Human Erythrocyte Sugar Transport is Incompatible with Available Carrier Models

Abstract

GLUT1-mediated, passive D-glucose transport in human erythrocytes is asymmetric, Vmax and K(m)(app) for D-glucose uptake at 4 degrees C are 10-fold lower than Vmax and K(m)(app) for D-glucose export. Transport asymmetry is not observed for GLUT1-mediated 3-O-methylglucose transport in rat, rabbit, and avian erythrocytes and rat adipocytes where Vmax for sugar uptake and exit are identical. This suggests that transport asymmetry is either an intrinsic catalytic property of human GLUT1 or that factors present in human erythrocytes affect GLUT1-mediated sugar transport. In the present study we assess human erythrocyte sugar transport asymmetry by direct measurement of sugar transport rates and by analysis of the effects of intra- and extracellular sugars on cytochalasin B binding to the sugar export site. We also perform internal consistency tests to determine whether the measured, steady-state 3-O-methylglucose transport properties of human erythrocytes agree with those expected of two hypothetical models for protein-mediated sugar transport. The simple-carrier hypothesis describes a transporter that alternately exposes sugar import and sugar export pathways. The fixed-site carrier hypothesis describes a sugar transporter that simultaneously exposes sugar import and sugar export pathways. Steady-state 3-O-methylglucose transport in human erythrocytes at 4 degrees C is asymmetric. Vmax and K(m)(app) for sugar uptake are 10-fold lower than Vmax and K(m)(app) for sugar export. Phloretin-inhibitable cytochalasin B binding to intact red cells is unaffected by extracellular D-glucose but is competitively inhibited by intracellular D-glucose. This inhibition is reduced by 13% +/- 4% when saturating extracellular D-glucose levels are also present. Assuming transport is mediated by a simple-carrier and that cytochalasin B and intracellular D-glucose binding sites are mutually exclusive, the cytochalasin B binding data are explained only if transport is almost symmetric (Vmax exit = 1.4 Vmax entry). The cytochalasin B binding data are consistent with both symmetric and asymmetric fixed-site carriers. Analysis of 3-O-methylglucose, 2-deoxy-D-glucose, and D-glucose uptake in the presence of intracellular 3-O-methylglucose, demonstrates significant divergence in experimental and theoretical transport behaviors. We conclude either that human erythrocyte sugar transport is mediated by a carrier mechanism that is fundamentally different from those considered previously or that human erythrocyte-specific factors prevent accurate determination of GLUT1-mediated sugar translocation across the cell membrane. We suggest that GLUT1-mediated sugar transport in all cells is an intrinsically symmetric process but that intracellular sugar complexation in human red cells prevents accurate determination of transport rates.

Steady‐State Cerebral Glucose Concentrations and Transport in the Human Brain

“…A similar analysis of tracer kinetic data reported also a millimolar K,, in rat brain (Cunningham et a!., 1986), but a different study suggested that analyzing brain glucose content as a function of plasma glucose content using the standard model gave a K~of 14 mM in rat brain (Mason et a!., i992). Such a high K, can be taken as an indication of an almost linear brain-blood glucose relationship.Some studies have suggested that although the human GLUT-i has anomalous asymmetric kinetic propefties, most mammalian erythrocyte kinetics are symmetric (Cloherty et al, 1996). Nevertheless, we show in Appendix that the linear relationship between brain and plasma glucose levels using reversible MichaelisMenten kinetics is preserved even when assuming asymmetric kinetic properties.…”

Supply and Demand in Cerebral Energy Metabolism: The Role of Nutrient Transporters

“…However, these transporters catalyze bi-directional fluxes, and the presence of intracellular and/or extracellular glucose alters the kinetics of transport both in and out of the cell. This bidirectional transport must be actively considered when modeling the flow of glucose from blood to the individual types of neural cells, primarily neurons and glial cells (see below Blomqvist et al, 1991;Carruthers, 1990;Choi et al, 2001;Cloherty et al, 1996;de Graaf et al, 2001; Gjedde, 1980; Gruetter et al, 1998; Hebert and Carruthers, 1991;Qutub and Hunt, 2005).The capacity for glucose transport depends on the concentration of the transporter proteins as well as their intrinsic catalytic turnover activity or number of transport cycles catalyzed per transporter per sec (k cat ) within the respective cellular compartments. It is relatively easy to determine the total concentration of GLUTs in brain microvessels, as these vessels can be readily isolated from whole brain (Vannucci, 1994).…”

“…Transport measurements in red cell ghosts show that the rate of net cellular export of 10 mmol/L glucose into saline containing 3 mmol/L glucose is inhibited 50% by intracellular ATP (i.e., when transport is asymmetric). Thus red cells serve as less efficient glucose carriers when metabolically replete, but efficiently transfer intracellular glucose to surrounding tissues when demand for glycolytic ATP is increased (Carruthers, 1986, Cloherty et al, 1996 Heard et al, 2000).Although not yet specifically shown, this may apply to other GLUT1-expressing cells, such as BBB endothelial cells and astrocytes. Glucose transporter1 transport asymmetry in erythrocytes declines from 12-fold at 41C to 4-fold at 241C to 1.3-fold at 371C (Lowe and Walmsley, 1986).…”

Docking Studies Show That D-Glucose and Quercetin Slide through the Transporter GLUT1

“…This new model of transport via GLUT1 built on previous transport, structural, and modeling studies (1,2,3,7,10,20,22,(23)(24)(25)(26)(27)(28) delineates how glucose and quercetin may slide via a pore structure and is clearly differentiated from the conventional alternating carrier description of glucose transport. It offers an explanation for the widely dispersed mutation sites affecting GLUT1-DS (30,31).…”