Glucose transporters: expression,regulation and cancer

Mammalian cells depend on glucose as a major substrate for energy production. Glucose is transported into the cell via facilitative glucose transporters (GLUT) present in all cell types. Many GLUT isoforms have been described and their expression is cell-specific and subject to hormonal and environmental control. The kinetic properties and substrate specificities of the different isoforms are specifically suited to the energy requirements of the particular cell types. Due to the ubiquitousness of these transporters, their differential expression is involved in various disease states such as diabetes, ischemia and cancer. The majority of cancers and isolated cancer cell lines over-express the GLUT family members which are present in the respective tissue of origin under non-cancerous conditions. Moreover, due to the requirement of energy to feed uncontrolled proliferation, cancer cells often express GLUTs which under normal conditions would not be present in these tissues. This over-expression is predominantly associated with the likelihood of metastasis and hence poor patient prognosis. This article presents a review of the current literature on the regulation and expression of GLUT family members and has compiled clinical and research data on GLUT expression in human cancers and in isolated human cancer cell lines.     

Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.     

ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors

ABSTRACT: BACKGROUND: Intrinsically fluorescent glucose derived carbon nanospheres (CSP) efficiently enter mammalian cells and also cross the blood brain barrier (BBB). However, the mechanistic details of CSP entry inside mammalian cells and its specificity are not known. RESULTS: In this report, the biochemical and cellular mechanism of CSP entry into the living cell have been investigated. We address the issue of mechanism of entry of CSP which provides further insights into its preference towards different cell types. By employing confocal imaging we show that CSP entry into the mammalian cells is an ATP-dependent clathrin mediated endocytosis process. Zeta potential studies suggest that it has a strong preference for cells which possess high levels of glucose transporters such as the glial cells, thereby enabling it to target individual organs/tissues such as the brain with increased specificity. CONCLUSION: The endocytosis of Glucose derived CSP into mammalian cells is an ATP dependent process mediated by clathrin coated pits. CSPs utilize the surface functional groups to target cells containing glucose transporters on its membrane thereby implicating a potential application for specific targeting of the brain or cancer cells.     

Occurrence of pancreatic lipase-related protein-2 in various species and its relationship with herbivore diet

Birds maintain higher plasma glucose concentrations (P(Glu)) than other vertebrates of similar body mass and, in most cases, appear to store comparatively very little glucose intracellularly as glycogen. In general, birds are insensitive to the regulation of P(Glu) by insulin. However, there appears to be no phylogenetic or dietary pattern in the avian response to exogenous insulin. Moreover, the high levels of P(Glu) do not appear to lead to significant oxidative stress as birds are longer-lived compared to mammals. Glucose is absorbed by the avian gastrointestinal tract by sodium-glucose co-transporters (SGLTs; apical side of cells) and glucose transport proteins (GLUTs; basolateral side of cells). In the kidney, both types of glucose transporters appear to be upregulated as no glucose appears in the urine. Data also indicate that the avian nervous system utilizes glucose as a metabolic substrate. In this review, we have attempted to bring together information from a variety of sources to portray how glucose serves as a metabolic substrate for birds by considering each organ system involved in glucose homeostasis.     

Characterization of low- and high-affinity glucose transports in the yeast Kluyveromyces marxianus

Glucose transport in the yeast Kluyveromyces marxianus proceeds by two functionally and presumably structurally distinct transporters depending on the carbon source of the culture medium. In lactose-grown cells, glucose was taken up through a high-affinity H+-sugar symporter (Km = 0.09 mM), whereas a low-affinity transporter (Km = 3.5 mM) was utilized in glucose-grown cells. The two transporters exhibited different substrate specificities. Galactose was demonstrated to be a selective substrate of the H+-glucose symporter (Km = 0.14 mM) and did not significantly enter glucose-grown cells. Fructose was a preferential substrate of the low-affinity carrier (Km = 3.5 mM), but it entered lactose-grown cells through a high-affinity H+-fructose symporter distinct from the H+-glucose one. Other putative substrates of the two glucose transporters were identified by competition experiments. 2-Deoxyglucose recognized both carriers with a similar affinity, while the non-phosphorylatable analogues 6-deoxyglucose, 3-O-methylglucose and D-fucose exhibited a 10-30 fold preference for the high-affinity transporter.     

