Monosaccharide Transport System Research Articles

Sigmoidal kinetics define porcine intestinal segregation of electrogenic monosaccharide transport systems as having multiple transporter population involvement.

Kinetic characterization of electrogenic sodium‐dependent transport in Ussing chambers of d‐glucose and d‐galactose demonstrated sigmoidal/Hill kinetics in the porcine jejunum and ileum, with the absence of transport in the distal colon. In the jejunum, a high‐affinity, super‐low‐capacity (Ha/sLc) kinetic system accounted for glucose transport, and a low‐affinity, low‐capacity (La/Lc) kinetic system accounted for galactose transport. In contrast, the ileum demonstrated a high‐affinity, super‐high‐capacity (Ha/sHc) glucose transport and a low‐affinity, high‐capacity (La/Hc) galactose transport systems. Jejunal glucose transport was not inhibited by dapagliflozin, but galactose transport was inhibited. Comparatively, ileal glucose and galactose transport were both sensitive to dapagliflozin. Genomic and gene expression analyses identified 10 of the 12 known SLC5A family members in the porcine jejunum, ileum, and distal colon. Dominant SGLT1 (SLC5A1) and SGLT3 (SLC5A4) expression was associated with the sigmoidal Ha/sLc glucose and La/Lc galactose transport systems in the jejunum. Comparatively, the dominant expression of SGLT1 (SLC5A1) in the ileum was only associated with Ha glucose and La galactose kinetic systems. However, the sigmoidal kinetics and overall high capacity (Hc) of transport is unlikely accounted for by SGLT1 (SLC5A1) alone. Finally, the absence of transport and lack of pharmacological inhibition in the colon was associated with the poor expression of SLC5A genes. Altogether, the results demonstrated intestinal segregation of monosaccharide transport fit different sigmoidal kinetic systems. This reveals multiple transporter populations in each system, supported by gene expression profiles and pharmacological inhibition. Overall, this work demonstrates a complexity to transporter involvement in intestinal electrogenic monosaccharide absorption systems not previously defined.

Transport-limited sucrose utilization and neokestose production by Phaffia rhodozyma

Growth of an astaxanthin hyper-producing strain of Phaffia rhodozyma on sucrose is accompanied by the accumulation of glucose and fructose in the medium due to the limited capacity of the corresponding monosaccharide transport system or systems. This is accompanied by the production of the trisaccharide neokestose by transglycosylation reactions.

Monosaccharide Transport by Eimeria tenella Sporozoites

Eimeria tenella sporozoites were incubated in the presence of 3 different [14C]-labeled sugars; D-glucose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose. The initial velocity, Vi, of uptake of D-glucose and 2-deoxy-D-glucose was similar, 41 micrograms/10(10) sporozoites/min and 46 micrograms/10(10) sporozoites/min, respectively; whereas that for 3-O-methyl-D-glucose was significantly lower, 17 micrograms/10(10) sporozoites/min. Initial velocity studies also revealed that glucose uptake was a saturable event, with an apparent KT of 20 mM and an apparent Vmax of 312 micrograms/10(10) sporozoites/min. Uptake was unaffected by exogenous sodium levels or the presence of ouabain. However, 0.1 mM phloretin significantly inhibited glucose uptake. Thus, it would appear that E. tenella sporozoites possess a Na-independent, phloretin-sensitive, carrier-mediated monosaccharide-transport system.

Monosaccharide transport into hemocytes of a sipunculan worm Themiste dyscrita.

The hemerythrin-containing blood cells, or hemocytes, of the sipunculan worm Themiste dyscrita were found to have a stereospecific and nonconcentrative monosaccharide transport system. The transport system transferred both D-glucose and 3-O-methyl-D-glucose (3-OMG), and transport into cells by this system was rapid, reaching 50% equilibrium in approximately 20 s at 10 degrees C with an initial concentration gradient of 0.1 mM; the contribution to total uptake by simple diffusion was very small. 3-OMG uptake showed saturation kinetics with a low half-saturation constant (Km less than or equal to 0.1 mM). The uptake of labeled 3-OMG by the hemocytes was strongly inhibited by unlabeled 3-OMG, 2-deoxy-D-glucose, alpha- and beta-D-glucose, D-galactose, and D-mannose. It was moderately inhibited by D-xylose, only slightly by alpha-methyl-D-glucoside and D-fructose, and uninhibited by sucrose, L-glucose, or D-sorbitol. Phloretin was more potent than phloridzin in blocking entry of 3-OMG. Cytochalasin B did not bind tightly to the T. dyscrita transporter and was not a potent inhibitor of transport; it half-maximally inhibited 3-OMG transport at 0.1 mM. Therefore, despite some differences the data suggest functional similarities in the mechanism of monosaccharide transport into blood cells of mammals and this invertebrate.

