Specific D-glucose transport in sarcolemma vesicles.

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate D-glucose in preference to L-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific D-glucose uptake by these vesicles is a saturable rpocess, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both D- and L-glucose into the vesicles. Stereospecific D-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca2+ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific D-glucose transport. Apparent uptake of both D- and L-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

The biguanide inhibition of d-glucose transport in membrane vesicles from small intestinal brush borders

Biguanides inhibit d-glucose uptake in vesicles from small-intestinal brush border membranes. Evidence is presented that this inhibition is due to a reduced concentration of Na⁺ in the microenvironment of the carrier(s) for d-glucose. Biguanides do not inhibit the uptake of either d-fructose or l-glucose.     

Interactions between Na+-dependent uptake of d-glucose,phosphate and l-alanine in rat renal brush border membrane vesicles

d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na⁺-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the $\text{V}$ value of the phosphate uptake was decreased, whereas the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na⁺-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na⁺ gradient insofar as the rate of the Na⁺ uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions.     

Glucose transport in isolated brush-border and lateral-basal plasma-membrane vesicles from intestinal epithelila cells

Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, i.e. alkaline phosphatase (EC 3.1.31), was clearly separated from the marker for the lateral-basal plasma membrane, i.e. the (Na⁺, K⁺)-ATPase (EC 3.6.1.3). A microvillus membrane fraction was obtained with a high specific activity of alkaline phosphatase (an 8-fold enrichement over the starting homogenate). The lateral-basal plasma membrane fraction contained (Na⁺, K⁺)-ATPase (5-fold over homogenate) with some alkaline phosphatase (2-fold over homogenate).Glucose transport was studied in both membrane fractions. The uptake of d-glucose was much faster than that of l-glucose in either plasma membrane, d-Glucose uptake could be accounted for completely by its transport into an osmotically active space. Interestingly, the characteristics of the glucose transport of the microvillus membrane were different from those of the lateral-basal plasma membrane. In particular: Na⁺ stimulated the d-glucose transport by the microvillus membrane, but not by the lateral-basal plasma membrane. In addition, the glucose transport of the microvillus membrane was much more sensitive to phlorizin inhibition than that of the lateral-basal plasma membrane.These experiments thus provide evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose between the two types of plasma membrane of the intestinal epithelial cell.     

Glucose transport in thymocyte plasma-membrane vesicles

Calf-thymocyte membrane vesicles, prepared by hypotonic lysis and homogenization, were isolated by standard centrifugal techniques designed for enrichment of plasma membrane. At 20°C, these vesicles equilibrated with d-glucose and 3-O-methyl-d-glucose more rapidly than with l-glucose. About 25% of the equilibrium d-sugar space (6 μl/mg protein) was very slowly penetrated by l-glucose ( ). The time course of d-sugar accumulation in excess of l-glucose accumulation indicated that this space equilibrated with d-glucose and 3-O-methyl-d-glucose with half-times of approximately 0.2–0.4 min. The remainder of the equilibrium d-sugar space (about 75%) appeared equally accessible to both glucose isomers ( to 5 min). This was confirmed in studies of efflux from preloaded vesicles, where the d-glucose space fell with a short half-time (0.2 min) to the l-glucose space, after which the two isomers exited with the same half-time. Addition of sucrose to increase osmolarity reduced both spaces (specific and non-specific) in a manner which indicated that little if any of the vesicle sugar was bound. This was confirmed by the fact that equilibrium glucose space was independent of glucose concentration and by the fact that vesicles immediately lost their sugar when diluted with water at 0°C. These data indicate the presence of two vesicle types, discriminant and indiscriminant as regards transport of the glucose isomers. Entry of d-glucose into the discriminant (stereospecific) vesicles was temperature sensitive (Q₁₀ > 2), saturable (K_m 2 mM), and was inhibited by phloretin (K_i < 200 μM), N-ethylmaleimide (K_i < 10 mM) and cytochalasin B (K_i < 2 μM), suggesting that these vesicles contain the plasma-membrane glucose carrier. Entry of l- and d-glucose into the indiscriminant vesicles showed none of these properties. The equilibrium-exchange K_m and V were about five times the entry K_m and V, indicating the substrate loading greatly facilitates carrier translocation, at least in the outward direction.     

