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Sample records for affinity glucose transporters

  1. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

    PubMed Central

    Kruckeberg, A L; Bisson, L F

    1990-01-01

    The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes. Images PMID:2233722

  2. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    PubMed Central

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  3. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  4. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis

    PubMed Central

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-01-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose–H+ symporter. Analyses of STP10 mRNA and STP10 promoter–reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  5. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis.

    PubMed

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-04-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  6. A MEMS Dielectric Affinity Glucose Biosensor.

    PubMed

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-06-20

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  7. A Differential Dielectric Affinity Glucose Sensor

    PubMed Central

    Huang, Xian; Leduc, Charles; Ravussin, Yann; Li, Siqi; Davis, Erin; Song, Bing; Li, Dachao; Xu, Kexin; Accili, Domenico; Wang, Qian; Leibel, Rudolph; Lin, Qiao

    2013-01-01

    A continuous glucose monitor with a differential dielectric sensor implanted within the subcutaneous tissue that determines the glucose in the interstitial fluid is presented. The device, created using microelectromechanical systems (MEMS) technology, consists of sensing and reference modules that are identical in design and placed in close proximity. Each module contains a microchamber housing a pair of capacitive electrodes residing on the device substrate and embedded in a suspended, perforated polymer diaphragm. The microchambers, enclosed in semi-permeable membranes, are filled with either a polymer solution that has specific affinity to glucose or a glucose-insensitive reference solution. To accurately determine the glucose concentration, changes in the permittivity of the sensing and the reference solutions induced by changes in glucose concentration are measured differentially. In vitro characterization demonstrated the sensor capable of measuring glucose concentrations from 0 to 500 mg/dL with resolution and accuracy of ∼1.7 μg/dL and ∼1.74 mg/dL, respectively. In addition, device drift was reduced to 1.4% (uncontrolled environment) and 11% (5 °C of temperature variation) of that from non-differential measurements, indicating significant stability improvements. Preliminary animal testing demonstrated that the differential sensor accurately tracks glucose concentration in blood. This sensor can potentially be used clinically as a subcutaneously implanted continuous monitoring device in diabetic patients. PMID:24220675

  8. Glucose transport in brain - effect of inflammation.

    PubMed

    Jurcovicova, J

    2014-01-01

    Glucose is transported across the cell membrane by specific saturable transport system, which includes two types of glucose transporters: 1) sodium dependent glucose transporters (SGLTs) which transport glucose against its concentration gradient and 2) sodium independent glucose transporters (GLUTs), which transport glucose by facilitative diffusion in its concentration gradient. In the brain, both types of transporters are present with different function, affinity, capacity, and tissue distribution. GLUT1 occurs in brain in two isoforms. The more glycosylated GLUT1 is produced in brain microvasculature and ensures glucose transport across the blood brain barrier (BBB). The less glycosylated form is localized in astrocytic end-feet and cell bodies and is not present in axons, neuronal synapses or microglia. Glucose transported to astrocytes by GLUT1 is metabolized to lactate serving to neurons as energy source. Proinflammatory cytokine interleukin (IL)-1β upregulates GLUT1 in endothelial cells and astrocytes, whereas it induces neuronal death in neuronal cell culture. GLUT2 is present in hypothalamic neurons and serves as a glucose sensor in regulation of food intake. In neurons of the hippocampus, GLUT2 is supposed to regulate synaptic activity and neurotransmitter release. GLUT3 is the most abundant glucose transporter in the brain having five times higher transport capacity than GLUT1. It is present in neuropil, mostly in axons and dendrites. Its density and distribution correlate well with the local cerebral glucose demands. GLUT5 is predominantly fructose transporter. In brain, GLUT5 is the only hexose transporter in microglia, whose regulation is not yet clear. It is not present in neurons. GLUT4 and GLUT8 are insulin-regulated glucose transporters in neuronal cell bodies in the cortex and cerebellum, but mainly in the hippocampus and amygdala, where they maintain hippocampus-dependent cognitive functions. Insulin translocates GLUT4 from cytosol to plasma

  9. Facilitated diffusion of 6-deoxy-D-glucose in bakers' yeast: evidence against phosphorylation-associated transport of glucose.

    PubMed Central

    Romano, A H

    1982-01-01

    6-Deoxy-D-glucose, a structural homomorph of D-glucose which lacks a hydroxyl group at carbon 6 and thus cannot be phosphorylated, is transported by Saccharomyces cerevisiae via a facilitated diffusion system with affinity equivalent to that shown with D-glucose. This finding supports the facilitated diffusion mechanism for glucose transport and contradicts theories of transport-associated phosphorylation which hold that sugar phosphorylation is necessary for high-affinity operation of the glucose carrier. PMID:6754704

  10. Identification of Glucose Transporters in Aspergillus nidulans

    PubMed Central

    dos Reis, Thaila Fernanda; Menino, João Filipe; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; Colabardini, Ana Cristina; Savoldi, Marcela; Goldman, Maria Helena S.; Rodrigues, Fernando; Goldman, Gustavo Henrique

