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(Stroke. 1998;29:844-849.)
© 1998 American Heart Association, Inc.

Original Contributions

Low Glucose Enhances Na⁺/Glucose Transport in Bovine Brain Artery Endothelial Cells

Tomoyuki Nishizaki, MD, PhD; Toshiyuki Matsuoka, MD

From the Department of Physiology, Kobe University School of Medicine, Kobe, Japan.

Correspondence to T. Nishizaki, MD, PhD, Department of Physiology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

	Abstract

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Background and Purpose—Brain arteries are structurally characterized by the tight junctions of the endothelium and by no vasa vasorum that feed arteries themselves. This raises the question of how brain arteries are provided with glucose. A possible explanation is that glucose uptake into arteries may be mediated by both GLUT1, a facilitative glucose transporter, and a Na⁺/glucose cotransporter (SGLT)-like glucose transporter. The functional role for the SGLT-like glucose transporter, however, is unknown. In the present study we investigated SGLT-like glucose transporter–operated glucose uptake into brain arterial endothelial cells by recording glucose-evoked Na⁺ currents and monitoring uptake of [³H]-2-deoxy-D-glucose ([³H]-2-DOG).

Methods—Endothelial cells were cultured from bovine cerebral cortical arteries. Whole-cell patches were made to cells, and glucose-evoked currents were recorded. Cells were incubated with [³H]-2-DOG, and the uptake was determined by a liquid scintillation counter.

Results—Glucose and -methyl-D-glucoside (MeDG), a specific compound for the SGLTs, evoked Na⁺ currents in a whole-cell voltage-clamp configuration, and the currents were enhanced in cells with over 30 minutes' preincubation in glucose-free media. Glucose-induced currents were inhibited by MeDG, by the selective SGLT inhibitor phlorizin, by dinitrophenol (DNP), an inhibitor of energy metabolism, or by deletion of Na⁺ from extracellular solution, which indicates that glucose uptake into endothelial cells was mediated by a Na⁺- and energy-dependent glucose transporter. Notably, the currents were desensitized, reduced in a glucose concentration–dependent manner, and markedly inhibited by either a second application of glucose or the addition of glucose to the patch electrode filling solution; they were potentiated, however, by treatment with cytochalasin B, a GLUT1 to GLUT5 inhibitor. Consistent with the results of patch-clamp recordings, uptake of [³H]-2-DOG into endothelial cells was enhanced by glucose-free insult, and the enhancement was mediated by an SGLT-like glucose transporter.

Conclusions— The results presented demonstrate that an SGLT-like glucose transporter takes part in glucose uptake into brain artery endothelial cells and that the uptake is regulated by intracellular glucose concentrations; glucose-free insult and the ensuing low cytosolic glucose enhance activity of the SGLT-like glucose transporter. The SGLT-like glucose transporter in the brain arterial endothelium thus may be important in the maintenance of an adequate glucose concentration in the arterial wall under conditions of stress, such as hypoglycemia.

Key Words: sodium-glucose transport system • cerebral arteries • endothelium • glucose

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Brain arteries exhibit an unique structure distinct from body arteries; they are lined with the tight junctions of the endothelium and have no vasa vasorum to feed the arterial wall.¹ One therefore assumes that a carrier protein for glucose, a glucose transporter, may provide brain arteries with the major energy source glucose. Indeed, GLUT1, a facilitative glucose transporter, is shown to be expressed in the rat brain capillary endothelium^{2 3} and in cultured endothelial cells from bovine cerebral cortical arteries,⁴ thus supporting this hypothesis. In addition, we earlier found that an SGLT-like glucose transporter is expressed in cultured brain artery endothelial cells as well as GLUT1.⁴ This suggests the possibility that this transporter may be also involved in glucose uptake into endothelial cells. It is unknown, however, whether glucose is actually taken up into endothelial cells by this transporter, how this transporter is regulated, or what functional role this transporter has.