A Glucose Transporter Can Mediate Ribose Uptake: DEFINITION OF RESIDUES THAT CONFER SUBSTRATE SPECIFICITY IN A SUGAR TRANSPORTER*

Sugars, the major energy source for many organisms, must be transported across biological membranes. Glucose is the most abundant sugar in human plasma and in many other biological systems and has been the primary focus of sugar transporter studies in eukaryotes. We have previously cloned and characterized a family of glucose transporter genes from the protozoan parasite Leishmania. These transporters, called LmGT1, LmGT2, and LmGT3, are homologous to the well characterized glucose transporter (GLUT) family of mammalian glucose transporters. We have demonstrated that LmGT proteins are important for parasite viability. Here we show that one of these transporters, LmGT2, is a more effective carrier of the pentose sugar d-ribose than LmGT3, which has a 6-fold lower relative specificity (V_max/K_m) for ribose. A pair of threonine residues, located in the putative extracellular loops joining transmembrane helices 3 to 4 and 7 to 8, define a filter that limits ribose approaching the exofacial substrate binding pocket in LmGT3. When these threonines are substituted by alanine residues, as found in LmGT2, the LmGT3 permease acquires ribose permease activity that is similar to that of LmGT2. The location of these residues in hydrophilic loops supports recent suggestions that substrate recognition is separated from substrate binding and translocation in this important group of transporters.     

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.     

Demonstration of an insulin-insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells.

The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose-sensitive cytochalasin B binding and by protein immunoblotting using isoform-specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5'-nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT-2 occurred in a low-density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT-2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT-1 and GLUT-2, the two isoforms showed distinct subcellular distribution patterns: GLUT-2 was highly concentrated in LDM while very little GLUT-1 was found in this fraction, indicating that a large portion of GLUT-2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT-2, but lack the insulin-responsive glucose transporter translocation mechanism.     

GLUT,SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters

Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. Three families of secondary glucose transporters have been identified in human, including the major facilitator superfamily glucose facilitators GLUTs, the sodium‐driven glucose symporters SGLTs, and the recently identified SWEETs. Structures of representative members or their prokaryotic homologs of all three families were obtained. This review focuses on the recent advances in the structural elucidation of the glucose transporters and the mechanistic insights derived from these structures, including the molecular basis for substrate recognition, alternating access, and stoichiometric coupling of co‐transport.     

Herbivory-induced glucose transporter gene expression in the brown planthopper,Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

A second Warburg-like effect in cancer metabolism: The metabolic shift of glutamine-derived nitrogen

Carbon and nitrogen are essential elements for life. Glucose as a carbon source and glutamine as a nitrogen source are important nutrients for cell proliferation. About 100 years ago, it was discovered that cancer cells that have acquired unlimited proliferative capacity and undergone malignant evolution in their host manifest a cancer-specific remodeling of glucose metabolism (the Warburg effect). Only recently, however, was it shown that the metabolism of glutamine-derived nitrogen is substantially shifted from glutaminolysis to nucleotide biosynthesis during malignant progression of cancer—which might be referred to as a “second” Warburg effect. In this review, address the mechanism and relevance of this metabolic shift of glutamine-derived nitrogen in human cancer. We also examine the clinical potential of anticancer therapies that modulate the metabolic pathways of glutamine-derived nitrogen. This shift may be as important as the shift in carbon metabolism, which has long been known as the Warburg effect.     

A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.     

Structural Events in a Bacterial Uniporter Leading to Translocation of Glucose to the Cytosol

Among classes of sugar transporters, there exists a comparatively new family of transporters named SWEET transporters (semi-SWEETS in bacteria) that are uniport transmembrane proteins. It is hypothesized that sugar is transported from the extracellular side (via outward-open state) to intracellular side (inward-open state) through intermediate occluded state (both extracellular and intracellular gates closed). In our study, extensive unbiased all-atom molecular dynamics simulations were carried out with the outward-open and inward-open conformations to study this transition mechanism. We find that after 100?ns, the outward-open structure without sugar bound starts changing to the occluded form leading to closure of extracellular gates stabilized by electrostatic and hydrophobic interactions. Further simulations (up to 7?μs) have led to a transition toward the inward-open form and suggest that there exists more than one intermediate occluded conformation. We have also performed 5-μs simulations on the glucose-docked structure to identify the putative substrate-bound translocation pathway. Glucose binds to semi-SWEET with strong hydrogen bonds to Asn66 and Trp50. Comparative simulations of substrate bound, and unbound forms suggested that glucose, the putative substrate, facilitates relatively rapid conformational transitions. For the first time, we captured the release of glucose to the cytosol, in this family of transporters. We find that prior to release of glucose, the glucose forms interactions with polar residues near the intracellular gate which may facilitate its release. The distance between the residues Asn31 and Gly34 of the other protomer was found to play a decisive role in the transport of glucose to the cytoplasmic side.     

