Abstract
GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). GLUT1, GLUT8, and GLUT9 mRNAs are detected in bEnd.3 cells where GLUT1 mRNA levels are 33-fold greater than levels of GLUT8 or GLUT9 mRNA. Neither GLUT1 mRNA nor total protein levels are affected by acute metabolic stress. Cell surface biotinylation reveals that plasma membrane GLUT1 levels are increased 2-3-fold by metabolic depletion, although cell surface Na(+),K(+)-ATPase levels remain unaffected by ATP depletion. Treatment with the AMP-activated kinase agonist, AICAR, increases V(max) for net 3-O-methylglucose uptake by 2-fold. Glucose depletion and treatment with potassium cyanide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and AICAR also increase AMP-dependent kinase phosphorylation in bEnd.3 cells. These results suggest that metabolic stress rapidly stimulates blood-brain barrier endothelial cell sugar transport by acute up-regulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.
Highlights
Grants DK 36081 and DK 44888. □S The on-line version of this article contains supplemental Fig. 1. 1 To whom correspondence should be addressed: 364 Plantation St., LRB Rm
We show that ATP depletion of bEnd.3 cells increases Vmax for sugar transport and increases plasma membrane GLUT1 levels without changing endothelial cell GLUT1 mRNA or total GLUT1 protein levels
Cells—ATP levels were measured in bEnd.3 cells treated with PBS containing 5 mM glucose in the absence and presence of either 5 mM KCN or 8 g/ml FCCP for up to 2 h (Fig. 1A)
Summary
Tissue Culture— bEnd. cells were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin solution (Invitrogen) at 37 °C in a humidified 5% CO2 incubator. Cells—Confluent bEnd. cells in 12-well dishes were washed twice with DPBS and treated with DPBS ϩ 5 mM glucose, DPBS ϩ glucose ϩ 5 mM KCN, or DPBS ϩ glucose ϩ 8 g/ml FCCP for 10 min at 37 °C. Cells were serum-depleted in DMEM containing 25 mM glucose for 2 h prior to measuring uptake, washed with 1.5 ml DPBS, and allowed to incubate at 37 °C for 15 min. A, time course of ATP depletion of bEnd. cells incubated with PBS containing 5 mM glucose (F), 5 mM glucose ϩ 5 mM KCN (XI), or 5 mM glucose ϩ 8 g/ml FCCP (Œ). Cells were treated with PBS containing 5 mM glucose (F), 5 mM glucose ϩ 5 mM KCN (XI), or 5 mM glucose ϩ 8 g/ml FCCP (Œ) for 10 min and restored to normal growth media (PBS plus 5 mM glucose) for the times indicated. Sugar uptake data were fitted to the Michaelis-Menten Equation 1, v ϭ
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