Sodium-dependent D-glucose Transport Research Articles

A 96-well automated method to study inhibitors of human sodium-dependent D-glucose transport

The sodium-dependent D-glucose transporter (SGLT) family is involved in glucose uptake via intestinal absorption (SGLT1) or renal reabsorption (SGLT1 and SGLT2). Current methods for the screening of inhibitors of SGLT transporters are complex, expensive and very labor intensive, and have not been applied to human SGLT transporters. The purpose of the present study was to develop an alternative 96-well automated method to study the activity of human SGLT1 and SGLT2. Chinese hamster ovary (CHO) Flp-In cells were stably transfected with pcDNA5-SGLT1 or pcDNA5-SGLT2 plasmid and maintained in hygromycin-selection Ham's F12 culture medium until hygromycin-resistant clones were developed. SGLT1 and SGLT2 gene expression was evaluated by relative real-time reverse transcription-polymerase chain reaction (RT-PCR) quantification, Western blotting, and immunocytochemical analysis. The clones with higher expression of SGLT1 and SGLT2 were used for transport studies using [14C]-methyl-alpha-D-glucopyranoside ([14C]AMG). The advantage of using the 96-well format is the low amount of radioactive compounds and inhibitory substances required, and its ability to establish reproducibility because repetition into the assay. This method represents an initial approach in the development of transport-based high-throughput screening in the search for inhibitors of glucose transport. The proposed method can easily be performed to yield quantitative data regarding key aspects of glucose membrane transport and kinetic studies of potential inhibitors of human SGLT1 and SGLT2.

Increased sodium-dependent D-glucose transport in the jejunal brush-border membrane of spontaneously hypertensive rat.

The current studies explore the effect of hypertension on D-glucose transport into jejunal brush-border membrane vesicles (BBMV). Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, as a control group, were used. The purity of the BBMV from both groups of animals was validated by the finding that the specific activity of brush-border enzyme marker, sucrase, was severalfold greater in membrane vesicles compared with corresponding values in mucosal homogenate. D-glucose uptake was Na+ dependent in both groups of animals, with a transient increase in the intravesicular concentration of D-glucose. However, the initial rate and the magnitude of the accumulation of Na+-dependent D-glucose was significantly higher in SHR compared with WKY rats. In order to investigate the mechanism(s) for the increase in Na+-dependent D-glucose transport in SHR, several experiments were performed: (1) an experiment that indicated 22Na uptake, as an indicator for Na+ permeability, was similar between SHR and WKY rats, (2) kinetic studies that indicated that Vmax values of SHR were significantly greater that those of WKY rats. In contrast, similar Km values for glucose were found between SHR and WKY rats, (3) Na+-dependent phlorizin binding measurements that were not altered by hypertension and (4) a study of the brush-border membrane lipid composition that showed a significant increase in the free cholesterol/phospholipid ratio in SHR. We conclude that altered membrane cholesterol content and consequently altered lipid fluidity could be, at least in part, responsible for the observed increase in Na+-dependent D-glucose transport in SHR.

Decreased sodium dependent D-glucose transport across renal brush-border membranes in cis-diamminedichloride platinum induced acute renal failure.

Na-coupled D-glucose transport in rabbits with cis-diamminedichloride platinum (CDDP; cisplatin) induced acute renal failure (ARF) has been studied. ARF occurred at 3 days after injection of CDDP (3 mg/kg i.v.). Na-coupled D-glucose transport into brush-border membrane vesicles (BBMV) from both outer cortex (OC) and outer medulla (OM) of ARF rabbits under zero-trans condition was decreased. Increased Km (i.e., decreased affinity of transport carrier for D-glucose) in OC and decreased Vmax (i.e., decreased number of glucose carrier) in OM were observed in CDDP-induced ARF rabbits. Decrease glucose transport was also observed under equilibrium exchange condition. Intravesicular volume of BBMV from OC and OM of ARF rabbits was decreased. In homogenate and BBMV from OC and OM of ARF rabbits, activities of gamma-glutamyl transpeptidase and alkaline phosphatase (marker enzymes of brush-border membrane) were decreased. Activities of succinate dehydrogenase, glucose-6-phosphatase, and Na-K ATPase (marker enzymes of mitochondria, endoplasmic reticulum, and basal lateral membrane, respectively) were not affected by CDDP administration. These results suggested that one of the main target sites of CDDP in kidney is brush-border membrane (BBM) along the proximal tubule, that is, not only Na-coupled D-glucose transport carrier protein but also other proteins in BBM.

