Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model (original) (raw)

Mechanisms of glucose transport at the blood–brain barrier: an in vitro study

Brain Research, 2001

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood–brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood–brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood–brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood–brain barrier model of approximately 0.85. Under conditions where [glucose]lumen was higher than [glucose]ablumen, influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

High and Low-Affinity Transport of D-Glucose from Blood to Brain

Journal of Neurochemistry, 1981

Abstract: Measurements of the unidirectional blood-brain glucose flux in rat were incompatible with a single set of kinetic constants for transendothelial transport. At least two transfer mechanisms were present: a high-affinity, low-capacity system, and a low-affinity, high-capacity system. The low-affinity system did not represent passive diffusion because it distinguished between D-and L-glucose. The Tmax and Km, for the high-affinity system were 0.16 mmol 100 g−1 min−1 and 1 mM; for the low-affinity system, ∼ 5 mmol 100 g−1 min−1 and ∼ 1 M. With these values, physiological glucose concentrations were not sufficient to saturate the low-affinity system. In normoglycemia, therefore, three independent pathways of glucose transport from blood to brain appear to exist: a high-affinity facilitated diffusion pathway of apparent permeability 235·10−7 cm s−1, a specific but nonsaturable diffusion pathway of permeability 85·10−7 cm s−l, and a nonspecifc passive diffusion pathway of permeability 2·10−7 cm s−1.

Gibbs Free-Energy Gradient along the Path of Glucose Transport through Human Glucose Transporter 3

ACS chemical neuroscience, 2018

Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a β-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis-Menten characteristics of GLUT3, low K and high V, specifically suitable for neurons' high and constant demand of energy from their low-glucose environments. We compute GLUT3's binding free...

Blood-Brain Barrier Glucose Transporter

Journal of Neurochemistry, 2002

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)[2-3 H]propyl]-1,3-bis(D-mannose-4yloxy)-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 14to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for D-[ 14 C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 Ϯ 4% increase in total GLUT1/mg of microvessel protein and a 52 Ϯ 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.

In Silico Kinetic Study of the Glucose Transporter

Journal of Biological Physics, 2007

Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through biological membranes, and transport in erythrocytes is the most widely studied. One of the oldest and simplest models describing the kinetics of the transport reaction is that of alternating conformers, schematized in a cycle of four partial reactions where glucose binds and dissociates at two opposite steps, and the transporter undergoes transconformations at the other two opposite steps. The transport kinetics is entirely defined by the forward and backward rate constants of the partial reactions and the glucose and transporter concentrations at each side of the membrane, related by the law of mass action. We studied, in silico, the effect of modifications of the variables on the transient kinetics of the transport reaction. The simulations took into account thermodynamic constraints and provided results regarding initial velocities of transport, maximal velocities in different conditions, apparent influx and efflux affinities, and the turnover number of the transporter. The results are in the range of those experimentally reported. Maximal initial velocities are obtained when the affinities of the ligand for the transporter are the same at the extraand intracellular binding sites and when the equilibrium constants of the transconformation steps are equal among them and equal to 1, independently of the obvious effect of the increase of the rate constant values. The results are well adjusted to Michaelis-Menten kinetics. A larger initial velocity for efflux than for uptake described in human erythrocytes is demonstrated in a model with the same dissociation constants at the outer and inner sites of the membrane. The larger velocities observed for uptake and efflux when transport occurs towards a glucose-containing trans side can also be reproduced with the alternating conformer model, depending on how transport velocities are measured.

Characterization and Modulation of Glucose Uptake in a Human Blood–Brain Barrier Model

The Journal of Membrane Biology, 2013

The blood-brain barrier (BBB) plays a key role in limiting and regulating glucose access to glial and neuronal cells. In this work glucose uptake on a human BBB cell model (the hCMEC/D3 cell line) was characterized. The influence of some hormones and diet components on glucose uptake was also studied. 3 H-2-deoxy-Dglucose ([ 3 H]-DG) uptake for hCMEC/D3 cells was evaluated in the presence or absence of tested compounds.

Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes

2009

The glucose transporter is the obligatory port of entry for glucose into astrocytes, neurons and most other mammalian cell types. The glucose transporter, also known as GLUT, is an integral membrane protein of which 13 human isoforms have been described to date (Uldry and Thorens 2004). Kinetically, it is a uniporter, i.e., a transporter with a single binding site, which facilitates the permeation of a molecule across the plasma membrane following a concentration gradient. In many tissues, including the brain, muscle and fat, the glucose gradient between blood and cells is near maximum, which gives the transporters the ability to control the rate of glycolysis (Barros et al. 2005). As expected, GLUTs are highly regulated proteins, being the targets of hormones, growth factors, cytokines, exercise, muscle contraction, neurotransmitters, metabolic stress, oxidative stress, viral infection, etc. (Baldwin et al. 1997; Behrooz and Ismail-Beigi 1999; Barros et al. 2007). Since their introduction over 50 years ago, radiolabeled sugars have been the main tools for the study of glucose transport. These tracers, which provide a high signal-to-noise ratio, have permitted the kinetic characterization of the transporters in many cell types, the determination of their substrate specificity and the initial description of their rich regulation. Most of this work was carried out in erythrocytes, isolated adipocytes and tumor cell lines, which provided reasonably homogenous cell populations (Carruthers 1990). After molecular identification of the first mammalian glucose transporter (Mueckler et al. 1985), which would be named GLUT1, it was possible to address the relationship between transporter structure and function, again using isotopelabeled glucose analogs, typically by measuring the behavior of recombinant transporters in frog oocytes or in cell lines.

Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

Journal of Biological Chemistry, 2001

The transport of glucose across the mammalian bloodbrain 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-(dmannos-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.