Ian A. Simpson

Glucose transporter proteins in brain: Delivery of glucose to neurons and glia

Glucose is the principle energy source for the mammalian brain. Delivery of glucose from the blood to the brain requires transport across the endothelial cells of the blood‐brain barrier and into the neurons and glia. The facilitative glucose transporter proteins mediate these processes. The primary isoforms in brain are GLUT1, detected at high concentrations as a highly glycosylated form, (55 kDa) in blood‐brain barrier, and also as a less glycosylated, 45 kDa form, present in parenchyma, predominantly glia; GLUT3 in neurons; and GLUT5 in microglia. The rest of the transporter family, GLUTs 2, 4, and 7, have also been detected in brain but at lower levels of expression and confined to more discrete regions. All of the transporters probably contribute to cerebral glucose utilization, as part of overall metabolism and metabolic interactions among cells. We discuss the properties, regulation, cell‐specific location, and kinetic characteristics of the isoforms, their potential contributions to cerebral metabolism, and several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. GLIA 21:2–21, 1997.

SUPPLY AND DEMAND IN CEREBRAL ENERGY METABOLISM: THE ROLE OF NUTRIENT TRANSPORTERS

Glucose is the obligate energetic fuel for the mammalian brain, and most studies of cerebral energy metabolism assume that the majority of cerebral glucose utilization fuels neuronal activity via oxidative metabolism, both in the basal and activated state. Glucose transporter (GLUT) proteins deliver glucose from the circulation to the brain: GLUT1 in the microvascular endothelial cells of the blood—brain barrier (BBB) and glia; GLUT3 in neurons. Lactate, the glycolytic product of glucose metabolism, is transported into and out of neural cells by the monocarboxylate transporters (MCT): MCT1 in the BBB and astrocytes and MCT2 in neurons. The proposal of the astrocyte—neuron lactate shuttle hypothesis suggested that astrocytes play the primary role in cerebral glucose utilization and generate lactate for neuronal energetics, especially during activation. Since the identification of the GLUTs and MCTs in brain, much has been learned about their transport properties, that is capacity and affinity for substrate, which must be considered in any model of cerebral glucose uptake and utilization. Using concentrations and kinetic parameters of GLUT1 and −3 in BBB endothelial cells, astrocytes, and neurons, along with the corresponding kinetic properties of the MCTs, we have successfully modeled brain glucose and lactate levels as well as lactate transients in response to neuronal stimulation. Simulations based on these parameters suggest that glucose readily diffuses through the basal lamina and interstitium to neurons, which are primarily responsible for glucose uptake, metabolism, and the generation of the lactate transients observed on neuronal activation.

Diabetic Retinopathy Seeing Beyond Glucose-Induced Microvascular Disease

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Glucose transporter proteins in brain.

Glucose is the principal energy source for the mammalian brain. The presence of glucose transport proteins is essential to supply glucose to the neurons and glia within the brain. At least three glucose transporter isoforms have now been identified, and are thought to play a significant role, in the brain. This review describes our current understanding of cell‐specific glucose transporter expression in brain, which includes GLUT1 (55‐kDa form) present at a high concentration at the blood‐brain barrier as well as in parenchymal cells (45‐kDa form), most likely in astrocytes, GLUT3 expressed in neurons, and GLUT5 in microglia. We discuss some potential implications of this glucose transporter heterogeneity for cerebral metabolic activity.—Maher, F., Vannucci, S. J., Simpson, I. A. Glucose transporter proteins in brain. FASEB J. 8: 1003‐1011; 1994.

Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1–5 and SGLT1. The apparentK m of DHA transport via GLUT1 and GLUT3 was 1.1 ± 0.2 and 1.7 ± 0.3 mm, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2–4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, d-glucose, and 3-O-methylglucose among other hexoses while fructose and l-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-α-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2–8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific. J Neurosci. Res. 66:1198–1207, 2001.

