Human Erythrocyte Glucose Transporter Research Articles

Hydration of AMP and ATP Molecules in Aqueous Solution and Solid Films

Water enables life and plays a critical role in biology. Considered as a versatile and adaptive component of the cell, water engages a wide range of biomolecular interactions. An organism can exist and function only if its self-assembled molecular structures are hydrated. It was shown recently that switching of AMP/ATP binding to the insulin-independent glucose transporter Human Erythrocyte Glucose Transport Protein (GLUT1) may greatly influence the ratio of bulk and bound water during regulation of glucose uptake by red blood cells. In this paper, we present the results on the hydration properties of AMP/ATP obtained by means of dielectric spectroscopy in aqueous solution and for fully ionized forms in solid amorphous films with the help of gravimetric studies.

Shuttling glucose across brain microvessels, with a little help from GLUT1 and AMP kinase. Focus on “AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress”

The Blood-Brain Barrier: a Gatekeeper of Brain “Individuality” The blood-brain barrier (BBB) maintains the individuality of brain fluid, while allowing it to selectively import nutrients and process toxic products. Its major constituent is a single layer of continuous, nonfenestrated endothelium lining the microvessels, whose unique features account in large part for the integrity of the barrier. The endothelial cell monolayer is the major determinant, but other cells in the central nervous system (e.g., pericytes and astrocytes) contribute to the integrity of the BBB. The interendothelial tight junctions of the BBB are one of its signature elements that greatly restrict paracellular permeability to solutes, fluid, and transmigrating cells. This is markedly distinct from the leakier, discontinuous, and fenestrated endothelium of liver microvessels (sinusoids). For example, the tight junctions of the cerebral endothelium (pial vessels) confer an electrical resistance (1,300 ·cm 2 ) far higher than other endothelia (6). Therefore, communication between blood and the brain interstitial fluid requires individually tailored molecular mechanisms. Transcytosis is the movement of molecules through a cellular barrier. Whereas proteins can traverse through vesicular carrier processes that could carry solutes through fluid phase endocytosis, the brain microvasculature is uniquely poor in plasmalemmal and cytoplasmic caveolae (1). Instead, bidirectional passage of small molecules across the BBB is stringently regulated by numerous transporters and receptors. These typically work in tandem across the luminal and abluminal membranes of the endothelial cell.

Next-generation Akt inhibitors provide greater specificity: effects on glucose metabolism in adipocytes

Many human tumours exhibit activation of the PI3K (phosphoinositide 3-kinase)/Akt pathway, and inhibition of this pathway slows tumour growth. This led to the development of specific Akt inhibitors for in vivo use. However, activation of Akt is also necessary for processes including glucose metabolism. Therefore a potential complication of such anticancer drugs is insulin resistance and/or diabetes. In the process of characterizing the metabolic effects of early-phase Akt inhibitors, we discovered an off-target inhibitory effect on mammalian facilitative glucose transporters. In view of the crucial role of glucose transport for all mammalian cells, such an off-target effect would have major implications for further development of this family of compounds. In the present study, we have characterized a next-generation Akt inhibitor, MK-2206. MK-2206 is an orally active allosteric Akt inhibitor under development for treating solid tumours. We report that MK-2206 potently inhibits Thr308Akt and Ser473Akt phosphorylation in 3T3-L1 adipocytes (IC50 0.11 and 0.18 μM respectively) as well as downstream effects of insulin on GLUT4 (glucose transporter 4) translocation (IC50 0.47 μM) and glucose transport (IC50 0.14 μM). Notably, the potency of MK-2206 is approximately 1 log higher than previous inhibitors and its specificity is significantly improved with modest inhibitory effects on glucose transport in GLUT4-expressing adipocytes and GLUT1-rich human erythrocytes, independently of Akt. Nevertheless, MK-2206 clearly has potent effects on Akt2, the principal isoform involved in peripheral insulin action, in which case insulin resistance will probably be a major complication following in vivo administration. We conclude that MK-2206 provides an optimal tool for studying the effects of Akt in vitro.

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

Synaptic Vesicle Protein 2 Binds Adenine Nucleotides

Synaptic vesicle protein 2 (SV2) is required for normal calcium-regulated secretion of hormones and neurotransmitters. Neurons lacking the two most widely expressed isoforms, SV2A and SV2B, have a reduced readily releasable pool of synaptic vesicles, indicating that SV2 contributes to vesicle priming. The presence of putative ATP-binding sites in SV2 suggested that SV2 might be an ATP-binding protein. To explore this, we examined the binding of the photoaffinity reagent 8-azido-ATP[gamma] biotin to purified, recombinant SV2 in the presence and absence of other nucleotides. Our results indicate that SV2A and SV2B bind nucleotides, with the highest affinity for adenine-containing nucleotides. SV2A contains two binding sites located in the cytoplasmic domains preceding the first and seventh transmembrane domains. These results suggest that SV2-mediated vesicle priming could be regulated by adenine nucleotides, which might provide a link between cellular energy levels and regulated secretion.

