Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors.

The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.

Human erythrocytes express GLUT5 and transport fructose.

Although erythrocytes readily metabolize fructose, it has not been known how this sugar gains entry to the red blood cell. We present evidence indicating that human erythrocytes express the fructose transporter GLUT5, which is the major means for transporting fructose into the cell. Immunoblotting and immunolocalization experiments identified the presence of GLUT1 and GLUT5 as the main facilitative hexose transporters expressed in human erythrocytes, with GLUT2 present in lower amounts. Functional studies allowed the identification of two transporters with different kinetic properties involved in the transport of fructose in human erythrocytes. The predominant transporter (GLUT5) showed an apparent Km for fructose of approximately 10 mmol/L. Transport of low concentrations of fructose was not affected by 2-deoxy-D-glucose, a glucose analog that is transported by GLUT1 and GLUT2. Similarly, cytochalasin B, a potent inhibitor of the functional activity of GLUT1 and GLUT2, did not affect the transport of fructose in human erythrocytes. The functional properties of the fructose transporter present in human erythrocytes are consistent with a central role for GLUT5 as the physiological transporter of fructose in these cells.

Increased uptake and accumulation of vitamin C in human immunodeficiency virus 1-infected hematopoietic cell lines.

Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.

Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, GLUT1.

Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein-tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose-dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell-independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a Ki of approximately 12 microM for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a Ki of 7 microM. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein-tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose.

Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid.

We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na+, whereas the intracellular accumulation of ascorbic acid was found to be a Na(+)-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na(+)-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation.