Developmental Expression of GLUT1 and GLUT3 Glucose Transporters in Rat Brain

Abstract: Two glucose transport proteins, GLUT1 and GLUT3, have been detected in brain. GLUT1 is concentrated in the endothelial cells of the blood-brain barrier and may be present in neurons and glia; GLUT3 is probably the major neuronal glucose transporter. Of the few studies of glucose transport in the immature brain, none has quantified GLUTS. This study used membrane isolation and immunoblotting techniques to examine the developmental expression of GLUT1 and GLUT3 in four forebrain regions, cerebral microvessels, and choroid plexus, from rats 1–30 days postnatally as compared with adults. The GLUT1 level in whole brain samples was low for 14 days, doubled by 21 days, and doubled again to attain adult levels by 30 days; there was no regional variation. The GLUT3 level in these samples was low during the first postnatal week, increased steadily to adult levels by 21–30 days, and demonstrated regional specificity. The concentration of GLUT1 in microvessels increased steadily after the first postnatal week; the GLUT1 level in choroid plexus was high at birth, decreased at 1 week, and then returned to near fetal levels. GLUT3 was not found in microvessels or choroid plexus. This study indicates that both GLUT1 and GLUT3 are developmentally regulated in rat brain: GLUT1 appears to relate to the nutrient supply and overall growth of the brain, whereas GLUT3 more closely relates to functional activity and neuronal maturation.     

Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression

In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.     

Measurement of Cerebral Glucose Utilization Using Washout After Carotid Injection in the Rat

Abstract: The carotid injection technique, used previously to quantitate the kinetics of blood-brain barrier transport of metabolic substrates, may be modified to analyze the rate of cerebral glucose utilization. A 0.2-ml solution of [¹⁴C]glucose (G_F) and [³H]methylglucose (M), an internal reference, is rapidly injected into the carotid artery, followed by microwave fixation of brain at various times up to 4 min after injection. The brain radioactivity is separated into a fraction containing neutral hexoses (G_F and M) and a fraction containing metabolites of glucose. The G_F/M ratio is related to the rate constant (k₃) of brain glucose utilization by the simple, linear equation: In(G_F/M) = In(G_F°/M°) –k₃t, where G_F°/M°= the brain uptake index of glucose, relative to methylglucose, at 5-15 s after injection, and t= the time after carotid injection, e.g., 1–4 min. It is assumed that (a) the rate of influx due to recirculation of label is minimal during the 4-min circulation period; and (b) the rate constants of glucose efflux (k₂) and methylglucose efflux (k₂*) are identical. Independent estimates of k₂ and k₂* showed these parameters to be identical: k₂= 0.14 + 0.08 min-I; k₂*= 0.14 ± 0.02 min-I. A logarithmic plot of G_F/M ratios versus time was linear (r = 0.99), and was described by the slope k₂= 0.21 ± 0.02 min^?1. Assuming glucose is uniformly distributed in brain, then the glycolytic rate = k₃× brain glucose = (0.21 min^?1) (2.6 μmol g^?1) = 0.55 μmol min^?1 g^?1 for the cortex of the barbiturate-anesthetized rat. These studies provide the basis for a simple method of measurement of regional brain glycolysis that does not require either the use of correction factors, e.g., the lumped constant, or the use of differentially labeled glucose.     

Glucose Transporter 1, Distribution in the Brain and in Neural Disorders: Its Relationship With Transport of Neuroactive Drugs Through the Blood-Brain Barrier

Facilitative glucose transport is mediated by one or more of the members of the closely related glucose transporter (GLUT) family. Thirteen members of the GLUT family have been described thus far. GLUT1 is a widely expressed isoform that provides many cells with their basic glucose requirement. It is also the primary transporter across the blood-brain barrier. This review describes the distribution and expression of GLUT1 in brain in different pathophysiological conditions including Alzheimers disease, epilepsy, ischemia, or traumatic brain injury. Recent investigations show that GLUT1 mediates the transport of some neuroactive drugs, such as glycosylated neuropeptides, low molecular weight heparin, and d-glucose derivatives, across the blood-brain barrier as a delivery system. By utilizing such highly specific transport mechanisms, it should be possible to establish strategies to regulate the entry of candidate drugs.     

Blood—Brain Barrier Glucose Transporter

Abstract : The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)[2-³H]propyl]-1,3-bis(d -mannose-4-yloxy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration ; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for d -[¹⁴C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 ± 4% increase in total GLUT1/mg of microvessel protein and a 52 ± 13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.     

Lack of effect of antenatal glucocorticoid therapy in the fetal baboon on cerebral cortical glucose transporter proteins

BACKGROUND: Maternal antenatal glucocorticoid therapy is used to accelerate lung maturation of immature babies at risk of preterm delivery. It acutely affects brain activity of the human fetus and reduces the immunoreactivity of neurocytoskeletal and synaptic proteins in the fetal baboon brain. These effects might be based on cerebral energy failure due to a decreased neuronal glucose uptake that has been shown in vitro. METHODS: Glucose uptake into the brain is selectively facilitated by GLUT1 expressed in the blood-brain barrier and GLUT3 expressed in the neuronal membrane. Immunohistochemical distribution of GLUT1 and GLUT3 were examined in the frontal neocortex of the fetal baboon brain at 0.73 gestation (i.e. similar to 28 weeks of human gestation) after maternal betamethasone administration, mimicking the clinical dose regimen. RESULTS: Betamethasone did not alter GLUT1 and GLUT3 immunoreactivity. CONCLUSIONS: The results suggest that inhibition of glucose uptake is not the mechanism for the cerebral effects of antenatal glucocorticoids.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood-brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (-23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 microM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [(3)H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood-brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Developmental switch in brain nutrient transporter expression in the rat

