Hypervariability in Gene Copy Number for the Glucose Transporter Genes in Trypanosomes

The copy number of the gene encoding the carrier protein responsible for the uptake of glucose in bloodstream form Trypanosoma brucei (THT1) was investigated in the genome of 55 different members of the subgenus Trypanozoon. The gene is present in multiple copies in tandem arrays on two homologous chromosomes in these organisms, and copy number varies both intra- and interspecifically. Variability is also apparent in the number of genes encoding a second hexose transporter (THT2) which is the only isoform expressed in procyclic organisms. Multiple copies of THT2 are conserved in representatives of the non-tsetse transmitted species, Trypanosoma evansi and Trypanosoma equiperdum.     

African trypanosome glucose transporter genes: organization and evolution of a multigene family

Trypanosoma brucei brucei (EATRO-164) contains a tandem array of six genes encoding a glucose transporter, THT1 (trypanosome hexose transporter), followed by five genes encoding a second isoform, THT2. Two distinct clusters containing THT1 and THT2 genes have been identified in the EATRO-164 clone and in most other African trypanosome clones analyzed. Analysis of progeny from crosses between clones of T. b. brucei displaying polymorphism in THT1 copy number per cluster suggests that the two clusters of THT genes are present on homologous chromosomes. In addition, analysis of 30 African trypanosome clones revealed a high degree of polymorphism in THT1 copy number per cluster. Sequence comparison of five THT1 and two and one-half THT2 unit repeats, present within a 20-kb region, provided information about the genesis and evolution of the THT multigene family. The most divergent regions between THT1 and THT2 unit repeats probably arose from insertion of DNA fragments into an ancestral THT region. Genes of each of the different families are almost identical, and there are large regions of identity shared between THT1 and THT2 members. A mosaic copy containing most of a THT1 gene with the 3' extremity of a THT2 gene is found within the cluster. These results suggest that THT1 and THT2 arose by modification (insertion, mutation, or conversion) of duplicated ancestral genes. Functional constraints and homologous recombination may be evoked to explain the maintenance of the conserved sequences of THT1 and THT2.      

The structure-function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense.

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Km D-glucose = 41 microM versus 64 microM). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Ki D-glucose = 0.8 mM). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for D-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.     

Differential regulation of glucose transporter isoforms by the src oncogene in chicken embryo fibroblasts.

The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms.     

Analysis of the glucose transporter content of islet cell lines: Implications for glucose-stimulated insulin release

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The β-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high K_m for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, β-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in β-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high K_m transporter GLUT 2, may account for the characteristics aberrant patterns of glucose-stimulated insulin release in cell lines derived from β-cells.     

Human immunodeficiency virus type 1 infection of H9 cells induces increased glucose transporter expression.

A clone obtained from a differential display screen for cellular genes with altered expression during human immunodeficiency virus (HIV) infection matched the sequence for the human GLUT3 facilitative glucose transporter, a high-velocity-high-affinity facilitative transporter commonly expressed in neurons of the central nervous system. Northern (RNA) analysis showed that GLUT3 expression increased during infection. Flow cytometry showed that GLUT3 protein expression increased specifically in the HIV-infected cells; this increase correlated with increased 2-deoxyglucose transport in the HIV-infected culture. HIV infection therefore leads to increased expression of a glucose transporter normally expressed at high levels in other cell types and a corresponding increase in glucose transport activity. If HIV infection places increased metabolic demands on the host cell, changes in the expression of a cellular gene that plays an important role in cellular metabolism might provide a more favorable environment for viral replication.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

Std1 and Mth1 Proteins Interact with the Glucose Sensors To Control Glucose-Regulated Gene Expression in Saccharomyces cerevisiae

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii.

More than 40 nitrate respiration-deficient mutants of Haloferax volcanii belonging to three different phenotypic classes were isolated. All 15 mutants of the null phenotype were complemented with a genomic library of the wild type. Wild-type copies of mutated genes were recovered from complemented mutants using two different approaches. The DNA sequences of 13 isolated fragments were determined. Five fragments were found to overlap; therefore nine different genomic regions containing genes essential for nitrate respiration could be identified. Three genomic regions containing genes coding for subunits of ABC transporters were further characterized. In two cases, genes coding for an ATP-binding subunit and a permease subunit were clustered and overlapped by four nucleotides. The third gene for a permease subunit had no additional ABC transporter gene in proximity. One ABC transporter was found to be glucose specific. The mutant reveals that the ABC transporter solely mediates anaerobic glucose transport. Based on sequence similarity, the second ABC transporter is proposed to be molybdate specific, explaining its essential role in nitrate respiration. The third ABC transporter is proposed to be anion specific. Genome sequencing has shown that ABC transporters are widespread in Archaea. Nevertheless, this study represents only the second example of a functional characterization.     