锌转运蛋白基因研究进展

锌作为一种重要的微量元素参与了植物体内广泛的生理和生化过程,本文详细介绍了涉及Zn^2＋吸收转运的ZIP基因家族（ZRT／IRT相关蛋白）和CDF（Cation　diffusion　facilitator）家族。ZIP家族转运蛋白主要负责将Zn^2＋等二价阳离子跨膜转运进细胞内,以完成细胞内多种生理生化反应。CDF家族转运蛋白主要负责将过量Zn^2＋运出细胞,或者将细胞内过量Zn^2＋进行区室化隔离,降低Zn^2＋对细胞的危害作用。ZIP家族转运蛋白和CDF家族转运蛋白的相互协调使得Zn^2＋在细胞和有机体水平上维持着稳态,进而为细胞内各种生理生化反应的进行供一种保障机制。     

Mathematical model of cooperative work of ion pump,symport and antiport in epithelial cells

Transcellular transport in epithelial cells plays an important role in providing such physiological functions as excretion of cytotoxic substances or reabsorption of metabolites useful for the body life activity. These functions have been shown to be performed by the mechanisms—symport, antiport, ion pumps, and channels—that often function cooperatively. Models for kinetic peculiarities of the substrate transport with the aid of the above mechanisms are widely described in the literature. Much less attention is paid to modeling of cooperative activity of transporters that have different transport mechanisms. In this work we propose a mathematical model for flux coupling of three transporters—the ion pump, symporter, and antiporter as well as of two substrates, one of which (A) can be transported simultaneously by the symport and antiport mechanisms, while the other (B)—only by the latter mechanism. Analysis of the model has shown that for the pair of substrates (A and B) the flux coupling becomes possible if the following conditions are met: (1) the substrate A flux into the internal cell volume using the symport mechanism is to exceed its antiporter-realized flux in the opposite direction; (2) probability of reorientation from one side of membrane to the other side for the antiporter loaded with the substrate is to be essentially higher than that for empty transporter. The proposed model can be used for comparing efficiency both of excretion and of reabsorption of cell metabolites in representatives of different taxa.     

The involvement of phosphatidylinositol 3-kinase /Akt signaling in high glucose-induced downregulation of GLUT-1 expression in ARPE cells

Glucose transporters have been reported to be associated with the development of diabetic retinopathy. Retinal pigment epithelial cells (RPEs) are believed to play an important role in the pathogenesis of diabetic retinopathy. However, the effect of hyperglycemia on glucose transporters in RPEs and the related signal pathways have not yet been elucidated. Therefore, we examined the effect of high glucose on the glucose transporter 1 in ARPEs and the related signal molecules. In the present study, high glucose decreased 2-deoxyglucose uptake in a time (>2 h) and dose dependent manner. In addition, we found that high glucose downregulated the expression of glucose transporter 1 (GLUT-1). The high glucose-induced downregulation of GLUT-1 was blocked by Wortmanin, LY 294002 (PI-3 kinase inhibitors) and Akt (Akt inhibitor). The high glucose increased stimulation of Akt activation in a time dependent manner. We also investigated the upstream regulator of Akt activation. The high glucose-induced phosphorylation of Akt was blocked by bisindolymaleimide I, H-7, staurosporine (protein kinase C [PKC] inhibitors), vitamin C and catalase (antioxidants). In addition, the high glucose-induced downregulation of GLUT-1 was also blocked by PKC inhibitors and antioxidants. Moreover, high glucose increased lipid peroxide formation, which was prevented by PKC inhibitors. In conclusion, high glucose downregulated GLUT-1 by Akt pathway activation mediated by the PKC-oxidative stress signaling pathway in ARPE cells.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.