Hamster intestinal disaccharide absorption: extracellular hydrolysis precedes transport of the monosaccharide products.

Hydrolase-related transport was re-investigated in hamster small intestine by the tissue accumulation method. The Na+-dependent, phlorizin-sensitive monosaccharide transport system saturates with 30 mM-D-glucose. According to the hydrolase-related transport hypothesis, additional glucose units will be taken up if they are given in the form of a disaccharide susceptible to hydrolysis. But in experiments with [14C]sucrose we found no evidence for any such surplus glucose uptake. The uptake of 14C label from sucrose was abolished by using Tris, a strong inhibitor of sucrase, by adding competitive inhibitors of the D-glucose transport system (D-glucose, beta-methyl-D-glucopyranoside or phlorizin), and by substituting Li+ for the Na+ in the incubation medium. Glucose and fructose derived from sucrose did not enter the tissues in equimolar amounts: the glucose moiety was taken up much faster. We conclude that in hamster intestine there is no evidence for the existence of hydrolase-related transport with sucrose as the monosaccharide donor. The enzymatic hydrolysis of sucrose and the transport of its products, glucose and fructose, are two distinct events, acting sequentially.

Mechanisms of Organic Acid and Monosaccharide Transport in the Kidney of the Brush-tailed Possum, Trichosurus vulpecula

The mechanisms of organic acid and monosaccharide transport in the kidney of T. vulpecula were investigated using the renal cortical slice preparation. The kinetics, the sensitivity to inhibitors of metabolism and sodium transport, and the specificity of the concentrative uptake of p-amino-hippurate and alpha-methyl-D-glucoside were examined and compared with published results from studies in eutherian mammals. Some minor differences between metatherians and eutherians were noted with regard to the specificities of the renal slice uptake systems. Penicillin G, a competitor of organic acid transport in eutherian kidneys, did not interact with the marsupial uptake system, and sodium acetate, which stimulates transport in other mammals, inhibited p-aminohippurate uptake in the slice of the possum kidney. 2-Deoxy-D-glucose, which interacts with the phlorizin-sensitive monosaccharide transport system in the dog, rat and rabbit kidney, had no effect on alpha-methyl-D-glucoside uptake in the possum, and the magnitude of the interaction of D-fructose resembled that reported in the dog and rat but was greater than the inhibition reported in the rabbit. D-Glucuronic acid and D-glucuronic acid lactone inhibited alpha-methyl-D-glucoside uptake in the possum but had no effect on uptake in rat renal slices. In consideration of the reported variability of these parameters between different classes of eutherians, it was concluded that the primary mechanisms involved in organic solute transport by the proximal nephron of metatherians and eutherians were not significantly different.

Sugar Transport by Larval and Adult Proterometra macrostoma (Digenea) in Relation to Environmental Factors

Monosaccharide transport systems were identified in rediae and cercariae of Proterometra macrostoma. Glucose transport by cercariae in vitro was accomplished by the bodies, but tails absorbed glucose by diffusion alone. No sugar transport system was detected in adults obtained from laboratory infections of sunfish. Temperature of Elkhorn Creek where infected snails (Goniobasis livescens) were found varies from 28 C in summer to 5 C in winter. Glucose transport by both larval stages was optimal between 30 and 25 C and was negligible below 15 C. Transport by rediae was activated by Na+ and was maximal at 50 mM, the approximate Na+ concentration of snail hemolymph. The redial glucose transport system was inhibited by various sugars and was more sensitive to phlorizin than to phloretin. Rediae accumulated glucose and nonmetabolized 3-O-methyl-glucose against apparent concentration gradients, indicating an active transport system. In contrast, glucose transport by "embryonic" cercariae was completely inhibited by 10 mM Na+. Transport by cercariae aged in creek, water 6 hr became relatively insensitive to Na+, and were less permeable to glucose by diffusion. The cercarial system differed in other characteristics from the redial system, including its high sensitivity to phloretin relative to phlorizin and its lower Vmax. The cercarial system apparently functioned only as a facilitated diffusion system that served to move sugar across the tegument down its chemical gradient. Development of P. macrostoma thus showed functional transformations of the tegument that may be subject to regulation by environmental factors.