    2013-01-01

    To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose. PMID:24282591

  11. A MEMS differential viscometric sensor for affinity glucose detection in continuous glucose monitoring.

    PubMed

    Huang, Xian; Li, Siqi; Davis, Erin; Leduc, Charles; Ravussin, Yann; Cai, Haogang; Song, Bing; Li, Dachao; Accili, Domenico; Leibel, Rudolph; Wang, Qian; Lin, Qiao

    2013-05-01

    Micromachined viscometric affinity glucose sensors have been previously demonstrated using vibrational cantilever and diaphragm. These devices featured a single glucose detection module that determines glucose concentrations through viscosity changes of glucose-sensitive polymer solutions. However, fluctuations in temperature and other environmental parameters might potentially affect the stability and reliability of these devices, creating complexity in their applications in subcutaneously implanted continuous glucose monitoring (CGM). To address these issues, we present a MEMS differential sensor that can effectively reject environmental disturbances while allowing accurate glucose detection. The sensor consists of two magnetically driven vibrating diaphragms situated inside microchambers filled with a boronic-acid based glucose-sensing solution and a reference solution insensitive to glucose. Glucose concentrations can be accurately determined by characteristics of the diaphragm vibration through differential capacitive detection. Our in-vitro and preliminary in-vivo experimental data demonstrate the potential of this sensor for highly stable subcutaneous CGM applications. PMID:23956499

  12. A MEMS differential viscometric sensor for affinity glucose detection in continuous glucose monitoring

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Davis, Erin; Leduc, Charles; Ravussin, Yann; Cai, Haogang; Song, Bing; Li, Dachao; Accili, Domenico; Leibel, Rudolph; Wang, Qian; Lin, Qiao

    2013-05-01

    Micromachined viscometric affinity glucose sensors have been previously demonstrated using vibrational cantilever and diaphragm. These devices featured a single glucose detection module that determines glucose concentrations through viscosity changes of glucose-sensitive polymer solutions. However, fluctuations in temperature and other environmental parameters might potentially affect the stability and reliability of these devices, creating complexity in their applications in subcutaneously implanted continuous glucose monitoring (CGM). To address these issues, we present a MEMS differential sensor that can effectively reject environmental disturbances while allowing accurate glucose detection. The sensor consists of two magnetically driven vibrating diaphragms situated inside microchambers filled with a boronic-acid based glucose-sensing solution and a reference solution insensitive to glucose. Glucose concentrations can be accurately determined by characteristics of the diaphragm vibration through differential capacitive detection. Our in vitro and preliminary in vivo experimental data demonstrate the potential of this sensor for highly stable subcutaneous CGM applications.

  13. A MEMS differential viscometric sensor for affinity glucose detection in continuous glucose monitoring

    PubMed Central

    Huang, Xian; Li, Siqi; Davis, Erin; Leduc, Charles; Ravussin, Yann; Cai, Haogang; Song, Bing; Li, Dachao; Accili, Domenico; Leibel, Rudolph; Wang, Qian; Lin, Qiao

    2013-01-01

    Micromachined viscometric affinity glucose sensors have been previously demonstrated using vibrational cantilever and diaphragm. These devices featured a single glucose detection module that determines glucose concentrations through viscosity changes of glucose-sensitive polymer solutions. However, fluctuations in temperature and other environmental parameters might potentially affect the stability and reliability of these devices, creating complexity in their applications in subcutaneously implanted continuous glucose monitoring (CGM). To address these issues, we present a MEMS differential sensor that can effectively reject environmental disturbances while allowing accurate glucose detection. The sensor consists of two magnetically driven vibrating diaphragms situated inside microchambers filled with a boronic-acid based glucose-sensing solution and a reference solution insensitive to glucose. Glucose concentrations can be accurately determined by characteristics of the diaphragm vibration through differential capacitive detection. Our in-vitro and preliminary in-vivo experimental data demonstrate the potential of this sensor for highly stable subcutaneous CGM applications. PMID:23956499

  14. A MEMS affinity glucose sensor using a biocompatible glucose-responsive polymer

    PubMed Central

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2013-01-01

    We present a MEMS affinity sensor that can potentially allow long-term continuous monitoring of glucose in subcutaneous tissue for diabetes management. The sensing principle is based on detection of viscosity changes due to affinity binding between glucose and poly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA), a biocompatible, glucose-specific polymer. The device uses a magnetically driven vibrating microcantilever as a sensing element, which is fabricated from Parylene and situated in a microchamber. A solution of PAA-ran-PAAPBA fills the microchamber, which is separated from the surroundings by a semi-permeable membrane. Glucose permeates through the membrane and binds reversibly to the phenylboronic acid moiety of the polymer. This results in a viscosity change of the sensing solution, which is obtained by measuring the damped cantilever vibration using an optical lever setup, allowing determination of the glucose concentration. Experimental results demonstrate that the device is capable of detecting glucose at physiologically relevant concentrations from 27 mg/dL to 324 mg/dL. The glucose response time constant of the sensor is approximately 3 min, which can be further improved with device design optimization. Excellent reversibility and stability are observed in sensor responses, as highly desired for long-term, stable continuous glucose monitoring. PMID:24511207