To address these questions, we monitored glucose-evoked Na⁺ currents in cultured endothelial cells from bovine cerebral cortical arteries and assayed uptake of [³H]-2-DOG into cells. We show here that low glucose enhances activity of the SGLT-like glucose transporter and that this transporter may have a crucial role in the maintenance of an adequate glucose concentration in the arterial wall under conditions of stress, such as hypoglycemia.

	Materials and Methods

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Cell Culture
Bovine brain endothelial cells were cultured as described previously.⁴ Briefly, endothelial cell sheets were mechanically isolated from bovine cerebral cortical arteries 300 to 500 µm in diameter. The explants were plated on collagen-coated dishes and grown in Dulbecco's modified Eagle's medium with 20% fetal bovine serum, 1 ng/mL bovine pituitary fibroblast growth factor, 50 IU/mL penicillin, and 50 mg/mL streptomycin. The third-passage cells grown in 6-well, collagen-coated dishes and in coverslips were used for assay of 2-DOG uptake and for patch-clamp recording, respectively.

Immunohistochemistry
The brain arteries, endothelial cell sheets, and cultured cells were fixed in 100% methanol, incubated in 0.3% H₂O₂/methanol, and blocked in 10% normal goat serum before incubation with anti-human factor VIII/von Willebrand factor antibody (1:1000). The cells were then stained with peroxidase-conjugated donkey anti-rabbit IgG and 3-amino-9-ethylcarbazole, followed by cell nucleus staining with hematoxylin.

Electrophysiology
Cells were transferred to the recording chamber and continuously superfused at room temperature (20°C to 22°C) with glucose-free extracellular solution ([mmol/L] 145 NaCl, 5 KCl, 2.4 CaCl₂, 10 HEPES, 0.3x10^-3 tetrodotoxin, pH 7.4) after treatment with 10 mmol/L glucose-containing or glucose-free extracellular solution at 37°C for more than 30 minutes. For Na⁺-free extracellular solution, 145 mmol/L NaCl was replaced with 145 mmol/L LiCl. Whole-cell patches were made to the cells with use of patch electrode filling solution consisted of (mmol/L) 150 KCl, 5 EGTA, 0.5 ATP, and 10 HEPES, pH 7.2. Membrane currents from whole-cell voltage-clamp were recorded by an Axopatch-200A amplifier (Axon Instrument Inc). After formation of whole-cell patches, series resistance compensation was made up to 95%. Glucose was bath applied to cells during recording. The currents were filtered at 5 kHz, stored on magneto optical disk, and analyzed on a microcomputer with pClamp software (version 6; Axon Instrument Inc).

Assay of [³H]-2-DOG Uptake
The confluent cells with or without glucose-free exposure at 37°C were washed twice with glucose-free extracellular solution. Subsequently, the cells were incubated for 10 minutes in extracellular solution containing [³H]-2-DOG (specific activity, 6.1 Ci/mL; Dupont) in the presence and absence of several kinds of inhibitors. In some cases, [³H]-2-DOG uptake was assayed in Na⁺-free extracellular solution. After incubation with [³H]-2-DOG, the cells were washed three times with glucose/ Na⁺-free solution and then lysed with 0.1% SDS. The levels of [³H]-2-DOG uptake into endothelial cells were detected by a liquid scintillation counter.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Cultured Endothelial Cells
The inner layer of the brain artery was positive against the marker for endothelial cells, anti-factor VIII antibody (Fig 1A). The endothelial cell sheet alone was isolated for cultures (Fig 1B). Cultured cells displayed uniform cobblestone appearance typical for endothelial cells (Fig 1C), and the cells were also positive against anti-factor VIII antibody (Fig 1D).

View larger version (98K): [in this window] [in a new window]   Figure 1. Cultured endothelial cells from brain cortical arteries. The coronal section of the brain artery (A) and the isolated endothelial cell sheet (B) were stained with factor VIII antibody. Cultured endothelial cells are shown in panel C; the cells were positive for factor VIII staining (D).