Glucose transport in murine macrophages: in vitro characterization of the monosaccharide transport system of the thioglycollate-elicited mouse peritoneal macrophage.

Glucose transport in murine macrophages: in vitro characterization of the monosaccharide transport system of the thioglycollate-elicited mouse peritoneal macrophage.

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain extemal.

In vitro actions of glucocorticoids on murine macrophages: effects on glucose transport and metabolism, growth in culture, and protein synthesis.

In vitro exposure of homogeneous monolayer cultures of thioglycollate-elicited mouse peritoneal macrophages to glucocorticoid hormones for up to 96 hr produced a progressive, dose-related inhibition of cell growth and protein synthesis. The reductions in cell protein, incorporation of radiolabeled leucine, and intracellular water space were specific to steroids with glucocorticoid action, with half-maximal inhibition occurring at about 10(-9) M for dexamethasone. Similar dose-related, glucocorticoid-specific decreases in net glucose uptake, lactate production, and 14CO2 production were observed that were only partially explained by the decrease in cell mass. A decrease in the monosaccharide transport system Vmax was also noted but could be completely accounted for by the reduction in cell surface area and the resulting proportional loss in transport sites. No effect on transport system affinity could be demonstrated. The suggestion was made that such long-term culture of macrophages in the presence of glucocorticoids reverses the biochemical and functional changes that comprise the "activated" state of mononuclear phagocytes.

Characterization and regulation of galactose transport in Neurospora crassa.

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.

Distribution of sulfhydryl groups in intestinal brush border membranes: Localization of side-chains essential for glucose transport and phlorizin binding

1. 1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4′-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5′-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4′-dithiodipyridine react with 5,5′-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 μM of the penetrating organomercurial p- chloromercuribenzoate , but was about ten times less sensitive to the poorly permeating p- chloromercuriphenylsulfonate . In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol l-glutathione (reduced form) could only revert the inhibition in leaky membranes.

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55 000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3·10 −7 M, a value which is similar to that reported for the transport system in the intact erythrocyte.

Movements of monosaccharides between blood and tissues of vascularly perfused small intestine.

1. A method involving the analysis of pulse transients of the vascular concentrations of test sugar and extracellular marker has been used to study the movements of non-metabolized sugars between the cells and vascular fluid of the vascularly perfused small intestine of R. ridibunda. Reasons are given for supposing that the method does properly measure the net entry of sugars into a cellular compartment that includes at least the epithelium. 2. It is found that while the glucose analogues, 3-O-methyl-D-glucose (3MG) and 2-deoxy-D-glucose (2)DG) are able to enter a cellular compartment when they are added to the vascular bed, alpha-methyl-D-glucoside (alpha MG) is able to enter the compartment only at a very slow rate from the vascular bed. In contrast, 3MG and alaph MG are well absorbed from the lumen whereas the inward permeability of 2DG across the lumen face of the epithelium is very low and, unlike 3MG and alpha MG, is not influenced by the presence in the lumen of other transported sugars. 3. The presence of phlorizin in the intestinal lumen increases the flux of alpha MG and of 3MG in the direction vascular bed--bulk phase of lumen. Reasons are given for supposing that the movement of sugars from the vascular bed into the lumen may involve a cellular route but occurs, at least in part, through a paracellular, extracellular route. 4. The exit of monosaccharides that have been loaded previously into the epithelium either from the lumen or from the vascular bed has been investigated. 3MG washes out rapidly into the vascular bed and the exit is stimulated by the addition of D-glucose to the intestinal lumen and by the addition of 2DG, but not alpha MG, to the arterial infusate. In contrast, alpha MG, loaded into the epithelium from the lumen, washes out of the cells only slowly into the vascular bed, so that even with high rates of vascular perfusion alpha MG accumulates within the tissue. The sustained accumulation of alpha MG implies that not only is the permeability for exit into the blood restricted for this sugar, but also the permeability is low across the brush border in an outward direction, cell to lumen ('lobster pot effect'). The wash-out of 3MG into the vascular effluent is sufficently rapid that only in the absence of vascular perfusion is 3MG accumulated within the tissue. 5. The contrasting properties of the monosaccharide transport systems accessible from the intestinal lumen and the vascular bed respectively are discussed in relation to the problems of epithelial transport of monosaccharide.