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility Analysis of Transmembrane Segment 4 of the Glut1 Glucose Transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.

Sclerosing perineurioma: tumor of the hand with a short T2

We present two cases of sclerosing perineurioma, a rare soft tissue tumor, in the palm and the ring finger respectively, presenting as a small, painless and subcutaneous mass. This tumor has a predilection for the digits and palms of young, predominantly male adults. In the present cases the tumors showed very low signal intensity on T2-weighted magnetic resonance (MR) images. Histologically they contained abundant collagen and hyalinized stroma, which would account for areas of low signal intensity on T2-weighted MR images. Immunohistochemically, the tumor cells were positive for vimentin, epithelial membrane antigen and human erythrocyte glucose transporter 1 and negative for S-100 protein. To the best of our knowledge, the appearance of sclerosing perineurioma on MR imaging has not been previously reported in the English-language literature. Sclerosing perineurioma should be considered in the differential diagnosis of hand tumors when the tumor shows low signal intensity on T2-weighted MR images.

Overexpression of glucose transporter 1 in esophageal squamous cell carcinomas: a marker for poor prognosis

Recently, Glut1 (human erythrocyte glucose transporter) expression has been demonstrated in various tumors. The aim of this study is to evaluate the prognostic utility of Glut1 expression in esophageal carcinomas. We studied Glut1 expression by immunohistochemistry of paraffin sections from 63 esophageal squamous cell carcinomas. All 63 carcinomas expressed Glut1. The mean percentage of positively stained tumor cells was 77.8% (median, 84.7%). There were two staining patterns in positive cells: 'strongly positive' and 'weakly positive'. The percentage of 'strongly positive' cells (%Glut1-SP) ranged from 0% to 95.6% (mean, 32.3%; median, 27.4%). The 5-year survival rate for patients with a high %Glut1-SP (> 30%) was significantly lower than that for patients with a low %Glut1-SP (< 30%) (P < 0.01). Statistical analysis revealed that the relative risk of death for patients with high %Glut1-SP was 2.02 times that for patients with low %Glut1-SP (P = 0.064), suggesting a possible independent predictive value for %Glut1-SP.

Properties of the Human Erythrocyte Glucose Transport Protein Are Determined by Cellular Context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.

Fatty acid synthase is a marker of increased risk of recurrence in endometrial carcinoma

Purpose. To explore the expression of fatty acid synthase (FAS) and human erythrocyte glucose transporter 1 (GLUT1) in endometrial carcinomas and to detect associations with clinicopathological features and prognosis. FAS and GLUT1 are two molecules involved in energy supply of normal cells. These markers are overexpressed in neoplastic tissues because of their increased necessity of energy. Methods. Ninety-five patients with endometrial carcinoma were followed-up for an average period of 5 years. FAS and GLUT1 expressions were evaluated by immunohistochemistry on formalin-fixed paraffin-embedded tissues. Staining was determined with a semiquantitative method. Negative controls were obtained from patients submitted to hysterectomy for uterine prolapse. Results. Eighty-five cases were endometrioid, 7 were serous, and 1 was a mucinous carcinoma. Seventy-two cases (75%) were stage I, 12 (13%) were stage II, and 11 (12%) were stage III carcinomas. Sixteen (15%) carcinomas recurred. Nine patients (8%) died for cancer during the follow-up period. FAS expression was observed in 53 cases (56%). GLUT1 expression was observed in 32 (43%) cases. Statistical analysis revealed that FAS ( P = 0.04) and stage ( P = 0.001) of the disease were the only two independent predictors of recurrence. GLUT1 and other clinicopathologic parameters had no prognostic association. Conclusions. FAS is a reliable marker of clinically aggressive endometrial carcinomas. The knowledge of FAS expression in endometrial carcinomas is an important finding that may stratify patients into selected groups and determine therapeutic approaches for patient care.

Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose-binding site of the human erythrocyte glucose transporter GLUT1.