  15. A MEMS affinity glucose sensor using a biocompatible glucose-responsive polymer.

    PubMed

    Huang, Xian; Li, Siqi; Schultz, Jerome S; Wang, Qian; Lin, Qiao

    2009-07-16

    We present a MEMS affinity sensor that can potentially allow long-term continuous monitoring of glucose in subcutaneous tissue for diabetes management. The sensing principle is based on detection of viscosity changes due to affinity binding between glucose and poly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA), a biocompatible, glucose-specific polymer. The device uses a magnetically driven vibrating microcantilever as a sensing element, which is fabricated from Parylene and situated in a microchamber. A solution of PAA-ran-PAAPBA fills the microchamber, which is separated from the surroundings by a semi-permeable membrane. Glucose permeates through the membrane and binds reversibly to the phenylboronic acid moiety of the polymer. This results in a viscosity change of the sensing solution, which is obtained by measuring the damped cantilever vibration using an optical lever setup, allowing determination of the glucose concentration. Experimental results demonstrate that the device is capable of detecting glucose at physiologically relevant concentrations from 27 mg/dL to 324 mg/dL. The glucose response time constant of the sensor is approximately 3 min, which can be further improved with device design optimization. Excellent reversibility and stability are observed in sensor responses, as highly desired for long-term, stable continuous glucose monitoring. PMID:24511207

  16. A Capacitive MEMS Viscometric Sensor for Affinity Detection of Glucose

    PubMed Central

    Huang, Xian; Li, Siqi; Schultz, Jerome; Wang, Qian; Lin, Qiao

    2013-01-01

    This paper presents a capacitively based microelectromechanical systems affinity sensor for continuous glucose monitoring (CGM) applications. This sensor consists of a vibrating Parylene diaphragm, which is remotely driven by a magnetic field and situated inside a microchamber. A solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA), a biocompatible glucose-sensitive polymer, fills the microchamber, which is separated from its surroundings by a semipermeable membrane. Glucose permeates through the membrane and binds reversibly to the phenylboronic acid moiety of the polymer. This results in a viscosity change of the sensing solution, causing a detectable change in the Parylene diaphragm vibration which can be measured capacitively. Experimental results demonstrate that the device is capable of detecting glucose at physiologically relevant concentrations ranging from 30 to 360 mg/dL. The response time of the sensor to glucose concentration changes is approximately 1.5 min, which can be further improved with optimized device designs. Excellent reversibility and stability are observed in sensor responses, as highly desired for long-term CGM. PMID:24511213

  17. Glucose Transport Machinery Reconstituted in Cell Models

    PubMed Central

    Hansen, Jesper S.; Elbing, Karin; Thompson, James R.; Malmstadt, Noah

    2015-01-01

    Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it. PMID:25562394

  18. Glucose transport machinery reconstituted in cell models.

    PubMed

    Hansen, Jesper S; Elbing, Karin; Thompson, James R; Malmstadt, Noah; Lindkvist-Petersson, Karin

    2015-02-11

    Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it. PMID:25562394

  19. Role of Secondary Transporters and Phosphotransferase Systems in Glucose Transport by Oenococcus oeni ▿

    PubMed Central

    Kim, Ok Bin; Richter, Hanno; Zaunmüller, Tanja; Graf, Sabrina; Unden, Gottfried

    2011-01-01

    Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [14C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH. PMID:22020640

  20. Boronate Affinity-Molecularly Imprinted Biocompatible Probe: An Alternative for Specific Glucose Monitoring.

    PubMed

    Chen, Guosheng; Qiu, Junlang; Fang, Xu'an; Xu, Jianqiao; Cai, Siying; Chen, Qing; Liu, Yan; Zhu, Fang; Ouyang, Gangfeng

    2016-08-19

    A biocompatible probe for specific glucose recognition is based on photoinitiated boronate affinity-molecular imprinted polymers (BA-MIPs). The unique pre-self-assembly between glucose and boronic acids creates glucose-specific memory cavities in the BA-MIPs coating. As a result, the binding constant toward glucose was enhanced by three orders of magnitude. The BA-MIPs probe was applied to glucose determination in serum and urine and implanted into plant tissues for low-destructive and long-term in vivo continuous glucose monitoring. PMID:27411946

  1. Immunological identification of the human erythrocyte glucose transporter.

    PubMed Central

    Sogin, D C; Hinkle, P C

    1980-01-01

    A rabbit antibody against the human erythrocyte glucose transporter was purified by affinity chromatography and used to determine the distribution of transporter on polyacrylamide gels after electrophoresis in sodium dodecyl sulfate. Fresh erythrocyte ghosts showed transporter only at the broad 55,000 Mr band, as did the isolated transporter. HeLa cell plasma membranes showed a similar band of crossreacting material at Mr 55,000. The amount of crossreacting material in human erythrocyte ghosts and in plasma membranes from human HeLa cells and mouse L-1210 cells was determined in an enzyme-linked immunosorbent assay which gave results consistent with the extent of glucose-reversible binding of cytochalasin B. PMID:6934506

  2. SNF3 as High Affinity Glucose Sensor and Its Function in Supporting the Viability of Candida glabrata under Glucose-Limited Environment

