Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT-1: Influence on 18 F-FDG PET Uptake.

Positron emission tomography (PET) using 2-deoxy-2-[18 F]fluoro-d-glucose ([18 F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both nondiabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter-1 (hGLUT-1) in FaDu and GIST-1 cells by measuring [3 H]2-deoxy-d-glucose ([3 H]2-DG) and [3 H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [3 H]2-DG uptake. In GIST-1 cells, [3 H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H-pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT-1. We hypothesize that inhibition of hGLUT-1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [18 F]FDG uptake, increasingly used as a marker of tumor response. The hGLUT-1 inhibition by TKIs may have implications for routine [18 F]FDG-PET monitoring of tumor response in patients.

Molecular determinants for the thermodynamic and functional divergence of uniporter GLUT1 and proton symporter XylE.

GLUT1 facilitates the down-gradient translocation of D-glucose across cell membrane in mammals. XylE, an Escherichia coli homolog of GLUT1, utilizes proton gradient as an energy source to drive uphill D-xylose transport. Previous studies of XylE and GLUT1 suggest that the variation between an acidic residue (Asp27 in XylE) and a neutral one (Asn29 in GLUT1) is a key element for their mechanistic divergence. In this work, we combined computational and biochemical approaches to investigate the mechanism of proton coupling by XylE and the functional divergence between GLUT1 and XylE. Using molecular dynamics simulations, we evaluated the free energy profiles of the transition between inward- and outward-facing conformations for the apo proteins. Our results revealed the correlation between the protonation state and conformational preference in XylE, which is supported by the crystal structures. In addition, our simulations suggested a thermodynamic difference between XylE and GLUT1 that cannot be explained by the single residue variation at the protonation site. To understand the molecular basis, we applied Bayesian network models to analyze the alteration in the architecture of the hydrogen bond networks during conformational transition. The models and subsequent experimental validation suggest that multiple residue substitutions are required to produce the thermodynamic and functional distinction between XylE and GLUT1. Despite the lack of simulation studies with substrates, these computational and biochemical characterizations provide unprecedented insight into the mechanistic difference between proton symporters and uniporters.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

The ultrastructure of perisynaptic glia in the nucleus tractus solitarii of the adult rat: Comparison between single synapses and multisynaptic arrangements.

Astrocytes are now considered as essential partners of neurons. In particular, they play important roles in glutamatergic transmission, including transmitter inactivation by uptake. Here, we investigated the organization of astroglia in the Nucleus Tractus Solitarii (NTS), a sensory nucleus located in the caudal medulla. Special attention was given to perisynaptic astroglial processes. Investigations were performed at the light and electron microscope levels, using immunodetection of glial glutamate transporters, stereological methods, and serial reconstruction. In the NTS, the main glutamate transporter expressed by astrocytes was GLT1. The volume fraction of astrocyte processes and the density of astrocyte membranes reached 15% and 2.8 µm(2) µm(-3) , respectively. In spite of the relative abundance of astrocyte processes, we found that NTS glutamatergic synapses were not entirely surrounded by glia. Measurements were performed on 43 reconstructed asymmetric junctions which were either single synapses (n = 22) or synapses involved in multisynaptic arrangements (n = 21). Single synapses had 58% of their perimeter contacted by astrocyte processes on average. In multisynaptic arrangement, glial coverage was restricted to the outer part of synaptic diameters and amounted to 50% of this outer part on average. Incomplete glial coverage of NTS synapses may allow glutamate to diffuse out of the synaptic cleft and to activate extrasynaptic receptors as well as receptors from neighboring synapses. Especially, in multisynaptic arrangements, the lack of intervening glia may favor functional coupling between individual contacts.

Ultrastructure of human erythrocyte GLUT1.

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.