This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea. Testosterone, 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA) and DHEA-3-acetate inhibit glucose exit from human erythrocytes with half-maximal inhibitions (Ki) of 39.2+/-8.9, 29.6+/-3.7, 48.1+/-10.2 and 4.8+/-0.98 microM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR). Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3-gallate (ECG) has a Ki ECG of 0.14+/-0.01 microM, and epigallocatechin 3-gallate (EGCG) has a Ki EGCG of 0.97+/-0.13 microM. Flutamide reverses these effects. Androgen-screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action. There are sequence homologies between GLUT1 and the ligand-binding domain (LBD) of hAR containing the amino-acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3-keto and 17-beta OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.

Sclerosing perineurioma: a clinicopathological study of five cases and diagnostic utility of immunohistochemical staining for GLUT1.

We reviewed five cases of sclerosing perineurial tumor of the hand. Four patients were male and one was female with ages ranging from 11 years to 49 years (mean 26 years). The predominant reason for consultation at the outpatient clinic was a slowly growing painless mass. The sites of involvement were the thumb in two cases, and the ring finger, middle finger and palm in one case each. The lesions were hard and firm, well-circumscribed white masses with a fibrous consistency ranging from 1.2 cm to 4.0 cm (mean 2.5 cm) in maximum dimension. Microscopically, all the tumors were composed of thick collagen and variable numbers of small, epithelioid cells exhibiting corded, trabecular and whorled growth patterns. Electron microscopy showed long cytoplasmic processes with a discontinuous basal lamina and occasional pinocytotic vesicles in the tumor cells. Immunohistochemically, most of the tumor cells were positive for epithelial membrane antigen, vimentin, collagen type IV and CD10, but not for S-100 protein, CD34, desmin and cytokeratin. We also observed that the tumor cells were positive for the human erythrocyte glucose transporter (GLUT1) antigen, suggesting that GLUT1 may be a useful marker for the identification of sclerosing perineurioma.

Cooperative nucleotide binding to the human erythrocyte sugar transporter.

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.

Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1.

17 beta-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (K(i(ED/FAS))=2.84+/-0.16 microM and K(i(ED/TAM))=100+/-2 nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (K(i(TAM))=300+/-100 nM). In ghosts, ATP (1-4 mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (K(i(ATP/ED))=2.5+/-0.23 mM, K(i(ATP/GEN))=0.99+/-0.17 mM and K(i(ATP/CB))=0.76+/-0.08 mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1 mM (K(i(ED/TAM))=130+/-5 nM and K(i(ED/FAS))=2.7+/-0.9 microM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor beta (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225-229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421-423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His(337), Arg(349) and Glu(249) at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His(475), Glu(305) and Arg(346) in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export.

Detection of two distinct transporter systems for 2-deoxyglucose uptake by the opportunistic pathogen Pneumocystis carinii

Since the opportunistic pathogen Pneumocystis carinii grows only slowly in vitro, the mechanism of glucose uptake was investigated to better understand how the organism transports nutrients. Using the non-metabolizable analogue 2-deoxyglucose, two uptake systems were detected with Q 10 values of 2.12 and 2.09, respectively. One had a high affinity ( K m=67.5 μM) and the other a low affinity ( K m=5.99 mM) for 2-deoxyglucose uptake. Glucose or deoxyglucose phosphate products from transported radiolabeled substrates were not detected during the incubation times used in this study. Both systems were inhibited by mannose, galactose, fructose, galactosamine, glucosamine, and glucose but not by allose, 5-thioglucose, xylose, glucose 6-phosphate and glucuronic acid. Salicylhydroxamate, KCN, iodoacetate, and 2,4-dinitrophenol inhibited the high-affinity transporter, suggesting it required ATP. Ouabain, monensin, carbonyl cyanide m-chlorophenylhydrazone, and N, N′-dicyclohexylcarbodiimide also inhibited deoxyglucose uptake, as did the replacement of Na + in the incubation medium with choline, indicating requirements for Na + and H +. The high-affinity system was also inhibited by the protein synthesis inhibitors cycloheximide and chloramphenicol. In contrast, the low-affinity system transported deoxyglucose by facilitated diffusion mechanisms. Unlike the human erythrocyte glucose transporter GLUT1, the P. carinii transporters recognized fructose and galactose and were relatively insensitive to cytochalasin B, suggesting that the P. carinii glucose transporters may be good drug targets.

Expression of the human erythrocyte glucose transporter Glut-1 in areas of sclerotic collagen in necrobiosis lipoidica.