    PubMed Central

    Ng, Tzu Shan; Chew, Shu Yih; Rangasamy, Premmala; Mohd Desa, Mohd N.; Sandai, Doblin; Chong, Pei Pei; Than, Leslie Thian Lung

    2015-01-01

    Candida glabrata is an emerging human fungal pathogen that has efficacious nutrient sensing and responsiveness ability. It can be seen through its ability to thrive in diverse range of nutrient limited-human anatomical sites. Therefore, nutrient sensing particularly glucose sensing is thought to be crucial in contributing to the development and fitness of the pathogen. This study aimed to elucidate the role of SNF3 (Sucrose Non Fermenting 3) as a glucose sensor and its possible role in contributing to the fitness and survivability of C. glabrata in glucose-limited environment. The SNF3 knockout strain was constructed and subjected to different glucose concentrations to evaluate its growth, biofilm formation, amphotericin B susceptibility, ex vivo survivability and effects on the transcriptional profiling of the sugar receptor repressor (SRR) pathway-related genes. The CgSNF3Δ strain showed a retarded growth in low glucose environments (0.01 and 0.1%) in both fermentation and respiration-preferred conditions but grew well in high glucose concentration environments (1 and 2%). It was also found to be more susceptible to amphotericin B in low glucose environment (0.1%) and macrophage engulfment but showed no difference in the biofilm formation capability. The deletion of SNF3 also resulted in the down-regulation of about half of hexose transporters genes (four out of nine). Overall, the deletion of SNF3 causes significant reduction in the ability of C. glabrata to sense limited surrounding glucose and consequently disrupts its competency to transport and perform the uptake of this critical nutrient. This study highlighted the role of SNF3 as a high affinity glucose sensor and its role in aiding the survivability of C. glabrata particularly in glucose limited environment. PMID:26648919

  3. Continuous Monitoring of Glucose in Subcutaneous Tissue Using Microfabricated Differential Affinity Sensors

    PubMed Central

    Huang, Xian; Leduc, Charles; Ravussin, Yann; Li, Siqi; Davis, Erin; Song, Bing; Wang, Qian; Accili, Domenico; Leibel, Rudolph; Lin, Qiao

    2012-01-01

    Objective We describe miniaturized differential glucose sensors based on affinity binding between glucose and a synthetic polymer. The sensors possess excellent resistance to environmental disturbances and can potentially allow wireless measurements of glucose concentrations within interstitial fluid in subcutaneous tissue for long-term, stable continuous glucose monitoring (CGM). Methods The sensors are constructed using microelectromechanical systems (MEMS) technology and exploit poly(N-hydroxy-ethyl acrylamide-ran-3-acrylamidophenylboronic acid) (PHEAA-ran-PAAPBA), a glucose-binding polymer with excellent specificity, reversibility, and stability. Two sensing approaches have been investigated, which respectively, use a pair of magnetically actuated diaphragms and perforated electrodes to differentially measure the glucose-binding-induced changes in the viscosity and permittivity of the PHEAA-ran-PAAPBA solution with respect to a reference, glucose-unresponsive polymer solution. Results In vivo characterization of the MEMS affinity sensors were performed by controlling blood glucose concentrations of laboratory mice by exogenous glucose and insulin administration. The sensors experienced an 8–30 min initialization period after implantation and then closely tracked commercial capillary glucose meter readings with time lags ranging from 0–15 min during rapid glucose concentration changes. Clarke error grid plots obtained from sensor calibration suggest that, for the viscometric and dielectric sensors, respectively, approximately 95% (in the hyperglycemic range) and 84% (ranging from hypoglycemic to hyperglycemic glucose concentrations) of measurement points were clinically accurate, while 5% and 16% of the points were clinically acceptable. Conclusions The miniaturized MEMS sensors explore differential measurements of affinity glucose recognition. In vivo testing demonstrated excellent accuracy and stability, suggesting that the devices hold the potential to

  4. Glucose Transporters in Cardiac Metabolism and Hypertrophy

    PubMed Central

    Shao, Dan; Tian, Rong

    2016-01-01

    The heart is adapted to utilize all classes of substrates to meet the high-energy demand, and it tightly regulates its substrate utilization in response to environmental changes. Although fatty acids are known as the predominant fuel for the adult heart at resting stage, the heart switches its substrate preference toward glucose during stress conditions such as ischemia and pathological hypertrophy. Notably, increasing evidence suggests that the loss of metabolic flexibility associated with increased reliance on glucose utilization contribute to the development of cardiac dysfunction. The changes in glucose metabolism in hypertrophied hearts include altered glucose transport and increased glycolysis. Despite the role of glucose as an energy source, changes in other nonenergy producing pathways related to glucose metabolism, such as hexosamine biosynthetic pathway and pentose phosphate pathway, are also observed in the diseased hearts. This article summarizes the current knowledge regarding the regulation of glucose transporter expression and translocation in the heart during physiological and pathological conditions. It also discusses the signaling mechanisms governing glucose uptake in cardiomyocytes, as well as the changes of cardiac glucose metabolism under disease conditions. PMID:26756635

