The Journal of Neuroscience, December 1990, fO(12): 36623672 lmmunocytochemical Localization of the Erythroid Glucose Transporter: Abundance in Tissues with Barrier Functions Sami I. Harik,’ Rajesh N. Kalaria,’ Lars Andersson,3 Per Lundahl,3 and George Perry2 Departments of ‘Neurology and zPathology, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and 3Department of Biochemistry, University of Uppsala, Uppsala, Sweden. We investigated the cellular localization and tissue clistri- the human erythrocyte GT is most extensively studied, and it bution of the glucose transporter protein in the nervous sys- is now accepted that it mediates glucosediffusion in a manner tem of the monkey and rat, and in other tissues of the rat, that is saturable, stereospecific, nonconcentrative, nonenergy by immunocytochemical methods with monoclonal and poly- requiring, and not influenced by sodium or insulin. clonal antibodies to the glucose transporter of human eryth- We previously demonstratedthat the density of GT in isolated rocytes. We found intense immunostaining, indicating a high rat and pig brain microvessels,assessed by the D-glucose-dis- density of the glucose transporter, in all intraparenchymal placeablespecific binding of cytochalasin B, was secondonly to blood vessels of the brain and spinal cord, in pial vessels, that of human erythrocyte membranesand about 1O-fold higher and in endoneurial capillaries of peripheral nerves, nerve than in brain homogenates(Dick et al., 1984; Dick and Harik, roots, and dorsal root ganglia. Larger blood vessels at the 1986). Membrane-enriched subcellular fractions of brain mi- base of the brain and in major fissures did not stain. The crovesselshad an even higher density of GT (maximal binding only intraparenchymal brain microvessels that did not immu- of cytochalasin B exceeded 90 pmol/mg protein; Dick et al., nostain were in circumventricular organs. There was no spe- 1984). Irreversible photoaffinity labeling of cytochalasin B to cific immunostaining of neurons or glia, except for tanycytes brain microvessels,followed by SDS-PAGE, allowed estimation in the floor of the third ventricle, which immunostained in- of the M, of brain microvessel GT at about 53 kDa, similar to tensely. Vessels of the choroid plexus did not stain, but the that of human erythrocytes (Dick et al., 1984). Furthermore, choroid epithelium, especially its basal membranes, stained. antiserum to purified GT of human erythrocytes reacted with The only non-neural organ where immunostaining was evi- a polypeptide in a band correspondingto an M, of about 53 kDa dent in its microvessels was the testis. In addition to the on immunoblots of brain microvessel preparations, indicating endothelium of neural and testicular tissues, there was im- similar antigenic properties (Dick et al., 1984). Subsequentwork munostaining in certain epithelial tissues such as the peri- confirmed and extended these findings to sheep(Baldwin et al., neurium of peripheral nerves and nerve roots, the epithelium 1985), cows (Kasanicki et al., 1987), and humans@alaria et al., of the ascending loop of Henle in the kidney, and the epi- 1988;Kalaria and Ha&, 1989).The availability of molecularprobes dermis of the skin. Based on these findings, we hypothesize for GTs allowed studies of their mRNA tissue distribution. that a high density of the erythroid-type glucose transporter Northern blots revealed high levels of the erythroid-type GT is inherent to many endothelial and epithelial cells that are mRNA in brain microvessels(Flier et al., 1987; Weiler-Gtittler joined by occluding junctions. However, other epithelial tis- et al., 1989) and consistent findings were obtained by in situ sues with known occluding intercellular junctions that lack hybridization (Kalaria et al., 1989). The recent availability of the erythroid-type of glucose transporter may have other antibodies to the human erythrocyte GT and to other GT types types of glucose transporter proteins. permits their preciselocalization and tissue distribution, which is not yet known, but which was surmisedfrom mRNA blotting Facilitative glucosetransporter (GT) proteins are ubiquitous in or in situ hybridization studies. Immunocytochemical studies cell membranesof tissuesthat use glucose, where they play a documented the presenceof a high density of the erythroid-type crucial role in transporting the water-soluble o-glucose across GT in human (Kalaria et al., 1988) and dog (Gerhart et al., the lipid bilayer of plasma membranes(see reviews by Bell et 1989) brain microvessels and peripheral nerve perineurium al., 1990; Kasanicki and Pilch, 1990; Thorens et al., 1990). Of (Froehner et al., 1988) and in certain endothelial and epithelial the 4 or 5 members of the GT