Structure of the Blood–Brain Barrier and Its Role in the Transport of Amino Acids

Brain capillary endothelial cells form the blood-brain barrier (BBB). They are connected by extensive tight junctions, and are polarized into luminal (blood-facing) and abluminal (brain-facing) plasma membrane domains. The polar distribution of transport proteins mediates amino acid (AA) homeostasis in the brain. The existence of two facilitative transporters for neutral amino acids (NAAs) on both membranes provides the brain access to essential AAs. Four Na(+)-dependent transporters of NAA exist in the abluminal membranes of the BBB. Together these systems have the capability to actively transfer every naturally occurring NAA from the extracellular fluid (ECF) to endothelial cells and from there into circulation. The presence of Na(+)-dependent carriers on the abluminal membrane provides a mechanism by which NAA concentrations in the ECF of brain are maintained at approximately 10% those of the plasma. Also present on the abluminal membrane are at least three Na(+)-dependent systems transporting acidic AAs (EAAT) and a Na(+)-dependent system transporting glutamine (N). Facilitative carriers for glutamine and glutamate are found only in the luminal membrane of the BBB. This organization promotes the net removal of acidic- and nitrogen-rich AAs from the brain and accounts for the low level of glutamate penetration into the central nervous system. The presence of a gamma-glutamyl cycle at the luminal membrane and Na(+)-dependent AA transporters at the abluminal membrane may serve to modulate movement of AAs from blood to the brain. The gamma-glutamyl cycle is expected to generate pyroglutamate (synonymous with oxyproline) within the endothelial cells. Pyroglutamate stimulates secondary active AA transporters at the abluminal membrane, thereby reducing the net influx of AAs to the brain. It is now clear that BBB participates in the active regulation of the AA content of the brain.

Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited.

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.

The facilitative glucose transporter GLUT3: 20 years of distinction.

Glucose metabolism is vital to most mammalian cells, and the passage of glucose across cell membranes is facilitated by a family of integral membrane transporter proteins, the GLUTs. There are currently 14 members of the SLC2 family of GLUTs, several of which have been the focus of this series of reviews. The subject of the present review is GLUT3, which, as implied by its name, was the third glucose transporter to be cloned (Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers MG, Shows TB, Bell GI. J Biol Chem 263: 15245-15248, 1988) and was originally designated as the neuronal GLUT. The overriding question that drove the early work on GLUT3 was why would neurons need a separate glucose transporter isoform? What is it about GLUT3 that specifically suits the needs of the highly metabolic and oxidative neuron with its high glucose demand? More recently, GLUT3 has been studied in other cell types with quite specific requirements for glucose, including sperm, preimplantation embryos, circulating white blood cells, and an array of carcinoma cell lines. The last are sufficiently varied and numerous to warrant a review of their own and will not be discussed here. However, for each of these cases, the same questions apply. Thus, the objective of this review is to discuss the properties and tissue and cellular localization of GLUT3 as well as the features of expression, function, and regulation that distinguish it from the rest of its family and make it uniquely suited as the mediator of glucose delivery to these specific cells.

Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain

Active neuronal-glial interaction is important in the maintenance of brain homeostasis and is vital for neuronal survival following brain injury. The time course of post-ischemic astroglial dysfunction and neuronal death was studied in the spontaneously hypertensive rat (SHR) brain following permanent middle cerebral artery occlusion (MCAO). In situ hybridization with 35S-labeled riboprobes for GFAP and GLUT3 was used to monitor mRNA expression in glia and neurons. Astrocytic proteins GFAP, vimentin, S100, Glutathione-S-Transferase Yb (GST Yb) and neuronal protein TG2 were detected by immunofluorescence. Cells were co-stained with in situ end labeling (ISEL) to detect DNA fragmentation, a hallmark of cell death. GFAP mRNA expression declined rapidly in the ischemic region of the cortex and was almost absent by 12 h. Immunohistochemical studies revealed a parallel decline in the corresponding protein: a reduction in GFAP staining was apparent in the infarct after 3 h and by 24 h, there was essentially no remaining GFAP. Three other glial proteins (vimentin, S100 and GST Yb) disappeared from infarct over a similar time course. A few ISEL positive cells were observed in the infarct at 6 h, but maximal detection was not seen until 24-48 h. Most of the ISEL-positive cells were neurons, identified by co-staining with the neuronal marker TG2. Few cells expressing GFAP or other glial markers were positive at any time point. Neuronal GLUT3 mRNA declined more slowly than GFAP mRNA in the ischemic core and disappeared during the period of neuronal death. Concurrent with the loss of GFAP mRNA and protein expression in the infarct, there was a rapid rise in GFAP mRNA in the peri-infarct region of ipsilateral hemisphere and proximal region of the contralateral hemisphere. This was followed by the enhanced GFAP protein expression characteristic of reactive astrocytes, but over a significantly slower time course. These studies show that MCAO leads to a rapid decline of GFAP mRNA and glial proteins, which appears to precede the decline in neuronal mRNA and neuronal death within the infarct. Early astroglial dysfunction may play a critical role in determining the outcome of acute hypoxic-ischemic injury by compromising neuronal-glial interactions.