HEXOSE UPTAKE IN PRIMARY CULTURES OF BOVINE BRAIN MICROVESSEL ENDOTHELIAL-CELLS .1. 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-[H-3]methyl-D-glucose ([H-3]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [H-3]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 [H-3]3MG uptake and the apparent K(m) and V(max) 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 [3-H]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 (3-H]3MG, whereas L-glucose, D-fructose, and sucrose did not affect [3-H]3MG uptake by BMECs. [3-H]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 [3-H]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 [3-H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [3-H]3MG uptake irrespective of exposure time. The increase in [3-H]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 [3-H]3MG uptake to control levels in 48 h. The D-glucose-starvation-induced increase in [H-3]3MG uptake was shown to result from an increase in V(max); the K(m) 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-mu-g/ml) for 20 min did not affect the uptake of [H-3]3MG or 2-deoxy-D-[H-3]glucose ([H-3]2DG). The present study demonstrated that hexoses can be taken up by cultured bovine BMECs by a carrier-mediated, facilitated diffusion mechanism, similar to the mechanism observed in the blood-brain barrier (BBB) and that the uptake by bovine BMECs was regulated by low levels of D-glucose but not by high levels of D-glucose or by insulin.