  5. Glucose Measurement by Affinity Sensor and Pulsed Measurements of Fluidic Resistances

    PubMed Central

    Wyss, Thomas; Robin, Franck; Heinemann, Lutz

    2014-01-01

    Affinity sensors for glucose are based on a different measuring principle than the commercially available amperometric needle type sensors: reversible affinity interaction of glucose with specific receptors is the primary recognition mechanism instead of an enzymatic glucose oxidation. A novel pulsed-flow micro-fluidic system was used to characterize first the viscosity of a sensitive liquid containing the glucose receptor Concanavalin A and dextran and in a second approach to characterize the geometry of a fluidic resistance. In the viscometric sensor, glucose of the sensitive liquid is equilibrated, while passing through a dialysis chamber, with the surrounding medium. With the membrane flow sensor, the viscosity of the liquid remains constant but the pores of the flow-resisting membrane contain a swellable hydrogel affecting the width of the pores. Two types of hydrogel were tested with the membrane flow sensor; one is highly sensitive to pH and salt concentration, the other contains receptors of phenyl boronic acids to obtain sensitivity to glucose. The viscometric affinity sensor (first approach) showed a linear response over 0 to 30 mmol/L glucose concentration range. The disturbing effect of air bubbles could be compensated for. The sensing proof of principle of the second approach could be demonstrated by its linear response to different saline concentrations; however, the glucose-sensitive membrane developed showed only a small response to glucose. Glucose monitoring based on this pulsed flow measuring principle offers interesting alternatives for the development of CGM systems with different options for the glucose sensing part. PMID:24876545

  6. Pulmonary glucose transport in the fetal sheep.

    PubMed Central

    Barker, P M; Boyd, C A; Ramsden, C A; Strang, L B; Walters, D V

    1989-01-01

    1. In the chronically catheterized sheep fetus between 122 and 143 days gestation the concentration of D-glucose in lung liquid was very low (usually less than 0.01 mM, the lower limit of detection of the analytical method) whereas the mean plasma concentration was 0.19 mM (S.E.M. 0.4, n = 13). 2. When the lung liquid concentration of D-glucose was raised to 1.67-5.00 mM, rapid uptake was observed until the concentration had fallen to its preceding low level. The uptake showed saturation kinetics (Vmax = 2.29-8.78 mumol/min, increasing with gestation; mean Km = 0.14 +/- 0.02 mM, n = 11, no change with gestation). This active uptake of glucose was blocked by phloridzin (10(-4) M). It was associated with a decrease in lung liquid secretion rate from which a change in net sodium flux could be inferred of an order suggesting one-to-one glucose-sodium co-transport. 3. Radiolabelled 3-O-methyl-D-glucose (3-O-meG) - a monosaccharide which is transported but not metabolized - was taken up rapidly from lung liquid and this rapid uptake was inhibited by D-glucose with 50% inhibition at 0.35 mM (+/- 0.08, n = 9). It was also inhibited by phloridzin (10(-4) M). 4. Radiolabelled 2-deoxy-D-glucose - a monosaccharide which is not a substrate for sodium-coupled transport - was taken up only very slowly from lung liquid; the rate of uptake was appropriate for passive diffusional transport and it was unaffected by the addition of D-glucose or phloridzin to lung liquid. 5. Intravenous infusion of D-glucose caused no detectable increase in the concentration of glucose in lung liquid unless phloridzin was added, when a slow increase was observed. 6. In two experiments with active transport blocked by phloridzin in lung liquid (10(-4) M), the rate of entry of labelled 3-O-meG from plasma to lung liquid was measured during intravenous infusion of this tracer for 29 and 23 h. The rates of entry were similar to the rate of efflux of the tracer from lung liquid when uptake was blocked by

  7. Affinities of methylphenidate derivatives for dopamine, norepinephrine and serotonin transporters.

    PubMed

    Gatley, S J; Pan, D; Chen, R; Chaturvedi, G; Ding, Y S

    1996-01-01

    We have synthesized several derivative of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC50 values for binding these compounds to rat brain monoamine transporters were assessed using [3H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [3H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [3H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET > 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC50 values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of o-bromomethylphenidate did not bind to the DAT (IC50 > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r2=0.90). Abilities of several methylphenidate derivatives to inhibit [3H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [3H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC50 > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [3H]SCH 23390 or [123I]epidepride, respectively, to striatal membranes. PMID:8786705

  8. Glucose transporter expression in rat mammary gland.

    PubMed Central

    Burnol, A F; Leturque, A; Loizeau, M; Postic, C; Girard, J

    1990-01-01

    The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland. Images Fig. 1. Fig. 2. PMID:2396989

