Blood-brain Barrier GLUT1 Glucose Transporter Research Articles

Evidence for translational control elements within the 5'-untranslated region of GLUT1 glucose transporter mRNA.

Recent studies have indicated that the blood-brain barrier GLUT1 glucose transporter is under post-transcriptional regulation. To begin functional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full-length synthetic GLUT1 mRNA, and both 5'- and 3'-untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5'- and 5'-/3'-UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5'-UTR cis-acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5'-UTR generated by PCR was subcloned at the HindIII site of the pGL2 located upstream of the luciferase 5'-UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5'-UTR (nucleotides 1-171) markedly increased the expression of luciferase, and disruption of luciferase-leading sequence with an unrelated 171-nucleotide fragment decreased its expression. Insertion of nucleotides 1-96 of the hGLUT1 5'-UTR retained most of the stimulatory effect, and nucleotides 123-171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79-171 and 154-171 of bGLUT1 5'-UTR had marginal effects on luciferase expression. The present data provide evidence suggesting that the 5'-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational control of the GLUT1 gene in mammalian cells.

Amplification of blood-brain barrier GLUT1 glucose transporter gene expression by brain-derived peptides.

Glucose is a critical nutrient for the brain, and the transport of this hexose from blood to brain is mediated by the blood-brain barrier (BBB) GLUT1 glucose transporter. The expression of the BBB-GLUT1 gene is compromised in different pathological conditions and it is modulated by brain trophic factors. The brain-derived peptide preparation Cerebrolysin (Cl, EBEWE, Austria) increases the expression of the BBB-GLUT1 via mRNA stabilization. In order to gain more insights into the mechanism of BBB-GLUT1 gene regulation, the present investigation studied the effect of Cl on the expression of both the GLUT1 protein and GLUT1 reporter genes in brain endothelial cultured cells (ECL). Cl markedly increased the expression of reporter genes containing GLUT1 translational control elements and cis-acting elements involved in the stabilization of the GLUT1 mRNA transcript in a dose dependent manner. Cl produced only marginal effects on the reporter gene control lacking the GLUT1 regulatory elements. In parallel experiments, Cl markedly increased the uptake of 3H-2-deoxy-D-glucose and the levels of the GLUT1 protein measured by ELISA. Data presented here demonstrate: (i) that Cl increases the expression of BBB-GLUT1 reporter genes containing regulatory cis-elements involved in the stabilization and translation of the GLUT1 transcript; (ii) that the effect on both regulatory elements cooperates to increase gene expression; and (iii) that the increased levels of the BBB-GLUT1 reporter genes in Cl-treated ECL cells are associated with an increase in the glucose uptake and in the expression of the GLUT1 protein.

Cerebrolysin ® improves memory performance and the brain bioelectrical activity pattern in elderly humans

The blood-brain barrier (BBB) GLUT1 glucose transporter gene expression is known to be regulated by putative brain trophic factors. Therefore, the present study investigated the effect of a brain-derived peptide rich preparation containing a neurotrophic factor-like action [Cerebrolysin (Cl), EBEWE, Austria]. In cultures of brain capillary endothelial cells, Cl induced a transient increase in the abundance of BBB-GLUT1 relative to actin measured by reverse transcription-polymerase chain reaction during the first 2 h of incubation, whereas a significant reduction in the GLUT1 transcript was observed at 20 and 48 h. In addition, Cl abolished the fall in GLUT1 levels induced by actinomycin D. The present data suggest that brain-derived factors in Cl are able to modulate the expression of the BBB-GLUT1 gene increasing the BBB-GLUT1 transcript stability.

Post-transcription modulation of the blood-brain barrier GLUT1 glucose transporter by brain-derived factors.