Temperature dependence of the apparent affinity and the maximum velocity of the membrane-bound monosaccharide transport system in the yeast Rhodotorula gracilis

Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with d-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant K T, increased when transport velocity was stimulated by temperature (15–30°C) and decreased when the rate of uptake was reduced at temperatures above 30°C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28–30°C this was not observed in heat-treated cells (at either 37 or 45°C). In heattreated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.

The monosaccharide transport system of the human erythrocyte. Solubilization and characterization on the basis of cytochalasin B binding.

The monosaccharide transport system of the human erythrocyte. Solubilization and characterization on the basis of cytochalasin B binding.

Monosaccharide transport system of the human erythrocyte. Identification of the cytochalasin B binding component.

Monosaccharide transport system of the human erythrocyte. Identification of the cytochalasin B binding component.

The activity of the erythrocyte monosaccharide transport system is not correlated with crosslinkage of band 3 protein

We have prepared sealed vesicles from erythrocyte membranes in which 90% of band 3, the predominant intrinsic polypeptide, had been crosslinked by Cu 2+/o-phenanthroline treatment. The initial rates of L-sorbose uptake via the monosaccharide transport system in the vesicles varied from 72 to 176% of the rates for the corresponding control vesicles with uncrosslinked band 3. These results suggest that band 3 does not participate in monosaccharide transport.

Monosaccharide transport in protein-depleted vesicles from erythrocyte membranes.

Treatment of human erythrocyte membranes with dilute alkali (pH 11.5) generates sealed, protein-depleted vesicles that can be isolated by density gradient centrifugation. The vesicles are 0.5 to 2.0 micrometers in diameter, and their membranes are predominantly oriented inside-out. The vesicles lack protein bands 1, 2, 5, and 6 (nomenclature of Steck, T.L. (1974) J. Cell Biol. 62, 1-19) of the erythrocyte membrane. L-Sorbose, a substrate of the monosaccharide transport system in erythrocytes, is transported by the vesicles. Based on comparisons between erythrocytes and vesicles with regard to specificity, temparture dependence, and effects of inhibitors, we conclude that sorbose uptake into the vesicles occurs by way of the monosaccharide transport system. The specific activity of the transport system in vesicles, as determined by initial rate measurements of sorbose uptake, averaged 58% of that in erythrocytes. This finding indicates that the major polypeptides of Bands 1, 2, 5, and 6 do not play an obligatory role in monosaccharide transport.

Sucrose absorption by the rat small intestine in vivo and in vitro.

1. The absorption of glucose and fructose derived from sucrose has been studied using in vitro and in vivo loops of the rat jejunum.2. At low sucrose concentrations (1 and 10 mM) glucose appeared in the serosal compartment of the in vitro preparation at a faster rate than fructose, but at high sucrose concentrations (50 and 100 mM) the rates of serosal transfer of the two sugars were similar. Glucose and fructose appeared in the mucosal compartment, with the rate of fructose appearance exceeding that of glucose, at all the sucrose concentrations studied.3. Phlorizin (5 x 10(-5)M) added to the mucosal medium of the in vitro preparation abolished the serosal transfer of glucose derived from 50 mM sucrose, and reduced that of fructose by 75%.4. In the absence of sodium ions, the in vitro preparation failed to transfer glucose and fructose derived from 50 mM sucrose, into the serosal compartment.5. Glucose was actively accumulated in the whole gut wall of the in vivo preparation to concentrations higher than those in the plasma at 50 and 100 mM, but not at 10 mM sucrose concentrations. Fructose was also actively accumulated to about half the extent of glucose, but reached tissue concentrations greater than those in the plasma, at each sucrose concentration.6. The whole wall concentrations of glucose and fructose derived from sucrose added to the lumen continued to rise when the blood supply to the in vivo preparation was terminated.7. No increase in the in vivo whole wall concentrations of glucose and fructose were detected when sucrose was added to the lumen together with concentrations of glucose sufficient to saturate the monosaccharide transport systems.8. The results favour the view that disaccharide hydrolysis and resulting hexose transfer are sequential, separate events.