Histochem Cell Biol (2004) 121:201–207 DOI 10.1007/s00418-004-0633-9 ORIGINAL PAPER Katrin Elfeber · Alwin Köhler · Michael Lutzenburg · Christina Osswald · Hans-Joachim Galla · Otto W. Witte · Hermann Koepsell Localization of the Na + -d-glucose cotransporter SGLT1 in the blood-brain barrier Accepted: 30 January 2004 / Published online: 19 February 2004  Springer-Verlag 2004 Abstract Immunoreactivity of the Na + -d-glucose co- transporter SGLT1 was demonstrated in intracerebral capillaries of rat and pig. Immunostaining suggested that SGLT1 is located in the luminal membrane of the endothelial cells and in intracellular vesicles. Using in situ hybridization, SGLT1 mRNA was not detectable in intracerebral capillaries of non-treated or sham-operated Wistar rats. However, 1 day after a transient occlusion of the right middle cerebral artery, SGLT1 mRNA was detected in capillaries of both brain hemispheres. Expres- sion of SGLT1 was also demonstrated in primary cultures of capillary endothelial cells from pig using polymerase chain reaction after reverse transcription and western blotting. The data suggest that SGLT1 participates in transport of d-glucose across the blood-brain barrier and is upregulated after brain ischemia and reperfusion. Keywords Sugar transport · Sodium d-glucose cotransporter · SGLT1 · Blood-brain barrier · Brain ischemia Introduction d-glucose is the major energy source of the brain. Its translocation across the blood-brain barrier is tightly linked to cerebral glucose utilization (Hawkins et al. 1983). Facilitative diffusion systems of the GLUT trans- porter family and Na + -d-glucose cotransporters of the SGLT family have been identified as glucose transporters in brain (Maher et al. 1994; Poppe et al. 1997). Previous studies showed that the transporters GLUT1, GLUT3, and SGLT1 are expressed in neurons, and GLUT1 in brain capillaries (Maher et al. 1994; Poppe et al. 1997). It has been also reported that GLUT1 at the blood-brain barrier and in neurons is upregulated during ischemia (Harik et al. 1994). In the present study, we demonstrate that SGLT1 is expressed in the blood-brain barrier and is upregulated 1 day after occlusion of the middle cerebral artery. Materials and methods Antibody generation and purification Antibody rSGLT1-Ab was raised in rabbits against a peptide comprising amino acids 582–600 of rat SGLT1 (Lee et al. 1994). This sequence contains one identical amino acid as compared to other SGLT subtypes. The antibody against the corresponding peptide of porcine SGLT1 (pSGLT1-Ab, previously called Ab1) was raised as described (Poppe et al. 1997). The antibodies were affinity purified on their respective antigenic peptides. The peptides were coupled via a C-terminal cysteine to polyacrylamide particles using the SulfoLink kit from Pierce (Bonn, Germany). Immunohistochemistry Five-micron-thin cryosections were fixed for 10 min with acetone cooled to 20C. After washing with phosphate-buffered saline (PBS), the sections were blocked with PBS containing 0.1% (w/v) BSA-C (Biotrend) plus 0.1% (w/v) Tween 20 and incubated for 16 h at 4C with affinity-purified rSGLT1-Ab (1:100), affinity- purified pSGLT1-Ab (1:50), or monoclonal antibody against van Willebrandt factor (vWF), a marker for vascular endothelial cells (12.5 mg/ml; Roche Diagnostics, Mannheim, Germany). After washing with PBS, the slides were covered for 1 h at room temperature with goat anti-rabbit IgG antibody coupled to indo- carbocyanine (Cy-3) or goat anti-mouse F(ab 0 ) 2 coupled to carbo- cyanine (Cy-2). Secondary antibodies were obtained from Dianova (Hamburg, Germany). Antibody binding was detected by epifluo- rescence or laser scanning microscopy. As controls, we performed K. Elfeber · A. Köhler · C. Osswald · H. Koepsell ( ) ) Institute of Anatomy and Cell Biology, University Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany e-mail: Hermann@Koepsell.de Tel.: +49-931-312700 Fax: +49-931-312087 M. Lutzenburg · O. W. Witte Clinic of Neurology, University Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany H.-J. Galla Institute of Biochemistry, Wilhelm-Klemmstrasse 2, 48149 Münster, Germany