Dependent Phlorizin Research Articles

Lactoferrin ameliorates prostaglandin E2-mediated inhibition of Na+-glucose cotransport in enterocytes

Various immunoinflammatory cytokines are produced during chronic intestinal inflammation, which inhibits Na(+)-glucose cotransport (SGLT1) in villus cells. Lactoferrin (Lf), abundantly present in colostrum, is a multifunctional glycoprotein that is absorbed by receptor-mediated transcytosis in humans and animals and has been shown to exert anti-inflammatory effects. Therefore, this study aimed to examine whether Lf would prevent PGE2 effect on SGLT1 for glucose absorption in enterocytes. Intestinal epithelial cells (IEC-6) were grown on transwell plates, treated with phlorizin, PGE2, AH6809, and Lf, and 3-O-methyl d-glucopyranose (OMG) uptake was measured in 10 days postconfluent. Na(+)-dependent OMG uptake, phlorizin, and immunoblotting studies established the activity and apical membrane localization of SGLT1 in IEC-6 cells. PGE2 inhibited SGLT1 in a concentration- and time-dependent manner with an inhibitory constant (Ki) of 50.0 nmol/L and that was antagonized by prostanoid receptor inhibitor, AH6809. PGE2 did not alter Na(+)/K(+)-ATPase activity. In contrast, quantitative real-time polymerase chain reaction and Western blot analyses revealed that SGLT1-specific transcripts and protein expression level were decreased 3-fold by PGE2. Furthermore, PGE2 treatment increased intracellular cyclic adenosine monophosphate (cAMP) and Ca(2+) concentrations and decreased SGLT1 expression on the apical membrane, and these effects were ameliorated by Lf. Therefore, we conclude that Lf ameliorates the PGE2 inhibition of SGLT1 most likely via the Ca(2+)- and cAMP-signaling pathways.

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.

Phlorizin binding to renal outer cortical brush-border membranes of cadmium-injected rabbits.

The effect of cadmium injection on the high-affinity phlorizin binding to renal outer cortical brush-border membranes was studied in rabbits. Animals received subcutaneous injections of CdCl2 at a dose of 3 mg Cd/kg.day for about 3 weeks. Brush-border membranes were isolated from renal outer cortex by Percoll gradient centrifugation and magnesium precipitation. Cadmium injection resulted in a marked increase in membrane cadmium content and decrease in Na(+)-dependent phlorizin binding. The latter effect was attributed to a reduction in the number of binding sites (N0) and not to changes in the dissociation constant (Kd). Similar results were observed in normal membrane vesicles directly exposed to cadmium in vitro. These results suggest that one mechanism of the cadmium-induced glycosuria is a loss of active Na(+)-glucose cotransport carrier in the proximal tubular brush-border membrane by direct interaction with free cadmium.

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter.

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.

Examination of sodium/glucose cotransport by using a visible glucose analog

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.

Isolation and reconstitution of the intestinal Na+/glucose cotransporter.

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter.

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation.

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na+ and glucose were omitted. Na+ could not be replaced by K+, Rb+, or Li+. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na+ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na+-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na+-induced active conformer of the Na+/glucose symport system.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Identification of D-glucose-binding polypeptides which are components of the renal Na+-D-glucose cotransporter.

D-Glucose-binding polypeptides in the Na+-D-glucose cotransporter from pig renal cortex were identified by affinity labeling with two D-glucose analogs, 10-N-(N-[4-azido-2-nitrophenyl]-beta-alanyl)amino-1-decyl-beta-D- glucopyranoside (NapADG) and 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG). During short-term incubation in the dark, NapADG and BADG are reversible inhibitors of Na+ gradient-dependent D-glucose uptake and Na+-dependent phlorizin binding with Ki values of about 40 and 400 microM, respectively. Irreversible inhibition of Na+-dependent phlorizin binding, which was prevented by D-glucose or phlorizin, was measured after a 1-h incubation with BADG. Both NapADG and BADG selectively labeled polypeptides with apparent molecular weights of 82,000, 75,000, 64,000, and 47,000. Since labeling of the Mr 82,000 and 75,000 polypeptides by both analogs was partially dependent on the presence of Na+ and was partially protected by D-glucose or phlorizin but not by L-glucose or D-mannose, these polypeptides are thought to be components of the renal Na+-D-glucose cotransporter which contain D-glucose-binding sites. For the Mr 64,000 and 47,000 polypeptides, Na+ dependence and D-glucose protection were not constantly observed. However, also, these polypeptides are thought to be components or proteolytic splitting products of the Na+-D-glucose cotransporter since we observed that three monoclonal antibodies showed cross-reaction with the BADG-labeled Mr 82,000, 64,000, and 47,000 polypeptides (K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, and H. Koepsell, manuscript in preparation). When the BADG-labeled Mr 82,000 and 75,000 polypeptides were analyzed after two-dimensional separation by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three-labeled, D-glucose-protectable polypeptides with the respective molecular weights and isoelectric points of 82,000 and 5.6, 75,000 and 5.4, and 75,000 and 6.9 were distinguished. The data indicate that renal brush-border membranes contain several polypeptides which are components of the Na+-D-glucose cotransporter and contain D-glucose-binding sites.

