific differences in their pH-dependence or that RSV triggered the same mechanisms in both segments.

Conclusion

• The Ussing chamber results demonstrate that the basal Isc and the tissue reactivity to RSV is pH sensitive but with different characteristics in the jejunum and the ileum.
• RSV not only inhibits intestinal glucose transport at all pH levels in a segment specific manner (which has already been shown for pH 7.4-8.4 [8,9]) but also has a substantial impact on the electrogenic transport of the dipeptide glygln (Figure 4).
• Segment specific differences were also observed regarding the correlation of the percentage inhibition of glucose and dipeptin transport.

Reference

10.1016/j.jnutbio.2020.108467