Brain-derived peptides or factors in the brain-derived preparation Cerebrolysin (Cl, EBEWE, Austria) increase the expression of the blood-brain barrier (BBB) GLUT1 glucose transporter via mRNA stabilization. The post-transcriptional regulation of the BBB-GLUT1 gene is principally exerted by interaction of cis-regulatory elements located in the 3'-untranslated region of GLUT1 mRNA with cellular trans-acting factors (TAF). UV-cross linking and RNase T1 protection studies demonstrated the presence of 2 major GLUT1 RNA-TAF complexes named p88 (stabilizing) and the p44 (destabilizing). The p88 TAF was detected in cytosol of brain endothelial cultured cells (ECL) as a duplex of molecular weight of approximately 88 kDa, which were defined A and B (high and low MW, respectively). Cl markedly increased the abundance of the BBB-GLUT1 p88 TAF (complex B) in ECL cells, without changes in the levels of the p88 complex A. This was also confirmed by antisense oligomer displacement of the GLUT1 RNA-TAF complex formation. Cl per se, did not bind to the GLUT1 mRNA, nor induced the expression of the destabilizing p44 TAF. Data presented here suggest that the increased stabilization of the GLUT1 transcript induced by Cl is associated with augmented levels of the GLUT1 stabilizing p88 TAF.

The drug cerebrolysin and its peptide fraction E021 increase the abundance of the blood-brain barrier GLUT1 glucose transporter in brains of young and old rats.

The brain-derived peptidergic drug Cerebrolysin has been found to support the survival of neurons in vitro and in vivo. In the present study, we investigated the effects of Cerebrolysin and its peptide preparation E021 on spatial learning and memory, as well as on the abundance of the blood-brain barrier GLUT1 glucose transporter (GLUT1) in 2-month-old and 24-month-old rats. Young rats were treated with the drugs or saline (2.5 ml/kg/day) daily on postnatal days 1-7, and old rats for 19 consecutive days. For behavioural testing the Morris water maze was used. The abundance of GLUT1 was determined in brain slices by immunocytochemistry. Quantification of the density of the GLUT1 immunostaining was performed using light microscopy and a computerised image analysing system. All drug-treated rats, young and old, exhibit shorter escape latencies in the water maze, on all testing days (p < 0.01), indicating improved cognitive performance. Immunohistochemical data show an age-related decrease of the density of GLUTI (p < 0.05). In young animals, the administration of the drugs led to an increase of the abundance of GLUT1 in all experimental groups (p < 0.01). In old rats, the treatment with Cerebrolysin, but not with E021, resulted in an increase in the immunoreactive GLUT1 (p < 0.01). The elevated abundance of GLUT1 after the administration of both peptidergic substances might be supportive for the cognitive effects of this drug, by causing an improved nutritional supply of glucose to the neurons.

In vivo upregulation of the blood-brain barrier GLUT1 glucose transporter by brain-derived peptides.

Glucose is the critical metabolic fluid for the brain, and the transport of this nutrient from blood to brain is limited by the blood-brain barrier (BBB) GLUT1 glucose transporter. The expression of the BBB-GLUT1 gene is augmented in brain endothelial cultured cells incubated with brain-derived trophic factors and the brain-derived peptide preparation Cerebrolysin (C1, EBEWE, Austria). The aim of the present investigation was to determine if C1 induces similar changes in the expression of the BBB-GLUT1 gene following its administration to rats in vivo. The BBB glucose transporter activity was investigated with the intracarotid artery perfusion technique using [3H]diazepam as cerebral blood flow marker. The acute or chronic administration of C1 markedly increased the brain permeability surface area of D-[14C]glucose compared to controls (D-[14C]glucose/[3H]diazepam ratio, 1.6- to 1.9-fold increase in frontal cortex, P < 0.05). Increased activity of the BBB glucose transporter was correlated with a significant rise in the abundance of the BBB-GLUT1 protein measured by both Western blot analysis and immunocytochemistry, and with a decrease in the transcript levels of this transporter. Data presented here demonstrate that the in vivo administration of Cl increases the transport of glucose from blood to brain via BBB-GLUT1 gene expression.

Brain-derived peptides increase blood–brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization

The present investigation studied the effect of the brain-derived peptide preparation Cerebrolysin (Cl, EBEWE, Austria) on the turnover rate and gene expression of the blood–brain barrier (BBB) GLUT1 glucose transporter mRNA. Studies were performed in brain endothelial cultured cells transfected with the human (h) GLUT1 transcript. In control cells, the full length 2.8 Kb hGLUT1 mRNA was rapidly degraded following transfection, and the abundance of this transcript at 4 and 6 h was comparable to background mRNA levels seen in cells transfected without hGLUT1 mRNA. On the contrary, the decay of the hGLUT1 mRNA was stabilized in Cl-treated cells resulting in a marked reduction in the fractional turnover rate (72.4 and 4.0%/h, control and Cl, respectively). In parallel experiments, Cl induced a significant increase in the levels of immunoreactive GLUT1 protein measured by enzyme-linked immunosorbent assay (ELISA). In conclusion, data presented here demonstrate that factors in Cl increase BBB-GLUT1 transcript stability, and that this is associated with an induction of BBB-GLUT1 gene expression in brain endothelial cultured cells.