Glucose transport into spinach chloroplasts

The uptake of radioactively labeled hexoses and pentoses into the sorbitol-impermeable (3)H(2)O space (the space surrounded by the inner envelope membrane) of spinach (Spinacia oleracea L.) chloroplasts has been studied using silicone layer filtering centrifugation. Of the compounds tested, d-xylose, d-mannose, l-arabinose, and d-glucose are transported most rapidly, followed by d-fructose and l-arabinose. The rate of l-glucose uptake is only about 5% of that of d-glucose.The transport of d-glucose and d-fructose shows saturation characteristics, the K(m) for d-glucose was found to be about 20 mm. All sugars transport and phloretin inhibit d-glucose transport. The temperature dependency of d-glucose transport appears to have an activation energy of 17 kcal/mol.With low external concentrations of d-glucose the transport into the chloroplasts proceeds until nearly the external concentration is reached inside the chloroplasts.d-glucose transport is inhibited by high d-glucose concentrations in the medium. It is concluded that d-glucose and other hexoses are transported by carrier-mediated diffusion across the inner envelope membrane. This transport is similar to the transport of d-glucose into erythrocytes.     

An improved method of reconstitution of adipocyte glucose transport activity

The glucose transport activity of rat epididymal fat cells was reconstituted into egg lecithin liposomes with a high degree of reproducibility. The activity was solubilized with 20 mm sodium cholate in Buffer B (10 mm Tris-HCl, pH 7.5). After elimination of small molecules by gel filtration, the transport activity was incorporated into egg lecithin liposomes (Sigma, Type IX-E, homogeneously dispersed into Buffer B) by sonication (5 s), freezing (?70°C), thawing, and a second sonication (5 s). The sonication was done in a 16.8-mm polystyrene test tube (Sarstedt, 55-468) placed in a cup horn (from Heat Systems Ultrasonics) connected to a Branson's sonicator (W-185) at setting No. 3 (70 W of output). The optimum sample size was 80 μl, and the optimum clearance between the test tube and the sonicator horn was 2–3 mm. The concentration of egg lecithin at the reconstitution step was 25 mg/ml, and that of the microsomal protein was approximately 0.3–0.5 mg/ml. The glucose transport activity of reconstituted liposomes was assayed by incubating the latter with a mixture of d-[³H]glucose and l-[¹⁴C]glucose. The incubation was terminated by the addition of HgCl₂, and the reaction mixture was filtered with a Millipore filter (GSWP). The difference in the rates of uptake of d-glucose and l-glucose was regarded as representing the carrier-mediated glucose transport activity. The results of the assay indicated that the glucose transport activity could be reconstituted in a highly reproducible manner. The reconstituted activity was proportional, within a limit of experimental error, to the amount of protein used for reconstitution and was almost completely blocked by cytochalasin B, phloretin, or HgCl₂. However, a small amount of d-glucose was found to bind with the egg lecithin preparation.     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Effect of hyperglycemia on d-glucose transport across the brush-border and basolateral membrane of rat small intestine

Experimental hyperglycemia leads to an increase in the capacity of the rat small intestine to absorb glucose. This effect occurs within hours from the onset of hyperglycemia and is thought to involve an induction of glucose transport in the brush-border and/or basolateral membrane of the intestinal epithelium. We devised a protocol for the simultaneous preparation of brush-border vesicles and basolateral vesicles from rat small intestine to determine the locus for the inductioof glucose transporter in hyperglycemic rats. A 6 h period of intravenous infusion with a 30% glucose solution had no effect on the initial rate of glucose uptake across jejunal or ileal brush-border vesicles when measured in the absence of a Na⁺ gradient, suggesting that enhanced glucose uptake is not dependent on an increase in the number of Na⁺-dependent secondary active glucose transporters in the brush-border. Hyperglycemia did not effect the rate of glucose uptake across ileal basolateral vesicles but did cause a 78% increase in the initial rate of carrier-mediated d-glucose uptake across jejunal basolateral vesicles. The induction of glucose transport in the jejunal basolateral membrane was characterized by a rapid rate of glucose equilibration across the vesicles ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 46}\text{s}$ sorbitol infused controls, 18 s hyperglycemia) and a 75% increase in the V_max for carrier-mediated glucose uptake with no significant change in K_t. When the rats were pretreated with cycloheximide prior to intravenous infusion, the initial rate of d-glucose uptake dropped to 13% of that seen in jejunal basolateral vesicles prepared from untreated rats. These results suggest a rapid turnover rate for the Na⁺-independent glucose transporter in the basolateral membrane of the enterocyte. An increase in the number of functioning glucose transporters in the basolateral membrane may play an important role in the short-term induction of glucose absorption by the jejunum of the hyperglycemic animal.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Decreased Na+-gradient-dependent d-glucose transport in brush-border membrane vesicles from rabbits with experimental fanconi syndrome