Glut-1 is the human erythrocyte glucose transporter which mediates facilitative transport of glucose across epithelial and endothelial barrier tissues. A primary abnormality in glucose transport and Glut-1 cell-surface content has been observed in fibroblasts from NIDDM and obese individuals. With the strong correlation between necrobiosis lipoidica and diabetes mellitus, we investigated the expression of Glut-1 in diabetic individuals with necrobiosis lipoidica (NL). A polyclonal anti-Glut-1 antibody was used with a standard immunoperoxidase technique to determine Glut-1 expression by fibroblasts in areas of sclerotic collagen from specimens taken from diabetic individuals with necrobiosis lipoidica,and non-diabetic individuals with scars and granuloma annulare (GA). Our results showed Glut-1 expression in the areas of sclerotic collagen in patients with NL, possibly contributing to insulin resistance in these tissues. Our findings raises the question as to whether abnormalities in glucose transport by fibroblasts in individuals with necrobiosis lipoidica contribute to the histopathologic changes.

Immunohistochemical expression of human erythrocyte glucose transporter and fatty acid synthase in infiltrating breast carcinomas and adjacent typical/atypical hyperplastic or normal breast tissue.

To evaluate the immunohistochemical expression of GLUT1, human erythrocyte glucose transporter 1, and fatty acid synthase (FAS), 66 human breast carcinomas and adjacent peritumoral tissue were studied. GLUT1 and FAS were expressed in 53 and 61 carcinomas, in 17 and 14 typical/atypical hyperplastic tissues, and in 16 and 13 tissues adjacent to tumor normal breast tissue, respectively. Statistical analysis revealed association between invasive carcinomas, invasive carcinomas with in situ component and GLUT1 immunostaining. GLUT1 staining was associated with tumor grade, FAS with tumor stage, and GLUT1 and FAS coexpression with tumor grade. Controls expressed no immunostaining. GLUT1 and FAS are new markers involved in the biologic activities of cancer cells. GLUT1 and FAS coexpression may indicate increased use of energy by the neoplastic cells correlated with poorly differentiated features and aggressive behavior. The innovative finding that GLUT1 and FAS are observed in mammary carcinoma adjacent nonneoplastic tissues may suggest a role in detecting initial phases of breast carcinogenesis.

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.

Clinical Significance of Human Erythrocyte Glucose Transporter 1 Expression at the Deepest Invasive Site of Advanced Colorectal Carcinoma

Objective: Malignant cells exhibit increased glucose uptake and utilization in vitro and in vivo. This process is thought to be mediated by the glucose transporter (Glut) family. The aim of this study was to elucidate the clinical significance of Glut1 expression at the site of deepest invasion as a predictor of the invasive/metastatic potential and prognosis of advanced colorectal carcinoma (CRC). Methods: One hundred and fifty-two patients who had undergone surgical resection for advanced CRC were entered in this study. Histologic subclassifications at the deepest invasive site included well-differentiated (W), moderately to well-differentiated (Mw), moderately to poorly differentiated (Mp), poorly differentiated (Por) and mucinous (Muc) adenocarcinomas. Glut1 expression was examined immunohistochemically with a labeled streptavidin-biotin kit using anti-Glut1 polyclonal antibody MYM. As a marker of cell proliferation, Ki-67 expression was also examined. All immunoreactivity was analyzed at the deepest invasive site, central portion and superficial part. The immunohistochemical expression of Glut1 was defined as positive if distinct staining of the membrane or cytoplasm was observed in at least 30% of tumor cells. Results: Glut1 expression was detected in 56 of 152 lesions (36.8%) at the deepest invasive site. The incidence of Glut1 expression at the deepest invasive site correlated significantly with histologic grade (W/Mw grade, 28% vs. Mp/Por/Muc grade, 48%), depth of invasion (invasion of muscularis propria/invasion of subserosa or subadventitia, 29% vs. invasion of serosa or adventitia/invasion of adjacent structures, 52%), lymphatic invasion (absence of lymphatic invasion, 19% vs. presence of lymphatic invasion, 40%), lymph node metastasis (absence of lymph node metastasis, 25% vs. presence of lymph node metastasis, 41%) and Duke’s stage (Duke’s <D, 32% vs. Duke’s D, 51%). In the central portion and superficial part, there were no significant differences between Glut1, Ki-67 expression and clinicopathological findings. Glut1 expression at the deepest invasive site correlated significantly with the Ki-67 labeling index. In cases of curative surgery, patients with Glut1-positive lesions at the deepest invasive site showed a significantly poorer prognosis than those with Glut1-negative lesions. Multivariate analysis with logistic regression for 5-year survival in patients who had undergone curative surgery showed that lymph node metastasis and Glut1 expression were significant risk factors. Conclusion: These results indicate that Glut1 expression at the deepest site of tumor invasion can be a useful predictor of a high malignant potential and poor prognosis in advanced CRC.