Molecular regulation of the blood-brain barrier GLUT1 glucose transporter by brain-derived factors.

Glucose is the crucial metabolic fluid for the brain, and the transport of this nutrient from blood to brain is limited by the blood-brain barrier (BBB) GLUT1 glucose transporter. The activity of this transporter is altered in different pathophysiological conditions including Alzheimer's disease. The expression of the BBB-GLUT1 gene is directed by brain trophic factors, and the brain-derived peptide preparation Cerebrolysin (Cl, EBEWE, Austria), used in the treatment of Alzheimer's disease, increases the BBB-GLUT1 mRNA stability and the expression of the BBB-GLUT1 gene. In the present investigation, Cl markedly increased (p < 0.001) the expression of a BBB-GLUT1 reporter gene, named clone 753, that contains an important regulatory cis-acting element involved in the stabilization of this transcript in brain endothelial cultured cells (ECL). In experiments with a reporter gene lacking this regulatory element, Cl produced only a minimal fraction of the effect observed with clone 753. UV-cross linking/PAGE experiments showed that the GLUT1 transcript reacts with ECL cytosolic proteins to form a RNA/protein complex of approximately 80 kDa. The abundance of this cis/trans acting complex was found to be increased in Cl-treated cells. Overall, data presented here demonstrate that i) Cl increases the expression of a BBB-GLUT1-luciferase reporter gene containing a region of the 3'-untranslated region of BBB-GLUT1 mRNA with important regulatory cis-acting elements involved in the stabilization of this transcript, and ii) the increased expression of this BBB-GLUT1 reporter gene was associated with augmented abundance of a transacting factor that binds to the cis-acting element described in (i), suggesting that this association may be involved in the stabilization of GLUT1 mRNA induced by Cl.

Interictal seizure resections show two configurations of endothelial Glut1 glucose transporter in the human blood-brain barrier.

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood-brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood-brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

The 5′-Untranslated Region of GLUT1 Glucose Transporter mRNA Causes Differential Regulation of the Translational Rate in Plant and Animal Systems

The blood–brain barrier GLUT1 glucose transporter is under post-transcriptional regulation, and the 5′-untranslated region (5′-UTR) of the GLUT1 mRNA increases its translational rate in mammalian cells. To obtain more insight into the mechanism of translational control of GLUT1, the present investigation studied the translational efficiency of capped full-length synthetic human (h) and rabbit (rab) GLUT1 mRNA and both 5′- and 3′-UTR deleted hGLUT1 mRNAs in both mammalian and plant cell free translation systems. Translation efficiency of both h- and rabGLUT1 mRNA was increased 3- to 6-fold in rabbit retyculocyte lysate (RRL) compared with wheat germ extract (WGE). Confirming previous observations, deletion of 5′- and 5′/-3′-UTR markedly reduced the translation efficiency of the h-GLUT1 transcript in RRL. On the contrary, these deletions markedly increased the translation of GLUT1 in WGE. The present data provide additional evidence suggesting that the 5′-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational activation of the GLUT1 gene in mammalian cells and that factors involved in this cis/trans-acting interaction are either absent or down-regulated in plant systems.

Brain-derived peptides increase the expression of a blood-brain barrier GLUT1 glucose transporter reporter gene

The brain-derived peptide preparation Cerebrolysin (Cl; EBEWE, Austria) increases the stability of blood-brain barrier (BBB)-GLUT1 transcript. To determine if the increase in BBB-GLUT1 mRNA stability is associated with an augmentation of gene expression, the present investigation studied the effect of Cl on the expression of a BBB-GLUTI-luciferase reporter gene in brain endothelial cultured (ECL) cells. Dose: response studies showed that Cl markedly increased the expression of luciferase when the BBB-GLUT1-reporter gene was used. On the contrary, Cl produced no changes in the expression pattern of the control reporter gene, which lacks the GLUT1 regulatory sequence. Desensitization of the protein kinase C (PKC) receptor with the phorbol ester TPA, or inhibition with either 1-(5-isoquinolinylsuIfonyl)-2-methylpiperazine (H7) or staurosporine, had no effect on the increased levels of luciferase induced by Cl. Transfection efficiency was determined by measuring intracellular levels of the expression vector using a quantitative polymerase chain reaction (PCR) assay. The data presented here demonstrate that Cl increases BBB-GLUT1 gene expression in ECL cells through a mechanism that appears to be independent of activation of PKC.