Effect of SGLT-like Glucose Transporter Operation on Brain Endothelial Cells
Transport of glucose (D-isomer) mediated by the SGLTs can be relatively estimated by monitoring Na⁺ currents because Na⁺ is cotransported with glucose into cells; otherwise, L-glucose evoked no current. We therefore used D-glucose in this and further experiments. Application of glucose (0.1 mmol/L) produced inward currents at a holding potential of -60 mV in the whole-cell voltage-clamp configuration (Fig 2). Notably, the currents were potentiated after more than 30 minutes' pretreatment with glucose-free extracellular solution (Fig 2). MeDG (0.1 mmol/L), a specific compound for the SGLTs,⁵ also produced currents to the same level as glucose (0.1 mmol/L) (Fig 2). Currents evoked by glucose (0.1 mmol/L) in cells with 1-hour glucose-free exposure were inhibited by either the selective SGLT inhibitor phlorizin (50 µmol/L) or the ATP uncoupler DNP (1 µmol/L) (Fig 3A). Glucose (0.1 mmol/L) never produced currents in the presence of MeDG (0.1 mmol/L) (Fig 3A) or in Na⁺-free extracellular solution (Fig 3B). Voltage pulses from -140 to +20 mV in 20-mV increments after application of glucose (0.1 mmol/L) or MeDG (0.1 mmol/L) generated voltage-dependent inward currents that were inhibited by phlorizin (50 µmol/L), although spontaneous currents were not observed (Fig 4A and 4B). These results indicate that a Na⁺/energy-dependent glucose transporter (SGLT-like glucose transporter) was involved in glucose uptake into brain endothelial cells and that the uptake was enhanced by glucose-free insult.

View larger version (20K): [in this window] [in a new window]   Figure 2. Glucose- and MeDG-evoked currents in brain endothelial cells. Whole-cell patches were made to cells with and without 1-hour exposure to glucose-free extracellular solution, and glucose (0.1 mmol/L; n=7) or MeDG (0.1 mmol/L; n=7) was applied to the cells. The holding potential was -60 mV. In this and following figures, inward currents correspond to downward deflections.

View larger version (33K): [in this window] [in a new window]   Figure 3. SGLT-like glucose transporter in brain endothelial cells. Whole-cell patches were made to cells with 1-hour incubation in glucose-free extracellular solution. In panel A, glucose (0.1 mmol/L) was applied to the cells in the presence of phlorizin (50 µmol/L; n=7), DNP (1 µmol/L; n=5), or MeDG (0.1 mmol/L; n=5); in this case, glucose was applied when MeDG-induced currents reversed. B, glucose (0.1 mmol/L) was applied to a single cell in Na+-free extracellular solution and, in turn, Na+-containing extracellular solution (n=5). C, glucose (0.1 mmol/L) was repetitively applied to a single cell at 15-minute intervals (n=7) or applied to cells 5 minutes after patch formation with the patch electrode filling solution containing 0.01 mmol/L glucose (n=7). D, glucose (1 mmol/L) was applied to a single cell, and a second application was carried out after treatment with 1 µmol/L cyto B (n=7). In some cells, 10 mmol/L glucose was applied to cells in the presence of cyto B (1 µmol/L; n=7). The holding potential was -60 mV.

View larger version (35K): [in this window] [in a new window]   Figure 4. Glucose- and MeDG-induced current/voltage relations. Voltage pulses from -140 to 20 mV in 20-mV increments were applied to cells with 1 hour of glucose-free exposure before and after application of glucose (0.1 mmol/L; n=5) (A) or MeDG (0.1 mmol/L; n=5) (B) in the presence and absence of phlorizin (50 µmol/L).