  9. Development of an on-column affinity smart polymer gel glucose sensor.

    PubMed

    Thammakhet, Chongdee; Thavarungkul, Panote; Kanatharana, Proespichaya

    2011-06-10

    An on-column affinity smart polymer gel glucose sensor was developed as a non-enzymatic glucose sensor. A copolymer of 3-acrylamidophenylboronic acid and acrylamide, the so called "smart polymer", was synthesized in situ in a 5 cm long capillary tube with a detection window to provide the on-column detection. The optical density of this semitransparent affinity smart polymer gel, coated inside the tube, decreased with increasing glucose concentration and was detected using a UV-vis detector at 500 nm. The capillary tube was incorporated into a flow injection system. Under optimum conditions, a linear dynamic range of 0.5-16.0mM with a limit of detection of 0.5mM (S/N ≥ 3) was obtained. A single coated affinity smart polymer gel had good stability for up to 250 consecutive injections with relative standard deviation of less than 5%. The analysis time for each injection was 6 min. Ten glucose samples prepared in distilled water were analyzed by the developed method and the results compared well with those obtained from the conventional dinitrosalicylic acid (DNS) method (P>0.05). Real urine samples with known glucose levels were analyzed and the developed sensor provided comparable results to those from the normal strip test technique. Acceptable percentage recoveries, ranging from 88 ± 2% to 103 ± 4% from the spiked urine sample, were obtained. PMID:21601037

  10. Effect of endurance training on glucose transport capacity and glucose transporter expression in rat skeletal muscle

    SciTech Connect

    Ploug, T.; Stallknecht, B.M.; Pedersen, O.; Kahn, B.B.; Ohkuwa, T.; Vinten, J.; Galbo, H. )

    1990-12-01

    The effect of 10 wk endurance swim training on 3-O-methylglucose (3-MG) uptake (at 40 mM 3-MG) in skeletal muscle was studied in the perfused rat hindquarter. Training resulted in an increase of approximately 33% for maximum insulin-stimulated 3-MG transport in fast-twitch red fibers and an increase of approximately 33% for contraction-stimulated transport in slow-twitch red fibers compared with nonexercised sedentary muscle. A fully additive effect of insulin and contractions was observed both in trained and untrained muscle. Compared with transport in control rats subjected to an almost exhaustive single exercise session the day before experiment both maximum insulin- and contraction-stimulated transport rates were increased in all muscle types in trained rats. Accordingly, the increased glucose transport capacity in trained muscle was not due to a residual effect of the last training session. Half-times for reversal of contraction-induced glucose transport were similar in trained and untrained muscles. The concentrations of mRNA for GLUT-1 (the erythrocyte-brain-Hep G2 glucose transporter) and GLUT-4 (the adipocyte-muscle glucose transporter) were increased approximately twofold by training in fast-twitch red muscle fibers. In parallel to this, Western blot demonstrated a approximately 47% increase in GLUT-1 protein and a approximately 31% increase in GLUT-4 protein. This indicates that the increases in maximum velocity for 3-MG transport in trained muscle is due to an increased number of glucose transporters.

  11. Thirty-fifth anniversary of the optical affinity sensor for glucose: a personal retrospective.

    PubMed

    Schultz, Jerome S

    2015-01-01

    Since 1962 when Clark introduced the enzyme electrode, research has been intense for a robust implantable glucose sensor. An alternative "optical affinity sensor" was introduced by Jerome Schultz in 1979. The evolution of this sensor technology into a new methodology is reviewed. The approach integrates a variety of disparate concepts: the selectivity of immunoassays-selectivity for glucose was obtained with concanavalin A, detection sensitivity was obtained with fluorescence (FITC-Dextran), and miniaturization was achieved by the use of an optical fiber readout system. Refinements of Schultz's optical affinity sensor approach over the past 35 years have led to a number of configurations that show great promise to meet the needs of a successful implantable continuous monitoring device for diabetics, some of which are currently being tested clinically. PMID:25269660

  12. Glucose transporter of the human brain and blood-brain barrier

    SciTech Connect

    Kalaria, R.N.; Gravina, S.A.; Schmidley, J.W.; Perry, G.; Harik, S.I.

    1988-12-01

    We identified and characterized the glucose transporter in the human cerebral cortex, cerebral microvessels, and choroid plexus by specific D-glucose-displaceable (3H)cytochalasin B binding. The binding was saturable, with a dissociation constant less than 1 microM. Maximal binding capacity was approximately 7 pmol/mg protein in the cerebral cortex, approximately 42 pmol/mg protein in brain microvessels, and approximately 27 pmol/mg protein in the choroid plexus. Several hexoses displaced specific (3H)cytochalasin B binding to microvessels in a rank-order that correlated well with their known ability to cross the blood-brain barrier; the only exception was 2-deoxy-D-glucose, which had much higher affinity for the glucose transporter than the natural substrate, D-glucose. Irreversible photoaffinity labeling of the glucose transporter of microvessels with (3H)cytochalasin B, followed by solubilization and polyacrylamide gel electrophoresis, labeled a protein band with an average molecular weight of approximately 55,000. Monoclonal and polyclonal antibodies specific to the human erythrocyte glucose transporter immunocytochemically stained brain blood vessels and the few trapped erythrocytes in situ, with minimal staining of the neuropil. In the choroid plexus, blood vessels did not stain, but the epithelium reacted positively. We conclude that human brain microvessels are richly endowed with a glucose transport moiety similar in molecular weight and antigenic characteristics to that of human erythrocytes and brain microvessels of other mammalian species.

