cDNA cloning and sequence analysis of the glucose transporter from porcine blood-brain barrier

A porcine brain microvessel-derived cDNA library enriched in blood-brain-barrier-specific sequences was constructed by a subtractive cloning procedure. Two cDNA clones from this library were found to encode a glucose transport protein. These clones were used to isolate a nearly full length cDNA from a representative brain microvessel library. Analysis of the amino-acid sequence deduced from the cDNA sequence revealed 97% identity with the human HepG2 glucose carrier. Amino-acid substitutions appear to be clustered in certain regions of the polypeptide. The nucleotide sequences of the 3'-noncoding regions close to the putative polyadenylation sites are highly conserved in glucose transporter mRNAs of different species. The expression of this mRNA has been investigated in various tissues and shown to be decreased in primary cultures of brain microvascular endothelial cells.     

Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization

The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.     

Inhibition of glucose transport in human erythrocytes by ubiquinone Q0

Searches of the protein data bases revealed limited homologies between several regions of the human erythrocyte glucose transporter containing a relative abundance of hydrogen-bonding amino-acid side chains, and proteins of the NADH-ubiquinone oxidoreductase family. This raised the possibility the binding sites for glucose and ubiquinone may be similar in the respective proteins. Experimental studies demonstrated that ubiquinone Q0 does in fact inhibit both glucose entry and glucose exit in human erythrocytes with kinetics consistent with the existence of ubiquinone binding sites at both the exofacial and endofacial sides of the transporter. Glucose transport was also inhibited by the water-soluble tryptophan-inactivating agent, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide, and this is consistent with the presence of tryptophan residues in two of the exofacial amino-acid sequences proposed as candidates for involvement in glucose binding sites.