Regulation of SGLT-like Glucose Transporter–Operated Currents by Cytosolic Glucose
Currents induced by glucose (0.1 mmol/L) or MeDG (0.1 mmol/L) were slowly desensitized, and a second application of glucose elicited a still lesser response after a 15-minute washing (Fig 3C). In addition, when glucose (0.01 mmol/L) was added to patch electrode filling solution, application of glucose (0.1 mmol/L) outside the patch pipette evoked very small currents (Fig 3C). Glucose-induced currents reduced in a dose-dependent manner and >10 mmol/L glucose produced no current (Fig 5). These results suggest that low cytosolic glucose enhanced glucose uptake mediated by the SGLT-like glucose transporter; otherwise, high cytosolic glucose inhibited the uptake.

View larger version (19K): [in this window] [in a new window]   Figure 5. Dose-response effect of glucose on the evoked currents. Glucose was applied to cells with 1 hour of glucose-free exposure at concentrations as indicated. The holding potential was -60 mV. Typical currents are illustrated in the upper panel, with results summarized in the lower panel. Each point represents the average percentage (±SD) of the current evoked by 0.1 mmol/L glucose (n=5 to n=7).

Cytochalasin B (1 µmol/L), a facilitative glucose transporter (GLUT1 to GLUT5) inhibitor, markedly enhanced currents induced by glucose (1 mmol/L), and 10 mmol/L glucose produced currents in the presence of cyto B (Fig 3D) although no current was evoked in the absence of cyto B (Fig 5), further supporting the idea that glucose uptake into endothelial cells mediated by the SGLT-like glucose transporter is regulated by intracellular glucose concentrations.

Uptake of [³H]-2-DOG Into Brain Endothelial Cells
Uptake of 2-DOG into endothelial cells was 37.1±1.8 pmol/mg protein per 10 minutes in cells without glucose-free exposure (Table 1). The facilitative glucose transporter inhibitors, such as phloretin (50 µmol/L) and cyto B (1 µmol/L), inhibited glucose uptake by 96% and 97%, respectively, whereas phlorizin (50 µmol/L), the energy metabolism inhibitors DNP (1 µmol/L) and iodoacetate (1 µmol/L), the Na⁺-K⁺/ATPase pump inhibitor ouabain (100 µmol/L), or deprivation of Na⁺ from extracellular solution blocked the uptake by only 4% to 6% (Table 1). Consistent with the result of glucose-induced currents, 2-DOG uptake was enhanced by 15.4 pmol/mg protein per 10 minutes in cells with 1-hour incubation in glucose-free extracellular solution ( increase) (Table 1). The enhancement was clearly inhibited by phlorizin, DNP, iodoacetate, ouabain, and deprivation of Na⁺, but, in contrast, it was not affected by either phloretin or cyto B (Table 1). This indicates that an enhancement in 2-DOG uptake by glucose-free insult resulted from activation of the SGLT-like glucose transporter.

View this table: [in this window] [in a new window]   Table 1. Uptake of [3H]-2-DOG Into Brain Endothelial Cells

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Plasma membranes are not permeable to a polar molecule glucose, and glucose is therefore taken up into cells by glucose transporters. Two families of glucose transporters are presently identified: facilitative glucose transporters such as GLUT1 (erythrocyte/HepG2), GLUT2 (liver), GLUT3 (brain), GLUT4 (muscle/fat), and GLUT5 (small intestine),^{6 7} and SGLTs (small intestine/kidney) such as SGLT1 and SGLT2.^{8 9 10 11 12 13} Extensive studies have been carried out to understand the distribution of these glucose transporters in a variety of tissues. Very little is known, however, about glucose transporters in brain arteries. Brain arteries are likely provided with glucose via glucose transporters, since they are not fed by vasa vasorum and have a barrier that is formed by the tight junctions of the endothelium. In an earlier study,⁴ through use of immunohistochemical analysis and Western immunoblot analysis, we found that the GLUT1 and SGLT-like glucose transporter are expressed in cultured endothelial cells from bovine brain cerebral cortical arteries, which suggests that glucose uptake into the endothelium is achieved by these transporters. The present study provides further evidence for the expression of the SGLT-like glucose transporter and makes clear its functional role in brain arteries.