  13. Gymnemic acids inhibit sodium-dependent glucose transporter 1.

    PubMed

    Wang, Yu; Dawid, Corinna; Kottra, Gabor; Daniel, Hannelore; Hofmann, Thomas

    2014-06-25

    To evaluate the activity of botanicals used in Chinese Traditional Medicine as hypoglycemic agents for diabetes type II prevention and/or treatment, extracts prepared from 26 medicinal herbs were screened for their inhibitory activity on sodium-dependent glucose transporter 1 (SGLT1) by using two-electrode voltage-clamp recording of glucose uptake in Xenopus laevis oocytes microinjected with cRNA for SGLT1. Showing by far the strongest SGLT1 inhibitory effect, the phytochemicals extracted from Gymnema sylvestre (Retz.) Schult were located by means of activity-guided fractionation and identified as 3-O-β-D-glucuronopyranosyl-21-O-2-tigloyl-22-O-2-tigloyl gymnemagenin (1) and 3-O-β-D-glucuronopyranosyl-21-O-2-methylbutyryl-22-O-2-tigloyl gymnemagenin (2) by means of LC-MS/MS, UPLC-TOF/MS, and 1D/2D-NMR experiments. Both saponins exhibited low IC50 values of 5.97 (1) and 0.17 μM (2), the latter of which was in the same range as found for the high-affinity inhibitor phlorizin (0.21 μM). As SGLT1 is found in high levels in brush-border membranes of intestinal epithelial cells, these findings demonstrate for the first time the potential of these saponins for inhibiting electrogenic glucose uptake in the gastrointestinal tract. PMID:24856809

  14. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    SciTech Connect

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increased in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.

  15. Mitogen-stimulated and rapamycin-sensitive glucose transporter 12 targeting and functional glucose transport in renal epithelial cells.

    PubMed

    Wilson-O'Brien, Amy L; Dehaan, Carrie L; Rogers, Suzanne

    2008-03-01

    We hypothesized that glucose transporter 12 (GLUT12) is involved in regulation of glucose flux in distal renal tubules in response to elevated glucose. We used the Madin-Darby canine kidney polarized epithelial cell model and neutralizing antibodies to analyze GLUT12 targeting and directional GLUT12-mediated glucose transport. At physiological glucose concentrations, GLUT12 was localized to a perinuclear position. High glucose and serum treatment resulted in GLUT12 localization to the apical membrane. This mitogen-stimulated targeting of GLUT12 was inhibited by rapamycin, the specific inhibitor of mammalian target of rapamycin (mTOR). The functional role of GLUT12 was also examined. We constructed a GLUT12 cDNA containing a c-Myc epitope tag in the fifth exofacial loop. Assays of glucose transport at the apical membrane were performed using Transwell filters. By comparing transport assays in the presence of neutralizing anti-c-Myc monoclonal antibody, we specifically measured GLUT12-mediated glucose transport at the apical surface. GLUT12-mediated glucose transport was mitogen dependent and rapamycin sensitive. Our results implicate mTOR signaling in a novel pathway of glucose transporter protein targeting and glucose transport. Activity of the mTOR pathway has been associated with diabetic kidney disease. Our results provide evidence for a link between GLUT12 protein trafficking, glucose transport and signaling molecules central to the control of metabolic disease processes. PMID:18039784

  16. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    SciTech Connect

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M. )

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub m}, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

  17. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    SciTech Connect

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-03-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect.

  18. Exercise training, glucose transporters, and glucose transport in rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Rodnick, K. J.; Henriksen, E. J.; James, D. E.; Holloszy, J. O.

    1992-01-01

    It was previously found that voluntary wheel running induces an increase in the insulin-sensitive glucose transporter, i.e., the GLUT4 isoform, in rat plantaris muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). The present study was undertaken to determine whether 1) the increase in muscle GLUT4 protein is associated with an increase in maximally stimulated glucose transport activity, 2) a conversion of type IIb to type IIa or type I muscle fibers plays a role in the increase in GLUT4 protein, and 3) an increase in the GLUT1 isoform is a component of the adaptation of muscle to endurance exercise. Five weeks of voluntary wheel running that resulted in a 33% increase in citrate synthase activity induced a 50% increase in GLUT4 protein in epitrochlearis muscles of female Sprague-Dawley rats. The rate of 2-deoxy-glucose transport maximally stimulated with insulin or insulin plus contractions was increased approximately 40% (P less than 0.05). There was no change in muscle fiber type composition, evaluated by myosin ATPase staining, in the epitrochlearis. There was also no change in GLUT1 protein concentration. We conclude that an increase in GLUT4, but not of GLUT1 protein, is a component of the adaptive response of muscle to endurance exercise and that the increase in GLUT4 protein is associated with an increased capacity for glucose transport.

  19. "On-off" switchable electrochemical affinity nanobiosensor based on graphene oxide for ultrasensitive glucose sensing.