Dietary triacylglycerol modulates sodium-dependent d-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent d-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as ( n − 3) unsaturated fatty acids and lower percentages of monounsaturated and ( n − 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0–20:5, 16:0–22:6 and 18:0–20:5 and lower levels of 18:0–18:2 and 16:0–18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na +-dependent d-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity ( V max) than butter fat membrane vesicles but a similar K m for glucose.

Sodium-dependent D-glucose transport after proximal small intestinal resection in rat.

Massive small intestinal resection results in both structural and functional changes in the residual small bowel. Sodium-dependent D-glucose transport was examined in brush-border membrane vesicles derived from the terminal 20-30 cm of ileal mucosa of male Sprague-Dawley rats, 2 and 6 wk after 66% proximal jejunoileal resection or jejunoileal transection. Kinetic characteristics for sodium-dependent D-glucose transport were investigated with rapid filtration under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Mucosal weight, protein, and DNA content were increased in the residual terminal intestinal segment compared with transected controls, whereas morphometric studies revealed increased villus and crypt heights as well as an increased mitotic index. Mean kinetic transport parameters at 6 wk after proximal small bowel resection revealed two saturable systems in the distal residual ileum: first, a low-affinity, high-capacity system with a Km of 0.19 +/- 0.03 mM and a Vmax of 0.48 +/- 0.04 nmol.mg protein-1.min-1; and second, a high-affinity, low-capacity system with a Km of 0.009 +/- 0.001 mM and a Vmax of 0.105 +/- 0.016 nmol.mg protein-1.min-1. In contrast, negligible sodium-dependent D-glucose transport was detected in the most distal ileum in control animals or animals 2 wk after resection or 2 and 6 wk after transection. Thus adaptational changes including mucosal hyperplasia and the appearance of two sodium-dependent D-glucose brush-border membrane vesicle transport systems occur in the residual distal intestine after massive proximal small bowel resection.

Evidence for a high-affinity sodium-dependent D-glucose transport system in the kidney

Renal brush-border membrane D-glucose transport was studied in vesicles prepared from early proximal tubules (outer cortical tissue) and late proximal tubules (outer medullary tissue). Kinetics of sodium-dependent D-glucose uptake, determined with zero trans sodium gradient of 200 mM at 21 degrees C demonstrated a curvilinear Woolf-Augustinsson-Hofstee plot in both cortical and medullary vesicles. Three sodium-dependent systems were discerned in the cortex, possessing Km values of 12.35 +/- 2.2 mM, 520 +/- 39 microM, and 12 +/- 1 microM, respectively. In contrast, only the high-affinity, low-capacity systems were present in the medullary tissue; with respective Km values of 484 +/- 95 microM and 11 +/- 1 microM and mean Vmax values of 2.4 +/- 0.1 and 0.44 +/- 0.08 nmol X mg protein-1 X min-1. The high-affinity systems were more abundant in the medulla than in the cortex. These studies support the notion of multiple transport processes for sodium-dependent D-glucose transport in the pig kidney. Further, they provide evidence for a third, high-affinity system located principally in the medullary tissue, perhaps the late proximal tubule.

Sodium-dependent d-glucose transport in brush-border membrane vesicles after massive distal small bowel resection in the rat