Molecular regulation of the blood-brain barrier GLUT1 glucose transporter by brain-derived peptides.

The blood-brain barrier (BBB) GLUT1 glucose transporter represents the limiting step in transport of glucose from blood to brain, and its activity has been shown to be altered in different pathophysiological conditions such as hypoglycemia, brain tumors, development, and Alzheimer’s disease. The molecular regulation of the BBB-GLUT1 gene is directed at both transcription and post-transcriptional stabilization of mRNA by brain-derived factors present in a bovine brain homogenate [Boado, et al. (1994) Mol Brain Res 22: 259] and in the brain-derived peptide rich preparation Cerebrolysin® (CL, EBEWE, Austria) [Boado (1994) Eur J Neurosci [Suppl] 7: 58], respectively. Since increased GLUT1 mRNA stability was correlated with augmented GLUT1 immunoreactive protein in hypoglycemia and with the antidiabetic agent pioglitazone, it is possible that Cl increases the translation efficiency of BBB-GLUT1 transcript. Therefore, in order to test this hypothesis, the present investigation studied the effect of CL on the expression of the BBB-GLUT1 gene in brain endothelial cultured cells transfected with a luciferase expression vector containing the complete 171 nucleotides of the 5′-untranslated region of the human GLUT1 mRNA, which possesses the GLUT1 translational control elements. Dose response studies showed that Cl at concentrations of 1–10µl/m1 culture media produced a marked increase in the levels of luciferase (122–324% of control), whereas no significant changes were observed with 25 or 50 µl/ml.

Upregulation of Blood-Brain Barrier GLUT1 Glucose Transporter Protein and mRNA in Experimental Chronic Hypoglycemia

An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.

Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia

Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia

Brain-derived peptides regulate the steady state levels and increase stability of the blood-brain barrier GLUT1 glucose transporter mRNA

The blood-brain barrier (BBB) GLUT1 glucose transporter gene expression is known to be regulated by putative brain trophic factors. Therefore, the present study investigated the effect of a brain-derived peptide rich preparation containing a neurotrophic factor-like action [Cerebrolysin (Cl), EBEWE, Austria]. In cultures of brain capillary endothelial cells, Cl induced a transient increase in the abundance of BBB-GLUT1 relative to actin measured by reverse transcription-polymerase chain reaction during the first 2 h of incubation, whereas a significant reduction in the GLUT1 transcript was observed at 20 and 48 h. In addition, Cl abolished the fall in GLUT1 levels induced by actinomycin D. The present data suggest that brain-derived factors in Cl are able to modulate the expression of the BBB-GLUT1 gene increasing the BBB-GLUT1 transcript stability.

Measurement of Blood‐Brain Barrier GLUT1 Glucose Transporter and Actin mRNA by a Quantitative Polymerase Chain Reaction Assay

The expression of the blood-brain barrier GLUT1 glucose transporter is down-regulated in brain capillary endothelial cells in tissue culture. Consequently, the study of the regulation of this low-abundance transcript requires the isolation of poly(A)+ mRNA from relatively large numbers of brain endothelial cells in culture (approximately 10(7)). Therefore, in order to facilitate studies with smaller amounts of cells, we describe here a quantitative polymerase chain reaction (PCR) assay to measure the mRNA of GLUT1 and the mRNA of the housekeeping gene, actin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endothelial cell line (ECL cells) in 25-mm 6-well cluster dishes, and total or poly(A)+ RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)18 primer, PCR was performed with sense and antisense primers for bovine GLUT1 and gamma-actin, respectively. Reactions were performed in the presence of 2.5 microCi of [alpha-32P]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship between RNA-cDNA and PCR products was observed for GLUT1 after 30 cycles, and for actin after 15 PCR cycles. The method was reproducible within specified ranges of starting RNA-derived cDNA, and the intraassay coefficient of variation averaged 7.2 +/- 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries >> EP > ECL. In addition, it is demonstrated that tumor necrosis factor-alpha induced a three- to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced expression of the blood-brain barrier GLUT1 glucose transporter gene by brain-derived factors