The SGLTs, which are identified in the small intestine and kidney, continuously carry glucose into cells against its concentration gradient as far as extracellular glucose is present; SGLT-operated currents are not desensitized and enhanced in a glucose concentration–dependent manner.¹⁴ In the present study, glucose-induced currents were inhibited by the selective SGLT inhibitor phlorizin, by the ATP uncoupler DNP, by deletion of Na⁺ from extracellular solution, or by MeDG, a specific agent for the SGLTs. MeDG produced currents in a fashion that mimics the effect of glucose, which indicates that an SGLT-like glucose transporter actually operates on the endothelium of brain arteries. Notably, the currents, inconsistent with those via the SGLTs in brush-border membranes, were desensitized. More striking are the observations that pretreatment with glucose-free media potentiated the currents and that otherwise, repetitive applications of glucose, higher concentrations of extracellular glucose, or addition of glucose to the patch electrode filling solution markedly reduced the currents. These findings suggest that the SGLT-like glucose transporter expressed in brain endothelial cells has a characteristic distinct from the SGLTs in brush-border membranes and is regulated by cytosolic glucose concentrations.

It is recognized that GLUT1 conveys glucose according to glucose concentration gradient and is characterized by faster glucose transport than the SGLTs. Interestingly, glucose-induced currents in brain endothelial cells were potentiated in the presence of cyto B, which suggests that cyto B produces the same condition as glucose-free insult; cyto B predominantly blocks glucose entry via GLUT1, and the eventual decrease in cytosolic glucose concentrations enhances activity of the SGLT-like glucose transporter.

Uptake of 2-DOG into brain endothelial cells was inhibited by over 95% by the facilitative glucose transporter inhibitors phloretin and cyto B in cells without glucose-free exposure, which indicates that GLUT1 is responsible for glucose uptake into the endothelium under normal conditions. On the other hand, the uptake was enhanced by pretreatment with glucose-free media and the enhancement was inhibited by the SGLT inhibitor, the energy metabolism inhibitors, and the Na⁺/K⁺-ATPase pump inhibitor, and by deprivation of extracellular Na⁺. This provides further evidence that glucose-free insult followed by low cytosolic glucose enhances activity of the SGLT-like glucose transporter, thus leading to an increase in glucose uptake. It is presently unknown by what mechanism glucose-free insult enhances activity of the SGLT-like glucose transporter. A study¹⁵ has demonstrated that high extracellular glucose inhibits insulin receptor kinase activity by serine/threonine phosphorylation, suggesting that glucose itself serves as a second messenger. The glucose signal may stimulate the expression of the SGLT-glucose transporter or the translocation of this transporter from cytosol to plasma membrane, or it may directly activate this transporter. To address this question, we are carrying out further experiments.

The GLUT1 and SGLT-like glucose transporter in the brain endothelium, thus, appear to supply brain arteries with glucose; the GLUT1 operates under normal conditions and, alternatively, the SGLT-like glucose transporter under conditions of stress such as hypoglycemia. The SGLT-like glucose transporter seems to be important in the maintenance of an adequate glucose concentration in the arterial wall. Another functional role of the SGLT-like glucose transporter may be its involvement in transport of glucose across the BBB. GLUT1 in brain capillaries is proposed to be the major glucose transporter at the BBB.² GLUT1 is preferentially localized on the abluminal membranes of the endothelium,³ and therefore transport of glucose across the BBB cannot be explained by GLUT1 alone. Considering that the BBB is composed of the endothelium, with the tight junctions and foot processes of the astrocytes, cultured brain endothelial cells here may not reflect the function at the BBB. Cultured brain endothelial cells, however, exhibited -glutamyl transpeptidase activity (205±7.9 nmol/mg protein per minute), a BBB enzymatic marker¹⁶ whereas no activity was obtained with cultured carotid artery endothelial cells, which suggests that cultured brain endothelial cells possess characteristics of the BBB. In addition, the findings that glucose was transported across the arterial wall by the SGLT-like glucose transporter in inverted cerebral cortical arteries but not in noninverted ones and that glucose transport was not observed in inverted carotid arteries⁴ indicate that the SGLT-like glucose transporter is selectively expressed in brain arteries and conveys glucose from the luminal membrane toward the abluminal side. The SGLT-like glucose transporter may thus be responsible for glucose uptake into the endothelium at the BBB and the GLUT1 for glucose transport toward astrocytes just as glucose is absorbed by the SGLT1/2 in the brush-border membranes and exits the small intestine or renal tubules across the basolateral membranes via GLUT2/5.¹⁴ To obtain further evidence for this, we are currently testing uptake of glucose using cocultures of astrocytes and endothelial cells.

