Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Domains responsible for the differential targeting of glucose transporter isoforms.

Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. In insulin-responsive tissues such as adipose tissues and muscle, GLUT4 protein resides mainly in the intracellular region in a basal condition and is translocated to the plasma membrane upon stimulation of insulin. In contrast, GLUT1 protein was distributed about equally between plasma membranes and low density microsomal membranes in 3T3-L1 adipocytes. Furthermore, GLUT1 and GLUT4 were reported to be differentially targeted to the plasma membrane and intracellular region, respectively, when expressed in Chinese hamster ovary cells and HepG2 cells. To elucidate the differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1 have been stably expressed in Chinese hamster ovary cells. Immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity revealed that two domains of GLUT4, which are not the NH2- or COOH-terminal domain, determine its targeting to the intracellular vesicles. The first domain contains the consensus sequence of the leucine zipper structure, suggesting that a dimer-forming structure of the glucose transporter might be required for its proper targeting. The other domain contains 28 amino acids, nine of which are different between GLUT1 and GLUT4. Immunoelectron microscopy revealed that the chimeric transporters containing both of these two domains of GLUT1, only the first domain of GLUT1, and none of the domains, exhibited a different cellular distribution with approximately 65, 30, and 15% of the transporters apparently on the plasma membrane, respectively. The addition of insulin did not alter the apparent cellular distributions of these chimeric transporters. These domains would be specifically recognized by intracellular targeting mechanisms in Chinese hamster ovary cells.     

PI 4,5-P2 stimulates glucose transport activity of GLUT4 in the plasma membrane of 3T3-L1 adipocytes

Insulin-stimulated glucose uptake through GLUT4 plays a pivotal role in maintaining normal blood glucose levels. Glucose transport through GLUT4 requires both GLUT4 translocation to the plasma membrane and GLUT4 activation at the plasma membrane. Here we report that a cell-permeable phosphoinositide-binding peptide, which induces GLUT4 translocation without activation, sequestered PI 4,5-P2 in the plasma membrane from its binding partners. Restoring PI 4,5-P2 to the plasma membrane after the peptide treatment increased glucose uptake. No additional glucose transporters were recruited to the plasma membrane, suggesting that the increased glucose uptake was attributable to GLUT4 activation. Cells overexpressing phosphatidylinositol-4-phosphate 5-kinase treated with the peptide followed by its removal exhibited a higher level of glucose transport than cells stimulated with a submaximal level of insulin. However, only cells treated with submaximal insulin exhibited translocation of the PH-domains of the general receptor for phosphoinositides (GRP1) to the plasma membrane. Thus, PI 4,5-P2, but not PI 3,4,5-P3 converted from PI 4,5-P2, induced GLUT4 activation. Inhibiting F-actin remodeling after the peptide treatment significantly impaired GLUT4 activation induced either by PI 4,5-P2 or by insulin. These results suggest that PI 4,5-P2 in the plasma membrane acts as a second messenger to activate GLUT4, possibly through F-actin remodeling.     

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.     

Acute and chronic signals controlling glucose transport in skeletal muscle.

Glucose transport into muscle cells occurs through facilitated diffusion mediated primarily by the GLUT1 and GLUT4 glucose transporters. These transporter proteins are controlled by acute and chronic exposure to insulin, glucose, muscle contraction, and hypoxia. We propose that acute responses occur through recruitment of pre-formed glucose transporters from an intracellular storage site to the plasma membrane. In contrast, chronic control is achieved by changes in transporter biosynthesis and protein stability. Using subcellular fractionation of rat skeletal muscle, recruitment of GLUT4 glucose transporters to the plasma membrane is demonstrated by acute exposure to insulin in vivo. The intracellular pool appears to arise from a unique organelle depleted of transverse tubule, plasma membrane, or sarcoplasmic reticulum markers. In diabetic rats, GLUT4 content in the plasma membranes and in the intracellular pool is reduced, and incomplete insulin-dependent GLUT4 recruitment is observed, possibly through a defective incorporation of transporters to the plasma membrane. The lower content of GLUT4 transporters in the muscle plasma membranes is reversed by restoration of normoglycemia with phlorizin treatment. In some muscle cells in culture, GLUT1 is the only transporter expressed yet they respond to insulin, suggesting that this transporter can also be regulated by acute mechanisms. In the L6 muscle cell line, GLUT1 transporter content diminishes during myogenesis and GLUT4 appears after cell fusion, reaching a molar ratio of about 1:1 in the plasma membrane. Prolonged exposure to high glucose diminishes the amount of GLUT1 protein in the plasma membrane by both endocytosis and reduced biosynthesis, and lowers GLUT4 protein content in the absence of changes in GLUT4 mRNA possibly through increased protein degradation. These studies suggest that the relative contribution of each transporter to transport activity, and the mechanisms by which glucose exerts control of the glucose transporters, will be key subjects of future investigations.     

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.     

The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame

The skeletal muscle cells are one of the main sites of glucose uptake through glucose transporter 4 (GLUT4) in response to insulin. In muscle cells, 5' adenosine monophosphate-activated protein kinase (AMPK) is known as another GLUT4 translocation promoter. Natural compounds that activate AMPK have a possibility to overcome insulin resistance in the diabetic state. Piceatannol is a natural analog and a metabolite of resveratrol, a known AMPK activator. In this study, we investigate the in vitro effect of piceatannol on glucose uptake, AMPK phosphorylation and GLUT4 translocation to plasma membrane in L6 myocytes, and its in vivo effect on blood glucose levels in type 2 diabetic model db/db mice. Piceatannol was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for type 2 diabetes and become an antidiabetic phytochemical.     

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

Fructose transporter in human spermatozoa and small intestine is GLUT5.

We recently reported that the glucose transporter isoform, GLUT5, is expressed on the brush border membrane of human small intestinal enterocytes (Davidson, N. O., Hausman, A. M. L., Ifkovits, C. A., Buse, J. B., Gould, G. W., Burant, C. F., and Bell, G. I. (1992) Am. J. Physiol. 262, C795-C800). To define its role in sugar transport, human GLUT5 was expressed in Xenopus oocytes and its substrate specificity and kinetic properties determined. GLUT5 exhibits selectivity for fructose transport, as determined by inhibition studies, with a Km of 6 mM. In addition, fructose transport by GLUT5 is not inhibited by cytochalasin B, a competitive inhibitor of facilitative glucose transporters. RNA and protein blotting studies showed the presence of high levels of GLUT5 mRNA and protein in human testis and spermatozoa, and immunocytochemical studies localize GLUT5 to the plasma membrane of mature spermatids and spermatozoa. The biochemical properties and tissue distribution of GLUT5 are consistent with a physiological role for this protein as a fructose transporter.     

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body¹. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes^2,3.The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.     

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.     

Ontogenic Changes of Villus Growth,Lactase Activity,and Intestinal Glucose Transporters in Preterm and Term Born Calves with or without Prolonged Colostrum Feeding

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.     

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.     

Role of clusters in insulin-regulated GLUT4 trafficking in adipose cells: A new paradigm?

Insulin stimulates glucose transport in muscle and adipose cells by stimulating translocation of glucose transporter 4 (GLUT4) to the plasma membrane. In a recent Cell Metabolism paper, Stenkula et al. found that insulin controls the spatial distribution of GLUT4 on the surface of isolated adipose cells through regulation of their post-fusion dispersal. The presence of GLUT4 in plasma membrane-associated clusters is suggestive of a new paradigm in membrane protein recycling.     

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.     

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.