Stephen A. Baldwin

The blood-nerve barrier is rich in glucose transporter

SummaryThe glucose transporter of the facilitated diffusion type has been localized in sections of innervated rat diaphragm muscle and sciatic nerve by immunofluorescence, using affinity-purified antibodies against both the entire transporter and the carboxy-terminal peptide. In both tissues the transporter was very abundant in the perineurial sheath of cells surrounding the nerve fibres. The transporter also appeared to be abundant in the endoneurial blood vessels of the sciatic nerve. The identity of the antigen as the glucose transporter was established by extracting sciatic nerve with sodium dodecylsulphate and immunoblotting the extract. A single reactive polypeptide with the expected molecular weight of 55 000 was found. The high concentration of glucose transporter in the cells of the blood—nerve barrier presumably ensures an adequate supply of glucose to the nerve fibres.

Hexose uptake in primary cultures of bovine brain microvessel endothetial cells. I. Basic characteristics and effects of d-glucose and insulin

The basic characteristics of hexose uptake and regulation of the glucose transporter (GLUT1) by D-glucose and insulin were studied in primary cultures of bovine brain microvessel endothelial cells (BMECs). A non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose [( 3H]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [3H]3MG was equilibrative, temperature-dependent, and independent of sodium. The uptake also decreased gradually with culture age from 7 to 13 days. Saturation kinetics were observed for [3H]3MG uptake and the apparent Km and Vmax values were determined to be 13.2 mM and 169 nmol/mg per min, respectively. Pre-incubation with high concentrations of D-glucose and 3MG accelerated [3H]3MG uptake by BMECs by a counter-transport mechanism. D-Glucose, 2-deoxy-D-glucose, D-mannose, D-xylose, D-galactose and D-ribose showed significant competitive inhibition with [3H]3MG, whereas L-glucose, D-fructose, and sucrose did not affect [3H]3MG uptake by BMECs. [3H]3MG uptake was inhibited significantly by cytochalasin B and phloretin but not by phlorizin, 2,4-dinitrophenol, or ouabain. D-Glucose starvation of BMECs by incubation with D-glucose-free media for 24 h resulted in a significant increase (40-70%) in uptake of [3H]3MG compared with control conditions (7.3 mM D-glucose). Low D-glucose treatments (2.43 and 1.83 mM) for 7 days induced a slight but significant increase (20%) in [3H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [3H]3MG uptake irrespective of exposure time. The increase in [3H]3MG accumulation following D-glucose starvation was dependent upon starvation time (12 to 48 hr) and protein synthesis. Refeeding of D-glucose (7.3 mM) to D-glucose-starved BMECs resulted in a return of [3H]3MG uptake to control levels in 48 h. The D-glucose-starvation-induced increase in [3H]3MG uptake was shown to result from an increase in Vmax; the Km remained constant. In addition, D-glucose-starved BMECs were shown to have an increased level of GLUT1 using an antibody against human GLUT1 and an enzyme-linked immunosorbent assay (ELISA). The increased uptake following D-glucose starvation was not significantly affected by the presence of L-glucose, was partially impaired by the presence of D-galactose, D-fructose, and D-xylose, and was completely inhibited by the presence of D-mannose and 3MG. Furthermore, preincubation of BMECs with insulin (10 micrograms/ml) for 20 min did not affect the uptake of [3H]3MG or 2-deoxy-D-[3H]glucose ([3H]2DG).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of the glucose-transport protein in mammalian brain capillaries

SummaryThe endothelial cells of mammalian brain capillaries, which form the anatomical basis of the blood-brain barrier, have been investigated by immunocytochemical methods to determine the distribution of the glucose-transport protein. A monoclonal antibody raised against the intact human erythrocyte glucose-transport protein and polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal sequence of the human erythrocyte glucose-transport protein were used for immunofluorescent staining of isolated human and bovine cerebral cortex microvessels. The pattern of fluorescence with both antibodies demonstrated the antigen to the distributed throughout the plasma membrane of the capillary endothelial cells. These results provide further evidence for the homology between the human erythrocyte and brain capillary glucose-transport protein, and confirm its abundance in brain capillaries.

Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. II: Effects of conditioned media from astroglial and glioma cells

Regulation of glucose uptake by an astroglial cell secreted factor(s) was studied in primary cultures of brain microvessel endothelial cells (BMECs). Uptake of a non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose ([3H]3MG), was measured after the BMECs were treated with media conditioned by primary cultures of rat astrocytes (Astrocyte Conditioned Media: ACM) or rat C6 glioma cells (Glioma Cell Conditioned Media: GCM). Uptake of [3H]3MG was significantly increased by ACM (30-50%) and GCM (60-200%) treatments, whereas conditioned medium from 3T3 fibroblasts (3T3) caused no significant effect. The elevation in [3H]3MG uptake increased with increasing time of exposure of BMECs to these conditioned media (CM), and the effect was shown to be reversible. Glucose depletion of CM was shown not to be a factor. The presence of cycloheximide, a protein synthesis inhibitor, during treatment of the BMECs with ACM and GCM blocked the increase in [3H]3MG uptake by the cells. These results suggested that ACM or GCM treatment elevated de novo synthesis of brain-type glucose transporter (GLUT1). Indeed, enhanced GLUT1 expression by these treatments in BMECs was demonstrated directly by enzyme-linked immunosorbent assay (ELISA) using antibodies against human GLUT1. After trypsinization of ACM and GCM, both conditioned media still induced significant stimulation of [3H]3MG uptake by BMECs. A significant increase in [3H]3MG uptake was also observed when ACM or GCM was exposed to BMECs through a dialysis membrane with a molecular weight cutoff of 1000. To examine whether the effects were specific to brain endothelial cells, [3H]3MG uptake experiments were performed employing aortic endothelial cells (AECs), pulmonary microvessel endothelial cells (PMECs), and 3T3 cells. ACM treatment did not alter 3MG uptake by these cells, suggesting that the ACM effect was specific to BMECs. On the other hand, [3H]3MG uptake by AECs and PMECs treated with GCM was significantly enhanced. The present study demonstrated that some factor(s) of relatively small molecular weight, which was released from astrocytes or glioma cells, stimulated glucose uptake by enhancing GLUT1 synthesis in BMECs.

Analysis of the glucose transporter content of islet cell lines: Implications for glucose-stimulated insulin release

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissues demand for glucose. The beta-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high Km for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, beta-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in beta-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high Km transporter GLUT 2, may account for the characteristic aberrant patterns of glucose-stimulated insulin release in cell lines derived from beta-cells.

A D‐Glucose‐Sensitive Cytochalasin B Binding Component of Cerebral Microvessels

Abstract: The technique of photoaffinity labelling with [4‐3H]cytochalasin B was applied to osmotically lysed cerebral microvessels isolated from sheep brain. Cytochalasin B was photo‐incorporated into a membrane protein of average apparent Mr 53,000. Incorporation of cytochalasin B was inhibited by D‐glucose, but not by L‐glucose, which strongly suggests that the labelled protein is, or is a component of, the glucose transporter of the blood–brain barrier. Investigation of noncovalent [4‐3H]cytochalasin B binding to cerebral microvessels by equilibrium dialysis indicated the presence of a single set of high‐affinity binding sites with an association constant of 9.8 ± 1.7 (SE) μM−1. This noncovalent binding was inhibited by D‐glucose, with a Ki of 23 mM. These results provide preliminary identification of the glucose transporter of the ovine blood–brain barrier, and reveal both structural and functional similarities to the glucose transport protein of the human erythrocyte.

Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure.

Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 10(7) cells (10 mg protein) yielded about 12 micrograms of vesicular protein and 11 micrograms of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypeptide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270,000, 245,000, 165,000 and 115,000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245,000, 190,000, 115,000 and 25,000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.

Antibodies as Probes of Nitrobenzylthioinosine-Sensitive Nucleoside Transporters

The passage of nucleosides across the membranes of mammalian cells is mediated by a variety of different transport systems [1]. In the kidney and small intestine active, sodium-linked uptake systems are present, at least four distinct transporters having been identified by virtue of their differing substrate specificities [2]. However, in most other mammalian cells nucleoside uptake and efflux occurs via the passive process of facilitated diffusion [1]. The passive transporters, like the active, can also be subdivided into classes, in this case by virtue of their sensitivity to inhibition by the 6-thiopurine analogue nitrobenzylthioinosine (NBMPR), to which the sodium-dependent transporters are insensitive. Passive transporters designated es (equilibrative-sensitive) bind and are inhibited by NBMPR with affinities in the 0.1–1 nM range, whereas transporters of the ei class (equilibrative-insensitive) are unaffected by micromolar concentrations of NBMPR [1,3,4].

ENT2, Equilibrative Nucleoside Transporter 2

Widely distributed in different cells and tissues, human ENT2 (hENT2 or SLC29A2) mediates equilibrative (Na+-independent) bidirectional plasma membrane transport of physiologic nucleosides (including …

ENT1, Equilibrative Nucleoside Transporter 1

Ubiquitously distributed in different cells and tissues, human ENT1 (hENT1 or SLC29A1) mediates equilibrative (Na+-independent) bidirectional plasma membrane transport of physiologic nucleosides (including …