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

Abstract

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 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 [ 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 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 μg/ml) for 20 min did not affect the uptake of [ 3H]3MG or 2-deoxy d-[ 3H]glucose ([ 3H]2DG). The present study demonstrated that hexoses can be taken up by cultured bovine BMECs by a carrier-mediated, facilitated diffusio 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.