Increased intestinal permeation and modulation of presystemic metabolism of resveratrol formulated into self-emulsifying drug delivery systems.

Despite various beneficial biological properties, resveratrol lacks therapeutic applications because of poor bioavailability due to variable absorption and extensive metabolism. The present study aims at evaluating the capability of self-emulsifying drug delivery systems (SEDDS) to enhance resveratrol permeation across rat intestine and to modulate its presystemic metabolism. For that purpose, semi-solid (SS) and liquid (L) SEDDS were prepared and dispersed in an aqueous buffer to produce nanoemulsions (NE). The jejunal absorptive transepithelial fluxes (Jms) of resveratrol elicited by these formulations (SS-NE and L-NE) and presystemic metabolization were determined on Ussing chambers. The absorptive fluxes through the intestinal epithelium from the nanoemulsions (Jms=20.5±3.1µgh-1cm-2 SS-NE; 28.9±2.9µgh-1cm-2 L-NE) were significantly increased compared to an ethanolic control solution (Jms=3.4±0.3µgh-1cm-2, p<0.05). No significant variations of conductance were observed after two hours of contact between the formulations and the mucosa. Simultaneously, the presystemic metabolization pattern was modified in the case of the nanoemulsions compared to the control solution. In conclusion, our data suggests that oil-in-water nanoemulsions prepared from SEDDS dispersions of medium-chain lipids could be promising formulations for enhancing oral delivery of resveratrol.

Effect of sorbin on electrolyte transport in rat and human intestine.

Stimulating water absorption in the colon represents an important target to reduce stool output in secretory diarrhea. Recently, a 153-amino-acid peptide was isolated from porcine upper small intestine and purified, taking into account the increase of water absorption in guinea pig gallbladder. Accordingly, this peptide was named sorbin. The aim of the present study was to determine if the COOH-terminal heptapeptide of sorbin (C7-sorbin) participates in the regulation of electrolyte transport in the colon. Different regions (from duodenum to colon) of stripped intestinal mucosa from rats or humans were mounted in Ussing chambers to measure the changes in short-circuit current (DeltaIsc) and net 22Na and 36Cl fluxes (JNanet and JClnet) after serosal exposure of 10(-7) to 10(-3) M C7-sorbin. In fasted rat intestine, C7-sorbin (10(-4) M) induced an immediate reduction in Isc in the distal ileum and proximal and distal colon but not in the duodenum and jejunum. In the colon, Isc reduction and JNanet and JClnet stimulation were dose dependent (EC50 = 2 x 10(-5) M). At 10(-3) M, maximal effect was observed (DeltaIsc = -1.14 +/- 0.05, DeltaJNanet = +4.97 +/- 1.38, and DeltaJClnet = +9.25 +/- 1.44 microeq. h-1. cm-2). C7-sorbin (10(-3) M) inhibited the increase in Isc induced by a series of 10 secretory agents such as secretin, vasoactive intestinal peptide, PGE2, and serotonin. In HT-29-Cl19A cells, C7-sorbin induced an increase in Isc, with a maximal effect at 10(-3) M (DeltaIsc = 0.29 +/- 0.10 microeq. h-1. cm-2). In human intestine, a dose-dependent decrease in Isc was observed in right and sigmoid colons in basal and stimulated conditions (EC50 congruent with 10(-5) M; at 10(-4) M, DeltaIsc = -2.66 +/- 0.17 microeq. h-1. cm-2) but not in the jejunum. The results indicate that C7-sorbin stimulated NaCl neutral absorption and inhibited electrogenic Cl- in rat and human intestinal epithelia. In addition, the antisecretory effect was essentially observed in the distal part of both rat and human intestine and the magnitude of the proabsorptive effect was directly related to the magnitude of the previously induced secretion.

Oxyntomodulin reduces hydromineral transport through rat small intestine.

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.

Comparison of the antisecretory effect of endogenous forms of peptide YY on fed and fasted rat jejunum.

It is intriguing that the antisecretory peptide YY is present in plasma in two forms: PYY1-36 and PYY3-36. PYY3-36 has been found in human and rabbit blood within 30 min of the beginning of the meal, when the peak of water and electrolyte secretion occurs in the duodeno-jejunum. The aim of this study was therefore to compare the antisecretory effect of PYY1-36 and PYY3-36 in fed and fasted rat jejunum. The variations in electrolyte secretion were assessed by measuring the variations in short-circuit current (delta Isc) and transepithelial isotopic chloride fluxes in jejunal mucosa isolated from fed and fasted animal, and mounted in Ussing Chambers. In fasted animals, 2 x 10(-7) M PYY3-36 induced a reduction in Isc of -0.50 +/- 0.01 microEq/hr.cm2, which was not statistically different from that induced by 2 x 10(-7) M PYY1-36 (-0.60 +/- 0.01 microEq/h cm2). In contrast, in fed animals, 2 x 10(-7) M PYY3-36 did not trigger a significant response on Isc and net chloride flux, while the response to PYY1-36 was present but blunted. The absence of response was probably not related to the presence of secretory peptides because PYY3-36 was still able to induce a reduction in Isc after stimulation by a series of 10 different secretory peptides. After 10(-8) M PYY3-36 addition to an epithelium from the fasted animal, response to 10(-7) M PYY3-36 was blunted for 30 min and returned to control value after 60 min. Plasma concentration of PYY was higher in the fed rats compared to fasted (213.78 +/- 38 vs. 53.62 +/- 11.47 pg/ml p < 0.01). After incubation of crypt cells with or without 0.1 microM of unlabeled PYY for 60 min, Scatchard analysis of equilibrium binding data show that binding capacity (Bmax) of receptors was reduced when crypt cells were previously incubated with unlabeled PYY without significant modification of dissociation constants. Bmax were 183 +/- 27 in control vs. 56 +/- 11 fmol/mg protein. These results confirm the antisecretory activity of PYY1-36 in the jejunum of fasted and fed rats. They further indicate that PYY3-36 displays similar activity to PYY1-36 in fasted animals, but lack of activity in fed animals. These results suggest that the two circulating forms of PYY act as antisecretory peptides by two different mechanisms, implying a C-terminal specificity.