The blood-brain barrier GLUT1 glucose transporter is localized in brain to the capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo. However, its expression is markedly downregulated in cultured bovine brain capillary endothelium (ECL cells), possibly due to the absence of brain-derived or astrocyte trophic factors in the tissue culture medium. To examine this hypothesis, we studied the effect of a bovine brain homogenate (BBH), and conditioned media and plasma membranes obtained from the rat C6 glioma cell line, on the abundance of the GLUT1 transcript in ECL cells. BBH induced a significant increase in the abundance of both GLUT1 and actin mRNAs, and this effect was dose and time dependent. The increase in the GLUT1 mRNA levels correlated with an increase in the transcriptional rate of this gene measured by nuclear run-on experiments. C6 conditioned media and C6 plasma membranes had no effect on the abundance of either GLUT1 or actin mRNA. To determine whether known growth factors cause BBH-like induction of GLUT1 and actin mRNAs, a series of growth factors was also tested. EGF and PDGF had no effect on the levels of these mRNAs. Basic FGF had a moderate effect and TNFα partially mimicked the effect of BBH on both GLUT1 and actin transcripts. The present data suggests that brain-derived trophic factors present in BBH stimulate BBB-GLUT1 glucose transporter gene expression in ECL cells through a transcriptional mechanism. Although this effect was partially mimicked by TNFα, C6 cell membranes or C6 conditioned media were unable to induce changes in the abundance of GLUT1 mRNA. Therefore, BBH may be a useful model to study the characterization of soluble brain-derived trophic factors involved in the induction of BBB-GLUT1 gene expression.

The Human Brain GLUT1 Glucose Transporter: Ultrastructural Localization to the Blood—Brain Barrier Endothelia

Immunogold electron microscopy was used to examine human brain resections to localize the GLUT1 glucose transporter. The tissue examined was obtained from a patient undergoing surgery for treatment of seizures, and the capillary profiles examined had characteristics identical to those described previously for active, epileptogenic sites (confirmed by EEG analyses). A rabbit polyclonal antiserum to the full-length human erythrocyte glucose transporter (GLUT1) was labeled with 10-nm gold particle-secondary antibody conjugates and localized immunoreactive GLUT1 molecules in human brain capillary endothelia, with < 0.25% of the particles beyond the capillary profile. Erythrocyte membranes were also highly immunoreactive, whereas macrophage membranes were GLUT1-negative. The number of immunoreactive sites per capillary profile was observed to be 10-fold greater in humans than in previous studies of rat and rabbit brain capillaries. In addition, half of the total number of immunoreactive gold particles were localized to the luminal capillary membrane. We suggest that the blood-brain barrier GLUT1 glucose transporter is up-regulated in seizures, and this elevated transporter activity is characterized by increased GLUT1 transporters, particularly on the luminal capillary membranes. In addition, acute modulation of glucose transporter activity is presumed to involve translocation of GLUT1 from cytoplasmic to luminal membrane sites, demonstrable with quantitative immunogold electron microscopy.

Developmental modulation of blood-brain barrier and choroid plexus GLUT1 glucose transporter messenger ribonucleic acid and immunoreactive protein in rabbits