Normal development of both human and rat brain is associated with a switch in metabolic fuel from a combination of glucose and ketone bodies in the immature brain to a nearly total reliance on glucose in the adult. The delivery of glucose, lactate, and ketone bodies from the blood to the brain requires specific transporter proteins, glucose and monocarboxylic acid transporter proteins (GLUTs and MCTs), respectively. Developmental expression of the GLUTs in rat brain, i.e., 55-kDa GLUT1 in the blood-brain barrier (BBB), 45-kDa GLUT1 and GLUT3 in vascular-free brain, corresponds to maturational increases in cerebral glucose uptake and utilization. It has been suggested that MCT expression peaks during suckling and sharply declines thereafter, although a comparable detailed study has not been done. This study investigated the temporal and regional expression of MCT1 and MCT2 mRNA and protein in the BBB and the nonvascular brain during postnatal development in the rat. The results confirmed maximal MCT1 mRNA and protein expression in the BBB during suckling and a decline with maturation, coincident with the switch to glucose as the predominant cerebral fuel. However, nonvascular MCT1 and MCT2 levels do not reflect changes in cerebral energy metabolism, suggesting a more complex regulation. Although MCT1 assumes a predominantly glial expression in postweanling brain, the concentration remains fairly constant, as does that of MCT2 in neurons. The maintenance of nonvascular MCT levels in the adult brain implies a major role for these proteins, in concert with the GLUTs in both neurons and astrocytes, to transfer glycolytic intermediates during cerebral energy metabolism.     

Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids

The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the k_cat of transport without changing the K_m values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

缺血性脑血管病变组织中葡萄糖转运蛋白1的改变

葡萄糖通过血脑屏障从血液中进入脑组织必须依赖葡萄糖转运蛋白(glucose transporter,GLUT)的帮助.GLUT1是血脑屏障上最主要的GLUT,也是脑毛细血管壁内皮细胞的分子标记.动物研究显示在急性脑缺血后脑内的GLUT1表达增加.检测了7例慢性微血管缺血性脑血管病变(ischemic cerebrovascular diseases,ICVD)的尸检脑组织中的GLUT1水平,并与11例同龄对照组比较.结果发现GLUT1水平在ICVD组中降低.其降低可能是由于低氧诱导因子-1α(hypoxia-induciblefactor-1α,HIF-1α)的下调所致.但是,在ICVD脑组织中的GLUT1水平降低不伴随有蛋白质O-GlcNAc糖基化水平的下降.上述结果为探讨脑缺血病变的机理提供了新线索.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Mammalian glucose permease GLUT1 facilitates transport of arsenic trioxide and methylarsonous acid

Arsenic exposure is associated with hypertension, diabetes, and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH)(3) uptake. Here, we report that mammalian glucose transporter GLUT1 catalyzes As(OH)(3) and CH(3)As(OH)(2) uptake in yeast or in Xenopus laevis oocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH)(3) and CH(3)As(OH)(2). The K(m) of GLUT1 is to 1.2mM for CH(3)As(OH)(2), compared to a K(m) of 3mM for glucose. Inhibition between glucose and CH(3)As(OH)(2) is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH)(3) and CH(3)As(OH)(2) in oocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.     

Glucose transporter asymmetries in the bovine blood-brain barrier

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.     

Sodium valproate inhibits glucose transport and exacerbates Glut1-deficiency in vitro

Anticonvulsant sodium valproate interferes with brain glucose metabolism. The mechanism underlying such metabolic disturbance is unclear. We tested the hypothesis that sodium valproate interferes with cellular glucose transport with a focus on Glut1 since glucose transport across the blood-brain barrier relies on this transporter. Cell types enriched with Glut1 expression including human erythrocytes, human skin fibroblasts, and rat astrocytes were used to study the effects of sodium valproate on glucose transport. Sodium valproate significantly inhibited Glut1 activity in normal and Glut1-deficient erythrocytes by 20%-30%, causing a corresponding reduction of Vmax of glucose transport. Similarly, in primary astrocytes as well as in normal and Glut1-deficient fibroblasts, sodium valproate inhibited glucose transport by 20%-40% (P < 0.05), accompanied by an up to 60% downregulation of GLUT1 mRNA expression (P < 0.05). In conclusion, sodium valproate inhibits glucose transport and exacerbates Glut1 deficiency in vitro. Our findings imply the importance of prudent use of sodium valproate for patients with compromised Glut1 function.     

A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing GLUT4-myc and AS160_v2

Muscle and fat cells translocate GLUT4 (glucose transporter 4) to the plasma membrane when stimulated by insulin. Usually, this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4 by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (substrate of 160 kDa), namely AS160_v2, and showed that its coexpression with GLUT4 in L6 myoblasts increased the insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which coexpress myc-tagged GLUT4 and AS160_v2, can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC₅₀ values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation, and an image-based assay increases our confidence in the mode of action of the compounds identified.