family that are thus far known, cells of the eye (Harik et al., 1990) with exquisite anatomical detail. About 25 yr ago, Crone predicted the existence of a GT in Received May 10, 1990; revised Aug. 16, 1990; accepted Aug. 17, 1990. endothelial cells of brain capillaries to allow the entry of glucose This work was supported by U.S. Public Health Service Grants HL-35617 and AG-00415 and bv the David S. Inealls Fund. Production of the monoclonal from the blood to the brain (Crone, 1961, 1963, 1965). His antibody was supported by the Swedish Natural Science Research Council. We opinions met with strong resistancebecause of the prevalent wish to thank Dr. Steven Emancipator for helpful discussions, Dr. Ora M. Rosen notion that glial processesformed the anatomical basis of the for donation of the GT antisera, and Dr. S. Ledbetter for donation of collagen type IV antiserum. We also thank Paul M. Whitney, Fares Abdallah, Peggy Richey, blood-brain barrier (BBB), and becausefew scientiststhen be- Sandra Siedlak, and Paul Mulvihill for technical assistance and Jeanette Barnhart lieved that the tiny endothelial cells of brain capillaries were for manuscrint oreoaration. Corresponhence’should be addressed to Dr. Sami I. Harik, Department of capableof undertaking so vital a task. In retrospect, the existence Neurology, University Hospitals of Cleveland, Cleveland, OH 44106. of a high density of the GT in brain endothelial cells is logical Copyright 0 1990 Society for Neuroscience 0270-6474/90/123862-l 1$03.00/O in view of the high brain metabolic rate for glucose,the isolation The Journal of Neuroscience, December 1990, 70(12) 3863 Figure 1. Immunocytochemically stained sections of monkey brain using monoclonal antibody to erythroid GT, later counterstained with hematoxylin. A, A section of the monkey occipital cerebral cortex. Roman numerals indicate the cortical layers. Pial vessels (arrowheads) were stained. Note the discrepancy in microvessel density among the cortical layers, which is highest in layers III and IV and lowest in layer I. The inset depicts a high-power view of a cortical microvessel. B, A section of the caudate nucleus, a subcortical gray matter region of the brain. C, A section of the internal capsule, a white matter region. Note the low vessel density in the white matter in comparison to that of gray matter regions depicted in A and B. D, A section through the cerebellum with its molecular (mol), Purkinje cell (PC) and granular layers (gr). There is an apparent lower density of microvessels in the molecular layer. E, A higher-power view through Sommer’s sector of the hippocampus. F, A similar view of the dentate gyrus. G, The nucleus locus coeruleus of the pons, with its dense vascular&y. H, A high-power view of the choroid plexus showing the brown reaction product mostly in the basal membranes of the choroid epithelium, but none in blood vessels (v) of the choroid. A-G were viewed under bright-field illumination, and H was viewed under differential interference contrast (Nomarski) illumination. Scale bars: A-G, 50 pm; inset in A and H, 10pm. The Journal of Neuroscience, December 1990, IO(12) 3865 of the brain behind capillaries with occluding interendothelial tical, thus reducing the possibility that the observed staining of a given junctions (i.e., the BBB), and the fact that brain capillaries, which structure is a cross-reaction artifact. Immunocytochemical stains. Paraffinsections (6 pm) werehydrated, constitute less than 1% of the brain weight, must transport glu- treated for 10 min with 10% normal goat serum in Tris-buffered saline cose for the much larger mass of surrounding neurons and glia. (pH, 7.6) and incubated overnight at 04°C with 1 of the primary The object of this work was to determine by immunocyto- antibodies in buffered saline containing 1% normal goat serum. Im- chemical methods the precise localization of the erythroid-type munocytochemical staining was performed according to the peroxi- dase-antiperoxidase procedure of Stemberger (1986) with 3’,3-di- GT in the monkey and rat brains and in various tissues of the aminobenzidine (Sigma) as cosubstrate. To appreciate histological rat, especially those that are known to possess barrier properties. detail, adjacent sections were stained with hemmatoxylin and eosin. Our hypothesis was that tissues that do not have barrier prop- Often, GT-immunostained sections were counterstained with hema- erties would allow easy penetration of the water-soluble glucose toxylin. Double-immunostaining for the GT (monoclonal antibody) and molecule and therefore should not have a high density of the type IV collagen was performed on sections of the rat brain and testis to determine the concordance between the 2 antigens in their