Isolation and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal tubule.

This paper describes a new method for solubilization and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal convoluted tubule. Selective solubilization is carried out with 0.1% Na+-deoxycholate followed by complete solubilization with 0.5% deoxycholate. The 100,000 X g supernatant of the deoxycholate extract is then subjected to a combination of chromatofocusing and gel exclusion chromatography. Purification is monitored by a new column assay which permits detection of the Na+-dependent high affinity phlorizin receptor in solubilized preparations. Na+-dependent phlorizin binding exhibits the same characteristics on the column assay as in intact brush border vesicles. Binding is temperature-dependent, inhibited by proteolytic agents, Na+-dependent, and inhibited by excess cold phlorizin and D-glucose but not L-glucose. Quantitation of specific binding at different stages of the isolation procedure indicates a final purification of approximately 80-140-fold compared to intact brush border membrane fragments. Enrichment of specific phlorizin binding is paralleled by enrichment of a 61-66-kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It is postulated that this polypeptide contains both the Na and the sugar specific binding site and represents a subunit of the intact Na+-dependent glucose transporter from dog kidney proximal tubule brush border membrane.

Evidence for tyrosyl residues at the Na+ site on the intestinal Na+/glucose cotransporter.

A tyrosine group has been identified at, or near, the Na+-binding site of the Na+/glucose and Na+/proline cotransporters of rabbit intestinal brush-borders. Three tyrosine group-specific reagents, n-acetylimidazole, tetranitromethane, and p-nitrobenzene sulfonyl fluoride, were used to evaluate the role of tyrosyl groups in Na+-dependent glucose transport, Na+-dependent phlorizin binding, and the Na+-induced fluorescence quenching of fluorescein isothiocyanate bound to the glucose site of the carrier. All three reagents inhibited glucose transport, phlorizin binding, and fluorescein isothiocyanate quenching by 50-85% with Ki values in the range 7-50 microM. The presence of Na+ during the exposure of membranes to the reagents completely protected against inhibition, the Na+ concentration required to produce 50% protection was 14-36 mM. Fluorescent derivatives of n-acetylimidazole were synthesized to identify the tyrosyl residues on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A total of five polypeptide bands were labeled with eosin or fluorescein n-acetylimidazole in a Na+-sensitive manner. Two of these bands, previously identified as the glucose (75,000-dalton) and proline (100,000-dalton) binding sites of the glucose and proline carriers, account for 50% of the Na+-sensitive tyrosyl residues. On the basis of these studies, we believe that the Na+/glucose cotransporter contains both the Na+ and glucose active sites on the same polypeptide or that the cotransporter consists of two similar polypeptides, each containing one substrate binding site.

Identification of the D-glucose binding polypeptide of the renal Na +-D-glucose cotransporter with a covalently binding D-glucose analog

The covalently binding D-glucose analog 10- N-(bromoacetyl)amino-1-decyl-β-D-glucopyranoside (BADG) was synthesised and shown to be a high-affinity inhibitor of the renal Na +-D-glucose contransporter. From renal brush-border membranes a protein fraction was isolated, in which the concentration of Na +-dependent phlorizin binding sites per mg protein was enriched 7-fold. In labeling experiments with this protein fraction a polypeptide of M r ~ 79000 was identified as containing the D-glucose binding site of the renal Na +-D-glucose cotransporter. Affinity labeling D-Glucose analog D-Glucose binding site Na±D-glucose cotransporter Brush-border membrane Kidney

Structural state of the Na + / d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na +-dependent phlorizin binding and Na +-dependent d-glucose transport

Target sizes of the renal sodium- d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at −50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na +-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na +-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium- d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding.

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.

Chemical modification of the small intestinal Na +/ d-glucose cotransporter by amino group reagents : Evidence for a role of amino group(s) in the binding of the sugar

Some amino group reagents inactivate the small-intestinal Na +/ d-glucose cotransporter, as measured either as a catalyst of Na +-dependent d-glucose transport or as a Na +-dependent phlorizin ligand. The amino group(s) studied in this paper are not identical with those investigated previously (Biber, J., Weber, J. and Semenza, G. (1983) Biochim. Biochim. Acta 728, 429–437): these are protected from inactivation by the simultaneous presence of Na + plus sugar substrates. They are thus likely to be located within the substrate-binding site.