Massive small intestinal resection in the rat results in both structural and functional changes in the residual small bowel. Sodium-dependent d-glucose transport was examined in brush-border membrane vesicles derived from the proximal small bowel mucosa of male Sprague-Dawley rats 2 and 6 wk after a 66% distal jejunoileal resection or jejunoileal transection. Kinetic characteristics for the low-affinity, high-capacity system and high-affinity, lowcapacity system were defined with rapid filtration under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Mucosal weight, protein, and deoxyribonucleic acid content were increased in the residual intestinal segment compared to transected controls and morphometric studies revealed increased villus and crypt heights as well as an increased mitotic index. Postresection mean kinetic parameters for d-glucose transport at 2 wk (low-affinity system: Km, 177.5 ± 45.1 μM; Vmax, 3.73 ± 0.99 nmol · mg protein−1 · min−1; and high-affinity system: Km, 6.2 ± 1.9 μM; Vmax, 0.12 ± 0.06 nmol · mg protein−1 · min−1) and 6 wk (low-affinity system: Km, 267.8 ± 83.1 μM; Vmax, 0.06 ± 0.01 nmol · mg protein−1 · min−1; and high-affinity system: Km, 6.5 ± 1.1 μM; Vmax, 0.06 ± 0.01 nmol · mg protein−1 · min−1) were similar to values posttransection at 2 wk (low-affinity system: Km, 280.4 ± 53.7 μM; Vmax, 3.05 ± 0.32 nmol · mg protein−1 · min−1; and high-affinity system: Km, 9.1 ± 1.3 μM; Vmax, 0.17 ± 0.01 nmol · mg protein−1 · min−1) and 6 wk (low-affinity system: Km, 271.7 ± 17.5 μM; Vmax, 4.69 ± 0.23 nmol · protein−1 · min−1; and high-affinity system: Km, 10.6 ± 4.2 μM; Vmax, 0.16 ± 0.09 nmol · mg protein−1 · min−1). These kinetic data suggest that the hyperplastic response in adapting proximal small bowel after distal resection is accompanied by a persistence of the membrane functional characteristics for both sodium-dependent d-glucose transport systems despite an altered pattern of enterocyte proliferation and differentiation.

Sodium-dependent D-glucose transport in brush-border membrane vesicles from isolated rat small intestinal villus and crypt epithelial cells.

Differentiation and maturation of enterocytes occur with migration from the crypt to villus compartments. To investigate the effect of epithelial cell differentiation on sodium-dependent D-glucose transport, brush-border membrane vesicles were prepared from small intestinal epithelial cell suspensions selectively isolated from villus and crypt populations. Enterocytes were isolated with a morphologically monitored sequential cell dissociation method. Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as differentiation markers of specific cell populations. Brush-border membrane vesicles were purified and their kinetic characteristics defined with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Typical "overshoot" phenomena characteristic of sodium D-glucose cotransport were observed for both villus (five- to eight-fold equilibrium values) and crypt brush-border membrane vesicles (two- to four-fold equilibrium values). Kinetics analyses of the initial D-glucose flux in brush-border membrane vesicles suggested the presence of at least two sodium-dependent D-glucose carriers in the villus and only a single carrier in the crypt compartments. These data indicate that sodium D-glucose cotransport occurs in brush-border membranes of both villus and crypt populations. Moreover, quantitative and qualitative differences between these two membrane populations suggest that epithelial D-glucose transport processes are differentiation dependent and reflect the degree of enterocyte development.

Isolation and Characterization of Brush Border Membrane Vesicles from Bovine Small Intestine

Intestinal brush border membrane vesicles were prepared from 21-wk-old Holstein bulls. The method of preparation included magnesium precipitation, differential centrifugation and sucrose density gradient centrifugation. Purification of brush border membranes was indicated by sevenfold enrichment of alkaline phosphatase activity. Uptake of D-[U-14C]glucose was sodium-stimulated and exhibited characteristic “overshoot” phenomenon. Sodium-dependent and sodium-independent D-glucose uptake was into an osmotically active space. Phloridzin (100 mM) completely inhibited sodium-dependent, but did not affect sodium-independent transport. Sodium-dependent D-glucose transport was inhibited more by D-glucose than by D-galactose, which inhibited more than D-xylose did. The sodium-dependent D-glucose transport was not inhibited by D-fructose, D-ribose or D-arabinose. Sodium-independent D-glucose transport was unaffected by the sugars tested. Glucose transport in bovine intestinal brush border is similar to that in monogastric species and is composed of two pathways: a sodium-dependent inhibitable system and a diffusional system.

Radiation inactivation studies on the rabbit kidney sodium-dependent glucose transporter.

