Acute Modulation of Sugar Transport in Brain Capillary Endothelial Cell Cultures during Activation of the Metabolic Stress Pathway

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-Omethylglucose 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 upregulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.The cells of the mammalian brain do not contain large stores of glycogen. It is therefore essential that glucose uptake by the brain exceeds glucose utilization to maintain proper brain function. To enter the brain, serum glucose must cross the bloodbrain barrier, an epithelium comprising endothelial cells connected by tight junctions that prevent paracellular diffusion of glucose and other nutrients. Thus, glucose transport into the brain requires trans-endothelial cell transport. This process is catalyzed by the glucose transport protein GLUT1, which is expressed at both luminal and abluminal membranes of the endothelium (1-5).Endothelial cells of the blood-brain barrier (bEND) 2 differ from those of the peripheral circulatory system (pEND) in several important ways. 1) bEND cells contain 2-5-fold more mitochondria than pEND cells (6). 2) Brain capillary walls (comprising bEND cells) are 40% thinner than capillary walls of the peripheral circulation (7). 3) pEND cells present significantly fewer tight junctions than bEND cells (8). 4) bEND cell tight junction complexes result in polarized cell surface protein expression that is less marked or absent in pEND cells (8). The resulting bEND cell architecture may give rise to behaviors that differ from those of pEND cells but that resemble those of other metabolically active cells a...

AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress

“…Several forms of acute metabolic stress, including glucose starvation, KCN or FCCP treatment induce AMPK phosphorylation and sugar transport stimulation (14) in the cultured brain microvascular endothelial cell line bEnd.3 (34). These observations establish that immortalized and primary cultures of cerebral microvasculature endothelial cells share a common response to cellular metabolic stress but do not directly address the role of AMPK activation in controlling GLUT1-mediated sugar uptake.…”

“…During chronic metabolic stress [e.g., hypoxia (24) and hypoglycemia (4,33)], endothelial cell GLUT1 gene and protein expression increases in vitro (3) and in vivo (5), thereby increasing blood-brain barrier sugar transport. In contrast, acute metabolic stress [e.g., short-lived hypoglycemia, hypoxia, metabolic poisoning (14), or seizures (10,35)] is without effect on GLUT1 expression but promotes recruitment of intracellular GLUT1 to the plasma membrane (16), thereby increasing blood-brain barrier sugar transport (14). The signals that regulate bloodbrain barrier sugar transport during acute metabolic stress are not known.…”

Proinflammatory Stimulants Promote the Expression of a Promiscuous G Protein-Coupled Receptor, mFPR2, in Microvascular Endothelial Cells

“…Murine microvascular endothelial cell line bEnd.3 [22] was obtained from cell bank of the Chinese Academy of Science. Dulbecco's modified eagle's medium (DMEM) was purchased from Gibco BRL (Burlington, ON, Canada).…”

Sequence Determinants of GLUT1 Oligomerization

“…These blots reveal an array of G4-Ab-reactive proteins of M r(app) 48,000 -75,000. This reflects GLUT1 heterogeneous glycosylation because GLUT1 treatment with peptide: N-glycosidase F causes the array to collapse to 1-2 more mobile bands (39). The 30 kDa band is an occasionally observed, proteolytic fragment of G1c4 and is also observed in non-transfected, GLUT1-expressing cells using GLUT1-specific antibodies (34).…”