The transport of glucose across the brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, is developmentally up-regulated in the postnatal period, as the brain switches from combustion of circulating ketone bodies to glucose. The principle transporter mediating the uptake of circulating glucose across the BBB is the GLUT1 isoform. To further define molecular mechanisms underlying developmental modulation of the BBB GLUT1 transporter, the amounts of brain microvessel GLUT1 mRNA and immunoreactive protein were quantitated. In addition, an immunocytochemical analysis of GLUT1 expression at the choroid plexus in developing brain was performed, since this transporter isoform is selectively expressed at the choroid plexus epithelium basolateral membrane. Quantitative Western blotting employing purified human erythrocyte glucose transporter as an assay standard showed that the concentration of immunoreactive GLUT1 protein in 70-day-old rabbit brain microvessels (111 +/- 3 pmol/mg protein) was not significantly different from the concentration of D-glucose-displaceable cytochalasin-B-binding sites (102 +/- 25 pmol/mg protein). Thus, GLUT1 is the principle isoform mediating glucose transport across the developing BBB. Quantitative Western blotting was performed on microvessels isolated from brains of rabbits on postnatal days 1, 14, 28, and 70. The concentrations of immunoreactive microvessel GLUT1 at these four stages of development were 13 +/- 2, 4 +/- 1, 49 +/- 2, and 111 +/- 3 pmol/mg protein, respectively. Capillary depletion analysis showed that essentially all of brain GLUT1 mRNA arises from the microvascular fraction, and Northern analysis of 10 micrograms poly(A)+ RNA from brains of rabbits 1, 14, 28, and 70 days postnatally showed a preferential stabilization of the GLUT1 mRNA compared to mRNA for two cytoskeletal proteins, actin and tubulin. Immunocytochemical analysis of immunoreactive GLUT1 in choroid plexus epithelia showed the following developmental modulation of the transporter protein: 1 day < 14 days < 28 days > 70 days. The concentration of immunoreactive GLUT1 at the basolateral membrane of choroid plexus epithelium at 28 days was much greater than the immunostaining of rabbit brain microvessels at the corresponding age. In conclusion, these studies show that immunoreactive GLUT1 protein initially undergoes down-regulation between birth and 14 days and then undergoes marked up-regulation between 14 and 70 days. Conversely, the concentration of GLUT1 mRNA is virtually unchanged in brain of rabbits at 70 vs. 14 days postnatally. These combined data suggest that a principle mechanism underlying the developmental regulation of GLUT1 at the BBB may be posttranscriptional.

Developmental modulation of blood-brain barrier and choroid plexus GLUT1 glucose transporter messenger ribonucleic acid and immunoreactive protein in rabbits.

The transport of glucose across the brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, is developmentally up-regulated in the postnatal period, as the brain switches from combustion of circulating ketone bodies to glucose. The principle transporter mediating the uptake of circulating glucose across the BBB is the GLUT1 isoform. To further define molecular mechanisms underlying developmental modulation of the BBB GLUT1 transporter, the amounts of brain microvessel GLUT1 mRNA and immunoreactive protein were quantitated. In addition, an immunocytochemical analysis of GLUT1 expression at the choroid plexus in developing brain was performed, since this transporter isoform is selectively expressed at the choroid plexus epithelium basolateral membrane. Quantitative Western blotting employing purified human erythrocyte glucose transporter as an assay standard showed that the concentration of immunoreactive GLUT1 protein in 70-day-old rabbit brain microvessels (111 +/- 3 pmol/mg protein) was not significantly different from the concentration of D-glucose-displaceable cytochalasin-B-binding sites (102 +/- 25 pmol/mg protein). Thus, GLUT1 is the principle isoform mediating glucose transport across the developing BBB. Quantitative Western blotting was performed on microvessels isolated from brains of rabbits on postnatal days 1, 14, 28, and 70. The concentrations of immunoreactive microvessel GLUT1 at these four stages of development were 13 +/- 2, 4 +/- 1, 49 +/- 2, and 111 +/- 3 pmol/mg protein, respectively. Capillary depletion analysis showed that essentially all of brain GLUT1 mRNA arises from the microvascular fraction, and Northern analysis of 10 micrograms poly(A)+ RNA from brains of rabbits 1, 14, 28, and 70 days postnatally showed a preferential stabilization of the GLUT1 mRNA compared to mRNA for two cytoskeletal proteins, actin and tubulin. Immunocytochemical analysis of immunoreactive GLUT1 in choroid plexus epithelia showed the following developmental modulation of the transporter protein: 1 day < 14 days < 28 days > 70 days. The concentration of immunoreactive GLUT1 at the basolateral membrane of choroid plexus epithelium at 28 days was much greater than the immunostaining of rabbit brain microvessels at the corresponding age. In conclusion, these studies show that immunoreactive GLUT1 protein initially undergoes down-regulation between birth and 14 days and then undergoes marked up-regulation between 14 and 70 days. Conversely, the concentration of GLUT1 mRNA is virtually unchanged in brain of rabbits at 70 vs. 14 days postnatally. These combined data suggest that a principle mechanism underlying the developmental regulation of GLUT1 at the BBB may be posttranscriptional.