Rabbit kidney cortical brush-border membrane vesicles were irradiated in the frozen state with increasing doses of high energy electrons from a Van de Graaff generator. Sodium-dependent D-glucose and L-alanine transport showed a simple exponential loss of activity with increasing radiation dosage. Target size calculation based on these data gives estimates of 1.0 X 10(6) daltons for the glucose transporter and 1.2 X 10(6) daltons for the alanine transporter. A highly purified glucose transport protein extracted from rabbit kidney cortex was similarly irradiated both before and after reconstitution into liposomes. The target size of this purified glucose transporter was 343,000 daltons, based on inactivation of transport. The intensity of the major 165,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation was decreased by radiation. The decrease in staining intensity was dose-dependent, yielding a target size of 298,000 daltons, in situ. We propose that the purified glucose transporter reconstituted into liposomes is a tetramer comprised of 85,000-dalton subunits.

Presence of a sodium-dependentd-glucose transport system in the kidney of the atlantic hagfish (Myxine glutinosa)

A membrane fraction, rich in brushborder membranes, was prepared from the archinephric duct of the atlantic hagfish (Myxine glutinosa) and the uptake ofd-glucose and other sugars into the membrane vesicles was investigated by a rapid filtration technique. Uptake ofd-glucose was found to be sodium-dependent, phloridzin-inhibitable and osmotically sensitive. A sodium gradient dependent ‘overshoot’ was demonstrated at 25° C as well as at the more physiological temperature of 4°C. The sodium dependentd-glucose transport was inhibited by α-methyl-d-glucoside, but not by 2-deoxy-d-glucose. Furthermore at the same concentration of sugars the initial uptake ofd-glucose was 7.2-fold higher thanl-glucose uptake.d-glucose transport across the membrane in the presence of a sodium gradient was stimulated when SCN− replaced Cl− and inhibited when gluconate replaced Cl−.d-glucose uptake in the presence of a sodium- and potassium gradient was decreased by the addition of valinomycin. In addition, the presence of ad-glucose gradient enhanced sodium uptake into the vesicles as compared to a mannitolgradient. Phloridzin inhibited thed-glucose dependent sodium flux. Thus an electrogenic stereospecific sodium glucose co-transport system, with properties similar to that found in the kidney of higher vertebrates is present in this primitive vertebrate and might participate in secondary-active sugar reabsorption in the archinephric duct.

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na +-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M r ≅ 165000 which is apparently a dimer of M r ≅ 85 000. When reconstituted and tested for transport, this protein showed Na +-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.

Influence of sodium ions on detergent solubilization of pig brush border D-glucose transport system for reconstitution experiments

The D-glucose uptake by liposomes resulting from the association of egg lecithins with solubilized membrane proteins was measured in order to assess their sodium dependent D-glucose transport activity. Membrane proteins were extracted by Triton X-100 solubilization of pig kidney brush border membrane vesicles, which were suspended either in KCl medium or in NaCl medium. When measured by equilibrium isotope exchange procedure in sodium conditions, the D-glucose uptake by sodium detergent extract associated to liposomes occurred with a higher velocity than that obtained with liposomes reconstituted from potassium detergent extract. No differences were observed in the permeability or in the protein content of two types of liposomes. These results are discussed in terms of activation of D-glucose transport system induced by sodium ions before membrane protein solubilization.

Synthesis of [ 3H]phlorizin and its binding behavior to renal brush-border membranes

Tetra- and tribromophlorizin have been synthesized under mild brominating conditions. With catalytic debromination in the presence of hydrogen or tritium gas, bromine atoms in the derivatives were completely substituted by hydrogen or tritium. The product was identical to the native phlorizin and was chemically pure. Tritiated phlorizin with extremely high specific radioactivity (45 Ci/mmol) was obtained when hydrogen gas was replaced by tritium gas. While the brominated compounds showed little inhibition of sodium D-glucose co-transport by isolated renal brush-border membranes. [3H]phlorizin had the same binding affinity to the brush-border membranes as native phlorizin and a Ki value of 1.2 microM for the sodium-dependent D-glucose transport. Binding studies performed using a flow-dialysis method resulted in 150 pmol of phlorizin-binding sites per milligram of membrane protein. This radioactive phlorizin can be a useful tool for determining D-glucose-(phlorizin) binding sites at a low phlorizin concentration in membranes, in nonvesicle forms such as collapsed membrane vesicles, and in purified protein fractions.