Translocation of the brain-type glucose transporter largely accounts for insulin stimulation of glucose transport in BC3H-1 myocytes.

Insulin-stimulated glucose transport was examined in BC3H-1 myocytes. Insulin treatment lead to a 2.7 +/- 0.3-fold increase in the rate of deoxyglucose transport and, under the same conditions, a 2.1 +/- 0.1-fold increase in the amount of the brain-type glucose transporter (GLUT 1) at the cell surface. It has been shown that some insulin-responsive tissues express a second, immunologically distinct, transporter, namely GLUT 4. We report here that BC3H-1 myocytes and C2 and G8 myotubes express only GLUT 1; in contrast, rat soleus muscle and heart express 3-4 times higher levels of GLUT 4 than GLUT 1. Thus translocation of GLUT 1 can account for most, if not all, of the insulin stimulation of glucose transport in BC3H-1 myocytes. On the other, hand, neither BC3H-1 myocytes nor the other muscle-cell lines are adequate as models for the study of insulin regulation of glucose transport in muscle tissue.     

Inhibition of glucose transporter gene expression by antisense nucleic acids in HL-60 leukemia cells.

Glucose is the basic source of energy for mammalian cells. The energy-independent transport of glucose down its concentration gradient is mediated by the facilitative glucose transporter family (GLUT). It has long been recognised that glucose transporter genes are overexpressed in many human cancer cells, to help provide extra energy for the rapid growth of cancer cells. In the present study, antisense oligonucleotides and plasmid-derived antisense RNA against GLUT-1 gene were synthesized and transfected into human leukemia HL-60 cells to investigate the effect of these antisense nucleic acids on tumour growth. Our results show that antisense nucleic acids inhibited the proliferation of HL-60 cells by 50-60% and the mRNA expression of GLUT-1 gene was suppressed as detected by Northern hybridization.     

The acquisition of increased insulin-responsive hexose transport in 3T3-L1 adipocytes correlates with expression of a novel transporter gene

The expression of two genes encoding facilitated glucose transporter proteins was studied during the differentiation of the 3T3-L1 fibroblastic cell line into adipocytes. The mRNA encoding the widely expressed HepG2/brain glucose transporter (GTI) is detectable in fibroblasts and its abundance remains unchanged during differentiation. On the other hand, the mRNA encoding a glucose transporter protein (GTIII) localized exclusively to muscle and adipose tissue is undetectable in fibroblasts but present in adipocytes. GTIII mRNA is first expressed three days after differentiation of 3T3-L1 cells has begun. Similarly, it is not until 3 days following the initiation of differentiation that GTIII protein can be detected, as assayed either by Western immunoblot or indirect immunofluorescence. The latter technique localizes GTIII predominantly to the perinuclear region of the adipocyte. The appearance of GTIII in developing fat cells correlates temporally with the acquisition of an increased stimulation of hexose uptake by maximal concentrations of insulin. These data support the concept that the marked increase in hexose transport in adipocytes in response to insulin is dependent on the expression in these cells of a specific, hormone-regulatable transport protein.     

The expression of vesicular glutamate transporters defines two classes of excitatory synapse

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.     

Is active glucose transport present in bovine ciliary body epithelium?

Hyperglycemia is a major risk factor for diabetic cataract formation. Effective regulation of glucose transport by the ciliary body epithelium (CBE) is pivotal to normal glycemic control in the anterior eye, which in turn affects the glucose level of the crystalline lens. The present study aimed to characterize the glucose transport mechanisms across the bovine blood-aqueous barrier (BAB) represented by the CBE. With an Ussing-type chamber, the glucose transport kinetics were measured and characterized in the presence and absence of various glucose transporter inhibitors. The saturation characteristics of the CBE to glucose were estimated from an Eadie-Hofstee plot. The mRNA expression of glucose transporters in specific regions of the bovine CBE was assessed using RT-PCR. The trans-CBE glucose flux was found to be sensitive to the glucose transporter inhibitors cytochalasin B, phloretin, and phlorizin. The transport system had a kinetic constant of 5.3 mM and a maximum velocity of 349.5 nmol.h(-1).cm(-2). Gene expression for GLUT1, GLUT3, GLUT4, GLUT5, and SGLT2 was observed in both the pars plana and pars plicata regions of the bovine CBE. This study demonstrates that glucose transport across the bovine CBE is primarily passive in nature. However, the novel findings of 1) the presence of a phlorizin-sensitive glucose flux and 2) gene expression for SGLT2 mean that a potential role for active glucose transport cannot be ruled out. The elucidation of the exact function of SGLT2 in the bovine CBE may shed important light on the glucose transport and physiology of the BAB and inform future studies of glycemic control in relation to diabetic cataract formation.