The effect of anhydro-4-epitetracycline on sodium gradient-dependent d-glucose transport of rabbit renal brush-border membrane vesicles was studied. The purity of isolated brush-border membrane vesicles as judged by enzyme activities was not different between normal control and anhydro-4-epitetracycline-administered rabbits. There was no difference in estimate of intravesicular volume, either. When NaCl was used for sodium gradient, the overshoot of d-glucose uptake into brush-border membrane vesicles isolated from anhydro-4-epitetracycline-treated rabbits was significantly smaller than that of normal control rabbits. In the cases of NaSCN or Na₂SO₄, the former was also smaller than the latter, but not significantly so. To avoid the possible effect of membrane potential on d-glucose uptake, the voltage-clamp method was applied. Even in the voltage-clamped condition, the overshoot of d-glucose uptake into vesicles from anhydro-4-epitetracycline-treated rabbits was decreased compared to that of normal rabbits. In vitro incubation of brush-border membrane vesicles with 20 mM anhydro-4-epitetracycline caused no alteration in sodium gradient-dependent d-glucose uptake. Our results demonstrate that there exists a disorder in sodium gradient-dependent d-glucose uptake of renal brush-border membrane in anhydro-4-epitetracycline-treated rabbits, and suggest that this disorder is one of the underlying mechanisms of experimental Fanconi syndrome.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Stereospecific D-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.     

Pathways of D-fructose transport in Arthrobacter pyridinolis

Previous work indicated that Arthrobacter pyridinolis can transport d-fructose by either a phosphoenolpyruvate: d-fructose phosphotransferase system or by a respiration-coupled system. The respiration-coupled transport system for d-fructose, which is stimulated by the addition of l-malate, has been characterized in membrane vesicles from d-fructose-grown cells. Such vesicles carry out malate-dependent uptake of d-fructose but not of d-glucose or l-rhamnose, indicating that there is a sugar-specific component to the respiration-coupled transport system. A mutant which is deficient in the d-fructose-specific component was isolated. Vesicles from fructose-glutamate-grown cells of a phosphotransferase-negative strain (AP100) exhibited malate-dependent d-fructose uptake, while phosphoenolpyruvate-dependent uptake was reduced to a small fraction of that seen with vesicles from wild-type cells. Inhibitors of electron transport, carbonyl cyanide m-chlorophenyl hydrazone, 2,4-dinitrophenol and N-ethylmaleimide caused marked inhibition of malate-dependent d-fructose uptake while exerting little or no effect on phosphoenolpyru-vate-dependent transport of the sugar in vesicles from wild-type cells. Activity of a flavin adenine dinucleotide-linked l-malic dehydrogenase was detected in membrane vesicles as well as in whole cells.     

Carrier-mediated transport of glycyl-L-proline in renal brush border vesicles

Renal brush border vesicles prepared from rabbit were shown to transport intact glycyl-L-proline into the intravesicular space by a Na⁺-independent, carrier-mediated process. The kinetics of hydrolysis of glycyl-L-proline by the vesicles showed that this dipeptide was extremely resistant to hydrolysis. The intravesicular concentration of glycyl-L-proline was the same in both NaCl and KCl media. Dipeptide uptake into the vesicles appeared to be via carrier-mediated transport down a concentration gradient.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled d-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na⁺ gradient yielded a saturable transport component. Kinetic analysis revealed a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na⁺ and bile salt across the ileal membrane in a manner analogous to d-glucose transport.