Phloretin-like Action of Bioflavonoids on Sugar Accumulation Capability of Isolated Intestinal Cells

Flavanones and flavones are structural analogues of phloretin. Like phloretin they inhibit the non-Na⁺-dependent, facilitated diffusion transport system for sugars associated with the lateral serosal boundary of intestinal epithelial cells. The degree of inhibition varies with the extent and position of hydroxylation of the flavonoid nucleus. Flavones are more potent than corresponding flavanones. Tri- and tetrahydroxylated forms are more inhibitory than similar penta- and hexahydroxylated molecules. With one exception, none of the 18 flavonoids tested has secondary effects as metabolic inhibitors, as does phloretin. Inhibition of the passive sugar transport system with flavonoids allows the concentrative Na⁺-dependent sugar transport system to establish a better concentration gradient than is observed in untreated cells. The degree of gradient enhancement is proportional to the degree of inhibition of the sugar “leak.” The flavonoid glycosides, which can be considered as phlorizin analogues, also inhibit the non-Na⁺-dependent sugar carrier, but less well than corresponding nonglycosylated agents. Only one of the glycosides inhibits the Na⁺-dependent transport system, and much less potently than phlorizin.     

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.     

Serosal and mucosal facilitated transport of urea in urinary bladder of of Bufo bufo: evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.     

Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Active transport of sugars by the intestine of snail (Cryptomphalus hortensis Müller).

Sugar transport by sacs of everted intestine of snail have been measured in vitro at 30 degrees C. D-galactose, D-glucose and 3-O-methylglucose were actively transported against a concentration gradient from the mucosal to the serosal compartment. The transport of these sugars was inhibited by 5 times 10(-8) to 10(-6) M phlorizin. L-arabinose was also accumulated in the serosal compartment against a concentration gradient; in this case, transport was not affected by phlorizin. The snail intestine did not show any ability for D-fructose active transport but there was a clear uptake of this sugar by the tissue. The O2 uptake of the snail intestine was not significantly affected by the presence of either sugars or phlorizin.     

The effect of BaCl2 on intestinal sugar transport in the rat in vitro

The effect of BaCl2 on galactose transport across isolated rat small intestine has been investigated. The addition of 5 mM BaCl2 or theophylline (3 mM) to the bathing solutions increased cell water free sugar accumulation and decreased mucosal to serosal sugar fluxes. However the effects of BaCl2 were smaller than those induced by theophylline. Removal of Ca2+ from the bathing solutions did not modify the response to BaCl2, though the response to theophylline was partially reduced. In the presence of 0.1 mM trifluoperazine, both theophylline and BaCl2 were without effect on sugar transport. These findings are discussed in terms of an effect of Ba2+ on intestinal smooth muscle tone.     

The action of inhibitors of sugar transport phlorizin, phloretin and cytochalasin B in model systems

Phlorizin, phloretin and cytochalasin B are known to be specific sugar transport inhibitors. A study was made of their effects on the carbohydrate-protein interaction in solution as a model system for examining the initial steps of sugar membrane transport. Glycogen precipitation by concanavalin A is inhibited only by alpha-methylmannoside, whereas both phlorizin and phloretin inhibit interactions between hexokinase and glucose, and between glucose-6-phosphate dehydrogenase and glucose-6-phosphate. Cytochalasin B was found to exert no effect on both the concanavalin A--glycogen interaction and the enzyme reactions investigated. The data obtained in the model system examination may suggest that the sites of glucose and cytochalasin binding are, respectively, spatially uncoupled.     

Inhibition of the intestinal transport of uracil by hexoses and amino acids

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.     

Transport of 3-O-methyl D-glucose and beta-methyl D-glucoside by rabbit ileum.

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereo-specific affinity and (b) are common. The apparent affinity constants at the brush-border indicate that sugars are selected in the order, beta-methyl glucose greater than D-galactose greater than 3-O-methyl glucose, (the Km values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the Kt values for Na+ activation of sugar entry across the brush-border are: 27, 25, and 140 mequiv. for beta-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na+, water, sugar and membrane-binding groups are all factors which determine selective affinity. In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer (Na) or (sugar) is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)2. This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion. Theophylline reduces the serosal border permeability to beta-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increase the intracellular accumulation of these sugars. The permeability of the serosal border to beta-methyl glucose entry is lower than permeability of the serosal border to beta-methyl glucose exit, which suggested that beta-methyl glucose may be convected out of the cell across the lateral serosal border.     

