Intestinal SGLT1-mediated absorption and metabolism of benzyl β-glucoside contained in Prunus mume: carrier-mediated transport increases intestinal availability

The intestinal absorption of benzyl β-glucoside (BNZβglc) contained in the fruit of Prunus mume SIEB. et ZUCC. (Rosaceae), which is traditionally used as a medicinal food in Japan, was studied in rat intestines. BNZβglc was absorbed from the mucosal to serosal sides. Its metabolite, benzyl alcohol (BAL), was also detected on both the mucosal and serosal sides. In the presence of phloridzin (Na⁺/glucose cotransporter (SGLT1) inhibitor) or in the absence of Na⁺ (driving force), BNZβglc absorption was significantly decreased. Transport clearance of BNZβglc across the brush border membrane decreased as its concentration increased. These results indicate that BNZβglc is transported by SGLT1. Metabolic clearance of BNZβglc also decreased as its concentration increased. The amount ratio of BNZβglc to BAL on the serosal side increased with the increase of BNZβglc concentration. The intestinal availability of BNZβglc was lower in the absence of Na⁺ than in the presence of Na⁺, indicating that the SGLT1-mediated transport of BNZβglc increases intestinal availability by decreasing the intestinal extraction ratio. This neutraceutical study concluded that intestinal carrier-mediated transport across the brush border membrane improves the intestinal availability of nutritionally, pharmacologically or physiologically active compounds that undergo intestinal metabolism (first-pass effect).     

Alanine Efflux across the Serosal Border of Turtle Intestine

The exit of alanine across the serosal border of the epithelial cells of turtle intestine was measured by direct and indirect techniques. A decrease or an increase in cell Na did not affect the amino acid flux from cell to serosal solution. Cells loaded with Na and alanine did not exhibit any extrusion of alanine when their serosal membranes were exposed to an Na-free medium containing alanine. However, substantial amino acid extrusion was observed across the mucosal cell border under similar conditions. Although alanine flux across the serosal membrane appeared to be Na-independent, it showed a tendency toward saturation as cellular alanine concentration was elevated. The results are consistent with the postulate that the serosal and mucosal membranes of intestinal cells are asymmetrical with respect to amino acid transport mechanisms. The serosal membrane appears to have an Na-independent carrier-mediated mechanism responsible for alanine transport while transport across the mucosal border involves an Na-dependent process.     

Effect of somatostatin on D-galactose transport across the small intestine of rats

Somatostatin was found to diminish control and theophylline-treated tissue sugar accumulation as well as control and also to diminish theophylline mucosal to serosal D-galactose fluxes. When Na+ was removed from the bath solution, sugar transport was unaltered by the presence of somatostatin. The same results were obtained with phlorizin in the medium. These results seem to suggest that the action of somatostatin is restricted to the Na+-dependent sugar carrier located on the brush border of the intestinal epithelium.     

Lysine Transport across Isolated Rabbit Ileum

Lysine transport by in vitro distal rabbit ileum has been investigated by determining (a) transmural fluxes across short-circuited segments of the tissue; (b) accumulation by mucosal strips; and (c) influx from the mucosal solution across the brush border into the epithelium. Net transmural flux of lysine is considerably smaller than that of alanine. However, lysine influx across the brush border and lysine accumulation by mucosal strips are quantitatively comparable to alanine influx and accumulation. Evidence is presented that the "low transport capacity" of rabbit ileum for lysine is due to: (a) a carrier-mediated process responsible for efflux of lysine out of the cell across the serosal and/or lateral membranes that is characterized by a low maximal velocity; and (b) a high "backflux" of lysine out of the cell across the mucosal membrane. A possible explanation for the latter observation is discussed with reference to the relatively low Na dependence of lysine transport across the intestinal brush border.     

Effect of various methods of oxygenation in the isolated small intestine on the tryptophan and glycine accumulation

The impact of various oxygenation types of intestinal preparations (mucosal, serosal and bilateral) on the transepithelial transport of amino acids and peptide according to the character of amino acids, their stereoisometry, incubation time, intestinal gradient and other factors, was investigated in fowl. Against-the-gradient-transport of L-tryptophan and glycine was observed at various oxygenation types, and its intensity decreased in the following sequence: bilateral, mucosal, serosal oxygenation. At various oxygenation types, the accumulation of L-tryptophan occurs more efficiently than that of DL-tryptophan and D-tryptophan. Using an in vitro bilateral intestinal oxygenation model system, we have demonstrated the plasma membrane of enterocyte microvilli to be rapidly impaired after oxidative stress. Glycine, but not L-tryptophan, attenuates oxidative injury in brush border membrane and alterations in amino acid transport activity. Overall, our data indicates that in vitro serosal oxygenation of the duodenum markedly improves glycine absorption, possibly involving the basolateral transporters.     

Intestinal transport and metabolism of glucose-conjugated kyotorphin and cyclic kyotorphin: metabolic degradation is crucial to intestinal absorption of peptide drugs

Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.     

Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption

The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis. The amino acid absorption in LPS treated rabbits was reduced compared to the control animals. The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity. The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine. The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect. However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment. Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Oxidative and drug-induced alterations in brush border membrane hemileaflet fluidity, functional consequences for glucose transport

Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na(+)-dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na(+)-dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A(2)C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity.     

Cytoskeleton involvement on intestinal absorption processes

It has been recently demonstrated in the laboratory that the cytoskeletal inhibitor cytochalasin E has an indirect inhibitory effect on the function of the intestinal Na+-sugar cotransporter (SGLT1). The present work confirms that cytochalasin E inhibits SGLT1 activity through cytoskeleton disruption, showing that in anaerobic conditions (N2 bubbling), which implies low cytosolic ATP levels, the inhibition is not observed. As it occurs in sugar transport, the Na+-dependent intestinal transport of phenylalanine decreases if cytochalasin E is present in the incubation medium. However, the activity of the brush border enzymes sucrase, amino peptidase N and gamma-glutamyl transferase is not affected by the inhibitor. These enzymes only have one transmembrane domain and the active center is projected to the intestinal lumen. Therefore, cytoskeleton changes that could modify the transmembrane enzyme segment do not alter the activity of these enzymes. Examination of the intestine morphology after 30 min incubation with cytochalasin E shows only light modifications which do not seem to explain the inhibitory effects of the toxin on Na+-sugar or Na+-phenylalanine cotransporters function. On the whole, these results indicate that the inhibition of cytochalasin E on galactose and phenylalanine intestinal transport is secondary to its action on cytoskeleton through protein structure modifications.     

Intestinal iodide secretion and its dependence upon mucosal I- permeability

Iodide secretion across different regions of rat small intestine has been investigated in vitro using the standard Wilson-Wiseman technique. Net I- secretion was observed along the entire small intestine, being significantly higher in the central region. Anaerobic conditions, ouabain (2 mM) and Na+ free Ringer solution prevented net I- secretion, whilst both theophylline (1 mM) and carbachol (0,1 mM) enhanced the observed basal intestinal I- secretion. Furthermore, Ca2+-deprived bathing solutions significantly reduced intestinal I- secretion. Epithelial I- uptake from both mucosal and serosal sides was measured by using a Ussing-type chamber technique. The initial rate of I- uptake across the mucosal membrane was significantly higher in the central region than in the proximal part of rat small intestine. No significant differences were observed in the rate of I- uptake from the serosal side. These studies suggest that mucosal I- permeability might determine the direction of net I- intestinal transport and that cytosolic Ca2+ may be a physiological regulator of intestinal I- transport.     

A soluble flavonoid-glycoside, alphaG-rutin, is absorbed as glycosides in the isolated gastric and intestinal mucosa

We investigated the absorption and metabolism of the highly soluble quercetin glycoside alphaG-rutin, a glucose adduct of insoluble rutin, using the isolated mucosa of the rat stomach and intestines equipped with the Ussing chamber. alphaG-rutin and rutin appeared in the serosal sides of the gastric body and all the intestinal mucosa after the addition of alphaG-rutin (1 mM) to the mucosal fluid. The degree of alphaG-rutin appearance was much lower in the gastric fundus than in the other parts. Quercetin was not found in the mucosal fluid of any mucosal specimen. The concentrations (microM) of alphaG-rutin and rutin in the serosal fluid as a result of transport from the mucosal side increased time-dependently and linearly with mucosal alphaG-rutin concentration (1, 10 or 100 mM). The highest transport was shown in the ileal mucosa. These results indicate that alphaG-rutin is partly hydrolyzed to rutin through the intestine and absorbed as such.     

Adriamycin-liposome interactions. A magnetic resonance study of the differential effects of cardiolipin on drug-induced fusion and permeability

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.     

The splanchnic metabolism of flavonoids highly differed according to the nature of the compound

The absorption and splanchnic metabolism of different flavonoids (namely quercetin, kaempferol, luteolin, eriodictyol, genistein, and catechin) were investigated in rats after an in situ perfusion of jejunum plus ileum (14 nmol/min). Net transfer across the brush border ranged widely according to the perfused compound (from 78% for kaempferol to 35% for catechin). This variation seems linked to the lipophilicity of a given flavonoid rather than to its three-dimensional structure. Except for catechin, conjugated forms of perfused flavonoids were also detected in the intestinal lumen, but the extent of this secretion depended on the nature of the perfused compounds (52% for quercetin to 11% for genistein). For some of the perfused aglycones, biliary secretion was an important excretion route: 30% of the perfused dose for genistein but only 1% for catechin. Thus the splanchnic metabolism of flavonoid is controlled by several factors: 1) the efficiency of their transfer through the brush border, 2) the intensity of the intestinal secretion of conjugates toward the mucosal and serosal sides, respectively, and 3) the biliary secretion of conjugates. These data suggested that the splanchnic metabolism of perfused flavonoids depends on the nature of the compound considered, which in turn influences their availability for peripheral tissues.     

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.