Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. 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-[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 Km and Vmax 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 Vmax; the Km 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 micrograms/ml) for 20 min did not affect the uptake of [3H]3MG or 2-deoxy-D-[3H]glucose ([3H]2DG).(ABSTRACT TRUNCATED AT 400 WORDS)
Takakura
Y
Y
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas, Lawrence 66045.
Kuentzel
S L
SL
Raub
T J
TJ
Davies
A
A
Baldwin
S A
SA
Borchardt
R T
RT
D019297:2,4-Dinitrophenol; D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D006601:Hexoses / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology
D004140:Dinitrophenols; D006601:Hexoses; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D003513:Cycloheximide; D010695:Phlorhizin; D005947:Glucose; D019297:2,4-Dinitrophenol; D010693:Phloretin