Regulation by Insulin and Insulin-Like Growth Factor of 2-Deoxyglucose Uptake in Primary Ependymal Cell Cultures

Ependymal cells have been reported to express the facilitative glucose carriers GLUT1, GLUT2, and GLUT4, as well as glucokinase. They are therefore speculated to be part of the cerebral glucose sensing system and may also respond to insulin with alterations in their glucose uptake rate. A cell culture model was employed to study the functional status of ependymal insulin-regulated glucose uptake in vitro. Insulin increased the uptake of the model substrate 2-deoxyglucose (2-DG) dependent on the insulin concentration. This was due to a near doubling of the maximal 2-DG uptake rate. Insulin-like growth factor (IGF-1) was at least 10 times more potent than insulin in stimulating the rate of ependymal 2-DG uptake, suggesting that IGF-1, rather than insulin, is the physiological agonist regulating glucose transport in ependymal cells. The predominant glucose transporter in ependymal cell cultures was found to be GLUT1, which is apparently regulated by IGF-1 in ependymal cells.     

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.     

Trafficking of glucose transporters-signals and mechanisms

The uptake of glucose into mammalian cells, catalysed by members of the GLUT family of glucose transporters, is regulated by a variety of hormones, growth factors and other agents. In adipocytes, skeletal muscle and heart the principal regulator is the hormone insulin, which rapidly stimulates glucose uptake by bringing about the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular compartment to the cell surface. Recent studies have implicated theC-terminal hydrophilic region of this protein as being primarily responsible for its insulin-regulated trafficking. In an attempt to identify the protein machinery involved in this trafficking, we have used glutathione S-transferase fusion proteins bearing hydrophilic domains of various GLUT transporters in affinity purification experiments on detergent-solubilized extracts of 3T3-L1 adipocyte intracellular membranes. TheC-terminal region of GLUT4 was found specifically to bind a number of polypeptides in these extracts, which are therefore candidates for components of the trafficking machinery. Although these proteins did not bind to the corresponding region of the more widely-distributed GLUT1 glucose transporter isoform, regulation of this transporter also appears to be of physiological importance in some cell types. To study such regulation we have used as a model system the interleukin-3 (IL-3)-dependent haemopoietic cell line IC.DP. These cells express a temperature-sensitive mutane of thev-abl tyrosine kinase, whose activation at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the kinase were found to stimulate glucose transport by promoting the translocation of GLUT1 to the cell surface. Moreover, inhibition of glucose uptake by addition of transport inhibitors markedly increased the rate of apoptosis, an effect which could be reversed by the provision of alternative energy sources. These observations suggest that the trafficking of GLUT1, regulated by growth factors or oncogenes, may play an important role in the suppression of apoptosis in haemopoietic cells.     

Cytochalisin D exerts stimulatory and inhibitory effects on insulin-induced glucokinase mRNA expression in hepatocytes

Developing rat brain undergoes a series of functional and anatomic changes which affect its rate of cerebral glucose utilization (CGU). These changes include increases in the levels of the glucose transporter proteins, GLUT1 and GLUT3, in the blood-brain barrier as well as in the neurons and glia. 55 kDa GLUT1 is concentrated in endothelial cells of the blood-brain barrier, whereas GLUT3 is the predominant neuronal transporter. 45 kDa GLUT1 is in non-vascular brain, probably glia. Studies of glucose utilization with the 2-¹⁴C-deoxyglucose method of Sokoloffet al., (1977), rely on glucose transport rate constants, k₁ and k₂, which have been determined in the adult rat brain. The determination of these constants directly in immature brain, in association with the measurement of GLUT1, GLUT3 and cerebral glucose utilization suggests that the observed increases in the rate constants for the transport of glucose into (k₁) and out of (k₂) brain correspond to the increases in 55 kDa GLUT1 in the blood-brain barrier. The maturational increases in cerebral glucose utilization, however, more closely relate to the pattern of expression of non-vascular GLUT1 (45 kDa), and more specifically GLUT3, suggesting that the cellular expression of the glucose transporter proteins is rate limiting for cerebral glucose utilization during early postnatal development in the rat.     

Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.     

Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxia-inducible factor-1

Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1 as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels. glucose transporter 1; glucose transporter 3; glucose transport     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential.Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1.These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.     

Glucose Transporter 1, Distribution in the Brain and in Neural Disorders: Its Relationship With Transport of Neuroactive Drugs Through the Blood-Brain Barrier

Facilitative glucose transport is mediated by one or more of the members of the closely related glucose transporter (GLUT) family. Thirteen members of the GLUT family have been described thus far. GLUT1 is a widely expressed isoform that provides many cells with their basic glucose requirement. It is also the primary transporter across the blood-brain barrier. This review describes the distribution and expression of GLUT1 in brain in different pathophysiological conditions including Alzheimers disease, epilepsy, ischemia, or traumatic brain injury. Recent investigations show that GLUT1 mediates the transport of some neuroactive drugs, such as glycosylated neuropeptides, low molecular weight heparin, and d-glucose derivatives, across the blood-brain barrier as a delivery system. By utilizing such highly specific transport mechanisms, it should be possible to establish strategies to regulate the entry of candidate drugs.     

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [³H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50–100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.     

Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression

In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.     

Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.     

Wnt1-inducible signaling protein 1 regulates laryngeal squamous cell carcinoma glycolysis and chemoresistance via the YAP1/TEAD1/GLUT1 pathway

Wnt1-inducible signaling protein 1 (WISP1) is a matricellular protein and downstream target of Wnt/β-catenin signaling. This study sought to determine the role of WISP1 in glucose metabolism and chemoresistance in laryngeal squamous cell carcinoma. WISP1 expression was silenced or upregulated in Hep-2 cells by the transfection of WISP1 siRNA or AdWISP1 vector. Ectopic WISP1 expression regulated glucose uptake and lactate production in Hep-2 cells. Subsequently, the expression of glucose transporter 1 (GLUT1) was significantly modulated by WISP1. Furthermore, WISP1 increased cell survival rates, diminished cell death rates, and suppressed ataxia-telangiectasia-mutated (ATM)-mediated DNA damage response pathway in cancer cells treated with cisplatin through GLUT1. WISP1 also promoted cancer cell tumorigenicity and growth in mice implanted with Hep-2 cells. Additionally, WISP1 activated the YAP1/TEAD1 pathway that consequently contributed to the regulation of GLUT1 expression. In summary, WISP1 regulated glucose metabolism and cisplatin resistance in laryngeal cancer by regulating GLUT1 expression. WISP1 may be used as a potential therapeutic target for laryngeal cancer.     

氯化钴增强培养海马神经元葡萄糖转运活性参与抗缺氧的作用

本文用培养新生大鼠海马神经元观察了氯化钴对葡萄糖转运活性的影响及其在神经元抗缺氧中的作用。结果表明,用CoCl2处理的培养海马神经元,24h后其2-脱氧-D-[1-^3H]葡萄糖摄取率和葡萄糖转运体GLUT1和GLUT3mRNA表达明显高于对照组,并且其在缺氧6或8h后的损伤也明显减轻,氯化钴对神经元缺氧损伤的保护作用被葡萄糖转运体抑制剂细胞松弛素B大部分消除,结果提示,氯化钴能够增强神经元GLUT1和GLUT3mRNA的表达和葡萄糖转运活性,CoCl2的这一作用可能是其增强神经元抗缺氧的重要机制。     

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.