In conclusion, the results presented here demonstrate that an SGLT-like glucose transporter is involved in uptake into brain endothelial cells as well as GLUT1 and that low glucose enhances activity of the SGLT-like glucose transporter. The SGLT-like glucose transporter seems to have a functional role in the maintenance of an adequate glucose concentration in the arterial wall under conditions of stress such as hypoglycemia.

	Selected Abbreviations and Acronyms

 

BBB = blood-brain barrier cyto B = cytochalasin B DNP = dinitrophenol 2-DOG = 2-deoxy-D-glucose GLUT = facilitative glucose transporter MeDG = -methyl-D-glucoside SGLT = Na+/glucose cotransporter

Received August 22, 1997; revision received January 14, 1998; accepted January 15, 1998.

	References

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1. Edvinsson L, MacKenzie ET, McCulloch J. Cerebral Blood Flow and Metabolism. New York, NY: Raven Press Publishers; 1993:40–56.

2. Pardridge WM, Boad RJ, Farrell CR. Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. J Biol Chem. 1990;265:18035–18040.[Abstract/Free Full Text]

3. Farrell CL, Pardridge WM. Blood-brain barrier glucose transporter is asymmetrically distributed on brain capillary endothelial luminal and abluminal membranes: an electron microscopic immunogold study. Proc Natl Acad Sci U S A. 1991;88:5779–5783.[Abstract/Free Full Text]

4. Nishizaki T, Kammesheidt A, Sumikawa K, Asada, T, Okada Y. A sodium- and energy-dependent glucose transporter with similarities to SGLT1–2 is expressed in bovine cortical vessels. Neurosci Res. 1995;22:13–22.[Medline] [Order article via Infotrieve]

5. Birnir B, Donald DDF, Wright EM. Voltage-clamp studies of the Na⁺/glucose cotransporter cloned from rabbit small intestine. Pflugers Arch. 1991;418:79–85.[Medline] [Order article via Infotrieve]

6. Bell GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care. 1990;13:198–208.[Abstract]

7. Thorens B. Facilitated glucose transporters in epithelial cells. Annu Rev Physiol. 1993;55:591–608.[Medline] [Order article via Infotrieve]

8. Hediger MA, Coady MJ, Ikeda TS, Wright EM. Expression cloning and cDNA sequencing of the Na⁺/glucose co-transporter. Nature. 1987;330:379–381.[Medline] [Order article via Infotrieve]

9. Hediger MA, Turk E, Wright EM. Homology of the human intestinal Na⁺/glucose and Escherichia coli Na⁺/proline co-transporters. Proc Natl Acad Sci U S A. 1989;86:5748–5752.[Abstract/Free Full Text]

10. Coady MJ, Pajor AM, Wright EM. Sequence homologies among intestinal and renal Na⁺/glucose co-transporters. Am J Physiol. 1990;259:C605–610.[Abstract/Free Full Text]

11. Ohta T, Isselbacher KJ, Rhoads DB. Regulation of glucose transporters in LLC-PK1 cells: effects of D-glucose and monosaccharides. Mol Cell Biol. 1990;10:6491–6499.[Abstract/Free Full Text]