Effect of serotonin on D-galactose transport across the rabbit jejunum

The patterns of storage and release of serotonin found in the enterochromaffin cells of the intestinal mucosa suggest that this hormone may be an important modulator of intestinal functions. Serotonin has been shown to produce secretion of water and electrolytes in rabbit ileum, but the hormone does not appear to interact significantly with other transport processes. The aim of the present study was to determine the effect of serotonin on D-galactose absorption in rabbit jejunum. The results obtained show that serotonin (10(-8) and 10(-6)M) partially reduced (by 20 and 40% respectively) D-galactose uptake across the mucosal border. This effect was concentration-dependent, and it seemed to be caused by the inhibition of Na+-dependent sugar transport. Methysergide, an antagonist of serotonin which binds with receptor 2 of serotonin, blocked the effect of serotonin. These findings suggest that serotonin may act as a regulator of sugar intestinal absorption, and that this serotonin regulation could be mediated by a direct or indirect action of the complex serotonin-receptor, which may inhibit the Na+-dependent transport system of sugars located in the brush-border membrane.     

Glucose transport across plasma membrane in human platelets

Studies have been carried out in the presence of 2-deoxyglucose, by utilizing a technique of platelet rapid filtration. Kinetic data suggest that glucose uptake across plasma membrane is the rate limiting step in its utilization. 2-deoxyglucose is transported by facilitated diffusion. L-glucose is transferred at only 1/1200 of the rate of glucose. Transport system shows high affinity for substrate. Transport is inhibited by cytochalasin B, phloretin and N-ethylmaleimide. Cytochalasin E does not affect 2-deoxyglucose uptake. Diamide can have activating or inhibitory effect. t-Butyl hydroperoxide is always activating. Insulin has no effect on rate transport. D-glucose, 3-O-methylglucose, non radioactive 2-deoxyglucose and D-mannose are strong competitors, whereas D-galactose and D-fructose compete weakly with 2-deoxyglucose transport.     

A Na+-independent,phloretin-sensitive monosaccharide transport system in isolated intestinal epithelial cells

A monosaccharide transport system in addition to the active Na+-dependent system characteristic of the brush border surface of vertebrate intestinal tissue has been identified in isolated chick intestinal epithelial cells. The newly described system differs in several characteristics from the Na+-dependent process, including function in the absence of Na+; a high sensitivity to phloretin, relative insensitivity to phlorizin; different substrate specificity; and a very high KT and Vmax. The system apparently functions only in a facilitated diffusion manner so that it serves to move monosaccharide across the cell membrane down its chemical gradient. An appreciable fraction of total sugar efflux occurs via the Na+-independent carrier from cells which have accumulated sugar to a steady state. Phloretin selectively blocks this efflux so that a normal steady-state sugar gradient of seven- to eightfold is transformed to a new steady-state gradient which is greater than 14-fold. Locus of the new system is tentatively ascribed to the serosal cell surface where it would serve for monosaccharide transfer between enterocyte and lamina propria of the villus.     

Effect of phloretin and phloridzin on properties of digestion and absorption in the rat small intestine

Experiments in vitro on everted sacs of rat small intestine have shown that phloretin (an inhibitor of basolatheral glucose GLUT2 transporter) added from mucosal side of the sacs decreases release of glucose from enterocytes into serosal fluid without changing glucose accumulation in tissue of the preparations. Addition of phloridzin (an inhibitor of Na⁺ and glucose co-transporter SGLT1) from mucosal side inhibited both glucose accumulation in the tissue and its release into serosal fluid. Unspecific effects of phloretin and phloridzin on activities of several digestive enzymes (in particular, alkaline phosphatase, amino peptidase, and glycyl-L-leucine dipeptidase) has been revealed in homogenates of the rat small intestine mucosa. In chronic experiments on rats, absorption of glycine from the isolated small intestinal loop was inhibited in the presence of phloretin in perfusate. The obtained results indicate that the experimental approach of inhibition of glucose absorption by phloretin used from mucosal side in vitro appears to give a significant overestimation of contribution of facilitated diffusion (with participation of the GLUT2 transporter inserted in the apical enterocyte membrane) to glucose transport across this membrane. Thus, the role of the GLUT2 transporter in the mechanism of glucose absorption in the small intestine under its physiological conditions does not seem to be as great as it is thought by the authors of the recently proposed hypothesis.     

Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues.

Cholecystokinin and analogues increased the uptake of 2-deoxy-D-glucose and 3-O-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The cholinergic analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretion, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca2+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.     

IC50 of trifluoperazine and RMI 12330A for the theophylline and phloretin-induced increase in intestinal D-galactose accumulation

The effect of trifluoperazine and RMI 12330A on D-Galactose accumulation was studied in isolated rat intestinal mucosa. Both drugs inhibited the theophylline and phloretin-induced increase in tissue sugar accumulation in a concentration-dependent fashion, with IC50 values close to 10(-6) M. These findings suggest that calmodulin might mediate the theophylline and phloretin actions on galactose transport in intact rat ileum.     

Active transport of D-xylose in the isolated small intestine of the bullfrog

Fluxes of D-xylose-1-C¹⁴ (xylose) across the wall of the isolated intestine of the bullfrog were studied. When sodium was the principal cation in the mucosal bathing fluid, the transport rate of xylose from the mucosa to the serosa was about 5 times greater than the transport rate from the serosa to the mucosa, indicating an active intestinal transport for this sugar. With potassium as the principal cation on the mucosal side, the transport rate of xylose from the mucosal to the serosal compartment is reduced about 5 to 6 times without appreciable change in the serosal to mucosal transport. The asymmetry was also considerably reduced when ouabain was added to the mucosal and serosal compartments. The data confirm the in vitro and in vivo observations indicating active transport of xylose and are also in accord with the earlier findings that active transport of sugars in the intestine is dependent upon the presence of sodium ions in the mucosal compartment and is inhibited by cardioactive steroids. Since the chemical constitution of xylose does not meet the requirements which were hitherto considered necessary for active transport of sugars in the intestine, this structural requirement has to be revised.     

Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses

Lysosomal membrane vesicles isolated from rat liver were exploited to analyze the mechanism of glucose transport across the lysosomal membrane. Uptake kinetics of [14C]D-glucose showed a concentration-dependent saturable process, typical of carrier-mediated facilitated transport, with a Kt of about 75 mM. Uptake was unaffected by Na+ and K+ ions, membrane potentials, and proton gradients but showed an acidic pH optimum. Lowering the pH from 7.4 to 5.5 had no effect on the affinity of the carrier for the substrate but increased the maximum rate of transport about 3-fold. As inferred from the linearity of Scatchard plots, a single transport mechanism could account for the uptake of glucose under all conditions tested. As indicated by the transstimulation properties of the carrier, other neutral monohexoses, including D-galactose, D-mannose, D- and L-fucose were transported by this carrier. The transport rates and affinities of these sugars, measured by the use of their radiolabeled counterparts, were in the same range as those for D-glucose. Pentoses, sialic acid, and other acidic monosaccharides including their lactones, aminosugars, N-acetyl-hexosamines, and most L-stereoisomers, particularly those not present in mammalian tissues, were not transported by this carrier. Glucose uptake and transstimulation were inhibited by cytochalasin B and phloretin. The biochemical properties of this transporter differentiate it from other well-characterized lysosomal sugar carriers, including those for sialic acid and N-acetylhexosamines. The acidic pH optimum of this glucose transporter is a unique feature not shared with any other known glucose carrier and is consistent with its lysosomal origin.     

Sodium dependence of intestinal active transport of sugars in snail (Cryptomphalus hortensis Müller).

Active transport of sugars (D-galactose, D-glucose, 3-0-methylglucose and L-arabinose) by sacs of everted intestine of snail (Cryptomphalus hortensis) was strongly inhibited, but not abolished, when all Na from the bathing solutions was substituted by K, Tris, Mg or Ca. Absence of Na produced also a marked inhibition of O2 consumption by the tissue. Omission of other cations (K, Ca, Mg), substituted by Tris, did not affect sugar transport or O2 uptake. Sodium seems to play a specific and important but not indispensable r?le in sugar active transport by snail intestine. Since anaerobiosis did not affect sugar transport, this Na role is independent of its effect on O2 uptake.     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.