Renal sodium- d-glucose cotransport system. Involvement of tyrosine residues in sodium-transporter interaction

Using brush-border membrane vesicles isolated from calf kidney cortex the effect of tyrosine-reactive reagents on sodium-dependent d-glucose transport was investigated. Treatment of the membranes for 60 min with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), N-acetylimidazole or tetranitromethane decreased d-glucose uptake 50, 70 and 40%, respectively. Tracer exchange experiments revealed that the inhibition of transport is due to a direct modification of the sodium- d-glucose cotransport system. The modification by NBD-Cl decreases the apparent V max of the transport system with respect to its interaction with sodium. In addition, the rate of inactivation of the transport system by NBD-Cl is reduced in the presence of high concentrations of sodium. The results indicate that tyrosine residues play an essential role in sodium- d-glucose cotransport and are probably involved in the binding and/or transport of sodium by the sodium- d-glucose cotransport system.

Heterogeneity of sodium-dependent D-glucose transport sites along the proximal tubule: evidence from vesicle studies

The glucose transport properties of brush border membrane vesicles from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) of rabbit kidney were studied. In the outer cortical preparation the behavior of the sodium-dependent component of D-glucose flux indicated the presence of a low-affinity transport system with Km congruent to 6 mM and Vmax congruent to 10 nmol.min-1.mg protein-1 as measured under zero trans conditions at 40 mM NaCl and 17 degrees C. By contrast, in the outer medullary preparation this component of flux behaved as a high-affinity system with Km congruent to 0.35 mM and Vmax congruent to 4 nmol.min-1.mg protein-1. Differences in transport specificity between the two preparations were also indicated and glucose uptake by the outer cortical vesicles was significantly more sensitive to inhibition by phlorizin. These results suggest the existence of two distinct sodium-dependent D-glucose transport systems in the renal proximal tubule brush border membrane. The kinetic studies presented here were done under zero trans sodium and glucose conditions. The rationale and methodology for carrying out these measurements reliably are discussed in detail.

Partial purification of hog kidney sodium- D-glucose cotransport system by affinity chromatography on a phlorizin polymer

A brush border membrane fraction isolated from hog kidney cortex was solubilized with 0.5% Triton X-100 and subjected to affinity chromatography on a phlorizin polymer. As demonstrated by transport studies with reconstituted proteoliposomes, the polymer adsorbs the sodium-dependent D-glucose transport system. The latter can be eluted from the polymer by 0.5 M D-glucose. The purified fraction contains 0.4% of the membrane protein extract and exhibits a 20–30-fold higher transport activity than the crude membrane extract. Other brush border membrane proteins such as alkaline phosphatase and aminopeptidase M are markedly reduced in the purified fraction. Thus, affinity chromatography on a phlorizin polymer is a suitable tool for the isolation of the sodium-glucose transport system present in brush border membranes.

The effect of harmaline on intestinal sodium transport and on sodium-dependent D-glucose transport in brush-border membrane vesicles from rabbit jejunum.

Harmaline inhibition of sodium uptake and of sodium-dependent D-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, "initial" D-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrations D-glucose uptake was stimulated by 10--15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations up to 2 mM, harmaline did not alter the equilibrium uptake of D-glucose or D-mannitol. After pre-equilibration with sodium (25 mM), G-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependent D-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane. Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependent D-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.

Sugar uptake into brush border vesicles from normal human kidney.

Uptake studies of simple sugars were performed on a membrane fractions containing osmotically active vesicles prepared from normal human kidney cortex. The uptake of D-glucose was found to be sodium-dependent and phlorizin-sensitive. The specificity of the D-glucose transport mechanism is such that it is shared by alpha-methyl-D-glucoside, D-galactose, and 5-thio-D-glucose, while 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-mannose, and D-fructose show little, if any, affinity. Measurement of the sodium-dependent component of the initial D-glucose uptake as a function of glucose concentration resulted in a curvilinear Scatchard plot, indicating the possibility of cooperative effects, or alternatively, the existence of two (or more) sodium-dependent D-glucose transporters. In the case of two transporters, we estimate that Km congruent to 0.3 mM and Vmax congruent to 2.5 nmol/min per mg of protein for the "high-affinity transporter," and Km approximately 6 mM and Vmax approximately 8 nmol/min per mg of protein for the "low-affinity transporter." These specificity and kinetic properties strongly suggest that the sodium-dependent D-glucose transport mechanism characterized in our studies is localized to the brush border of the proximal tubule.