Sodium fluoride inhibits the antisecretory effect of peptide YY and its analog in rabbit jejunum.

The antisecretory peptide YY (PYY) inhibits jejunal secretion through and inhibitory protein (Gi), whereas sodium fluoride (NaF) is a potent activator of G-proteins. This work was conducted to characterize the role of NaF in the antisecretory effect of PYY. For this purpose, electrogenic chloride secretion was assessed by measuring the in vitro variations in short-circuit current (delta Isc) due to alterations in ionic transport, using Ussing chambers Results: 1) NaF induced a transient increase in Isc at concentrations exceeding 5 mM. 2) 2 mM NaF inhibited the antisecretory effect of 0.1 microM PYY and of its analog P915. 3) stimulation of secretion by forskolin and dbcAMP was halved in the presence of 2 mM NaF. 4) Inhibition of protein kinase C by 0.1 mM bisindolylmaleimide caused a sustained increase in Isc in the presence of 5 mM NaF. In conclusion, these results confirm that PYY inhibits electrogenic chloride secretion and show that NaF stimulates it, and suggest that NaF reduces PYY-induced inhibition via a G-dependent and a G-independent functional pathway.

Evaluation of intestinal antisecretory peptides coefficient of dissociation: a functional approach.

The aim of this study was to determine the coefficient of dissociation (Kd) of three intestinal antisecretory peptides, peptide YY (PYY), galanin (GAL), and somatostatin-28 (SRIF) by a functional approach. The variations in short-circuit current (DeltaIsc) due to the modification of ionic transport across rat and rabbit small intestine were assessed in vitro, using Ussing chambers. Determination of Kd was made by mathematical analysis of concentration-response relationships. We found Kd values for GAL and SRIF of 1.6 and 3.67 nM, respectively, in rabbit ileum. In rat jejunum, we found Kd of 0.44 nM for PYY and 1.75 for SRIF. Our data are in close agreement with those derived from binding studies suggesting that evaluation of Kd by this functional approach can be used to assess the antisecretory potency of peptides or of analogs whose receptors in intestine have not yet been characterized.

Glutamate-arginine salts and hormonal responses to exercise.

Hormonal changes during exercise is of growing interest because of their role in adaptation, and performance. The production of amino acids (AA) due to the degradation of muscle protein increases during exercise and some AA may be utilized for energy expenditure or as hormonal secretagogues. Thus, one can propose a strategy to reduce muscle protein breakdown and regulate hormones involved in energy metabolism by dietary AA supplementation. We assessed the effects of glutamate-arginine salt (AGs) ingestion on exercise-induced hormonal alterations in highly trained cyclists (age 18-22 yrs). Using an indwelling catheter, we collected multiple blood samples at rest, during warm up, during and after an intense exercise session. Plasma growth hormone (hGH), insulin and cortisol were measured by radioimmunoassay. As reported in previous studies, we observed a marked increase in plasma hGH and cortisol levels during and after exercise in the placebo (Pl) condition as well as a slight decrease in insulin concentration. In addition, we found that the ingestion of AGs had significant effects on some dynamic hormonal changes. AGs had no effect on resting plasma levels of hGH, insulin or cortisol. However, the marked elevation in cortisol and hGH during and after exercise in the placebo condition, was greatly diminished when subjects ingested AGs. Our results show that AGs can modify exercise-induced hormonal changes and raise the possibility that it may be used to alter energy metabolism during endurance exercise.

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum.

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22-36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 x 10(-11) M (delta Isc -2 +/- 0.5 microA/cm2). The reduction was maximal at 10(-7) M (Isc -23 +/- 2 microA/cm2), and the concentration producing half-maximal inhibition was 10(-9) M. At 10(-7) M, reduction of 1sc by PYY reached 90% of response to 5 x 10(-5) M bumetanide. The PYY effect was partly reversed by 10(-5) M forskolin (Isc + 13.43 +/- 2.91 microA/h.cm2, p < 0.05) or 10(-5) M dibutyryl adenosine 3',5' cyclic monophosphate (Isc + 12 +/- 1.69 microA/cm2, p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 microEq/cm2 (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22-36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10(-7) M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.

Effects of an ingested glutamate arginine salt on ammonemia during and after long lasting cycling.

The purpose of this study was to examine the effect of glutamate-arginine salt (AGs) or placebo (P1) on ammonemia during and after 1 hour exercise on sporting event bicycle under ergonomic device at 80% VO2max in 3 healthy male volunteers (age 18-25 years). Subjects were tested in three sessions, at rest after AGs and during exercise with placebo (Pl) or AGs. The subjects were given 20 g of AGs or Pl orally and 30 min later, exercised at 75-80% VO2max for 30 min. Blood samples were taken at 0, +30, +60, +90, +120 min after AGs and analyzed for ammonemia. Our results show a highly significant increase in plasma ammonia concentration during exercise. The magnitude of this increase was diminished when subjects were given AGs before the exercise session, suggesting that AGs may help reduce physiologic fatigue.