12. Morrison AI, Panayotova HM, Feigl G, Scholermann B, Kinne RK. Sequence comparison of the sodium-D-glucose cotransport systems in rabbit renal and intestinal epithelia. Biochim Biophys Acta. 1991;1089:121–123.[Medline] [Order article via Infotrieve]

13. Wells RG, Mohandas TK, Hediger MA. Localization of the Na⁺/glucose co-transporter gene SGLT2 to human chromosome 16 close to the centromere. Genomics. 1993;17:787–789.[Medline] [Order article via Infotrieve]

14. Wright EM. The intestinal Na⁺/glucose cotransporter. Annu Rev Physiol. 1993;55:575–589.[Medline] [Order article via Infotrieve]

15. Pillay TS, Xiao S, Olefsky JM. Glucose induced phosphorylation of the insulin receptor-functional effects and characterization of phosphorylation sites. J Clin Inves. 1996;97:613–620.[Medline] [Order article via Infotrieve]

16. Orlowski M, Sessa G, Green JP. -glutamyl transpeptidase in brain capillaries: possible site of a blood-brain barrier for amino acids. Science. 1974;184:66–68.[Abstract/Free Full Text]

Editorial Comment

Nabil J. Alkayed, MD, PhD; Patricia D. Hurn, PhD

Guest Editors, Anesthesiology/Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Glucose is the source of metabolic energy in brain and requires continuous transport from plasma to parenchyma. Two classes of glucose carriers have been described in mammalian cell membranes: facilitative glucose transporters, which transport glucose down its concentration gradient, and Na⁺/glucose cotransporters, which couple the downhill transport of Na⁺ with an uphill uptake of glucose. However, it is unclear precisely which carriers are present in arterial endothelial cells. GLUT1, a facilitative glucose transporter, and SGLT, a type of Na⁺/glucose cotransporter, are known to be present in cultured brain artery endothelial cells (BAEC). The present study provides electrophysiological and biochemical evidence supporting a role for SGLT in cultured BAEC under conditions of low glucose concentration.

The key finding is that SGLT activity is enhanced by low extracellular glucose, which could serve as compensation for decreased glucose uptake via GLUT1 under these conditions. In a series of well-designed experiments, the authors demonstrate that SGLT activity is regulated by cytosolic glucose. Addition of glucose to the pipette solution, which represents the intracellular compartment, inhibited SGLT activity. A dose-dependent inverse relationship was present between extracellular glucose concentration and SGLT activity, an effect that is presumably mediated by cytosolic glucose, because activation of SGLT by low glucose is quickly desensitized. Regulation of SGLT by cytosolic glucose is further supported by the observation that inhibiting GLUT1-mediated glucose uptake (potentially depriving the cell of glucose) leads to potentiation of SGLT activity. One major strength of the study is its confirmation of electrophysiological findings by a biochemical assay of glucose uptake with use of radiolabeled glucose. The assay shows that depletion of cytosolic glucose by preincubation in glucose-free solution enhances glucose uptake, and the increment is abolished by inhibitors of SGLT or Na⁺/K⁺-ATPase, which have little effect on baseline uptake under normal glucose concentration.

The reader should be reminded that the main pathway for glucose uptake into BAEC under normal glucose concentration is GLUT1-facilitated diffusion. During normoglycemia, SGLT contributes very little to glucose uptake. It must also be emphasized that the endothelial cells of this study were isolated from large cerebral arteries, not from brain capillaries that carry out the bulk of glucose transport across the blood-brain barrier. The importance of SGLT in these cells remains to be demonstrated. From a clinical perspective, it will be important to compare these results in normal endothelial cells with those obtained from diabetic or atherosclerotic vessels.

	Selected Abbreviations and Acronyms

 

BBB = blood-brain barrier cyto B = cytochalasin B DNP = dinitrophenol 2-DOG = 2-deoxy-D-glucose GLUT = facilitative glucose transporter MeDG = -methyl-D-glucoside SGLT = Na+/glucose cotransporter

Received August 22, 1997; revision received January 14, 1998; accepted January 15, 1998.

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