    PubMed

    Huang, Jing; Zhang, Li; Liang, Ru-Ping; Qiu, Jian-Ding

    2013-03-15

    A novel "smart" electrochemical affinity nanobiosensor with "on-off" switchable property was designed for the ultrasensitive determination of glucose. The sensing approach was based on the glucose-ConA-dextran competitive system induced charge evolution in the use of graphene oxide (GO) as transducer element, resulting in the enhancement of interfacial electron transfer kinetics between the redox probe and the electrode. As concanavalin A (ConA) constituent was pH-sensitive, when the ConA-DexP/GO film electrode switched in probe Fe(CN)(6)(3-/4-) solution between pH 4.0 and 8.0, the film was cycled between the "on" and "off" states by the electrostatic attraction and repulsion of Fe(CN)(6)(3-/4-) to and from the electrode surface. Upon introduction of glucose into the ConA-DexP/GO complex at the "off" state, glucose competed with DexP for ConA and displaced ConA from the GO platform, resulting in gradual decrease of the surface negative charge as well as the resistance of probe for electron communication on the sensor surface, and making the switching from "off" state to "on" state simultaneously. This ultrasensitive glucose nanobiosensor had a broad linearity between the decrease in electron transfer resistance (ΔR) and the glucose concentration over a range from 5.0 μM to 9.0 mM with a detection limit as low as 0.34 μM. The proposed method showed potential application for fabricating novel biosensors and bioelectronic devices. PMID:23026685

  20. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

    PubMed Central

    Celenza, J L; Marshall-Carlson, L; Carlson, M

    1988-01-01

    The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression. Images PMID:3281163

  1. Molecular basis of ligand recognition and transport by glucose transporters.

    PubMed

    Deng, Dong; Sun, Pengcheng; Yan, Chuangye; Ke, Meng; Jiang, Xin; Xiong, Lei; Ren, Wenlin; Hirata, Kunio; Yamamoto, Masaki; Fan, Shilong; Yan, Nieng

    2015-10-15

    The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design. PMID:26176916

  2. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    SciTech Connect

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.

  3. ADP modifies the function of the glucose transporter: studies with reconstituted liposomes.

    PubMed Central

    Sofue, M; Yoshimura, Y; Nishida, M; Kawada, J

    1993-01-01

    Modification of function of the glucose transporter by nucleotides was studied by using liposomes reconstituted with the human erythrocyte glucose transporter. ADP enclosed in the liposomes inhibited the uptake of D-glucose and nicotinamide in a dose-dependent manner, but other enclosed nucleotides (ATP, AMP, CDP, GDP, UDP) showed no effect on the uptake of both. Only intraliposomal ADP was effective, and extra-liposomal ADP was not, under our experimental conditions. Intraliposomal ADP did not change Km, but decreased Vmax to approximately one-third of control for uptake of both D-glucose and nicotinamide. However, the binding and the affinity of cytochalasin B to the reconstituted liposomes were not affected by intraliposomal ADP. The uptake of uridine was not changed in the presence of ADP, indicating that the nucleoside transporter co-existing in the liposomal membranes is not regulated by ADP. Human erythrocytes whose intracellular ATP was decreased by Ca2+ ionophore A23187 also showed decreased uptake of 2-deoxy-D-glucose and nicotinamide. This phenomenon was very similar to that found in the liposomes. These findings suggest the possibility that the function of the glucose transporter is directly and negatively modified by an increased concentration of intracellular ADP. PMID:8318016

  4. Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4)

    NASA Technical Reports Server (NTRS)

    Hardy, R. W.; Ladenson, J. H.; Henriksen, E. J.; Holloszy, J. O.; McDonald, J. M.

    1991-01-01

    In rat adipocytes, palmitate: a) increases basal 2-deoxyglucose transport 129 +/- 27% (p less than 0.02), b) decreases the insulin sensitive glucose transporter (GLUT4) in low density microsomes and increases GLUT4 in plasma membranes and c) increases the activity of the insulin receptor tyrosine kinase. Palmitate-stimulated glucose transport is not additive with the effect of insulin and is not inhibited by the protein kinase C inhibitors staurosporine and sphingosine. In rat muscle, palmitate: a) does not affect basal glucose transport in either the soleus or epitrochlearis and b) inhibits insulin-stimulated glucose transport by 28% (p less than 0.005) in soleus but not in epitrochlearis muscle. These studies demonstrate a potentially important differential role for fatty acids in the regulation of glucose transport in different insulin target tissues.

  5. Glucose transporter expression differs between bovine monocyte and macrophage subsets and is influenced by milk production.

    PubMed

    Eger, M; Hussen, J; Koy, M; Dänicke, S; Schuberth, H-J; Breves, G

    2016-03-01

    The peripartal period of dairy cows is characterized by negative energy balance and higher incidences of infectious diseases such as mastitis or metritis. With the onset of lactation, milk production is prioritized and large amounts of glucose are transported into the mammary gland. Decreased overall energy availability might impair the function of monocytes acting as key innate immune cells, which give rise to macrophages and dendritic cells and link innate and adaptive immunity. Information on glucose requirements of bovine immune cells is rare. Therefore, this study aims to evaluate glucose transporter expression of the 3 bovine monocyte subsets (classical, intermediate, and nonclassical monocytes) and monocyte-derived macrophages and to identify influences of the peripartal period. Blood samples were either collected from nonpregnant healthy cows or from 16 peripartal German Holstein cows at d -14, +7, and +21 relative to parturition. Quantitative real-time PCR was applied to determine mRNA expression of glucose transporters (GLUT) 1, GLUT3, and GLUT4 in monocyte subsets and monocyte-derived macrophages. The low GLUT1 and GLUT3 expression in nonclassical monocytes was unaltered during differentiation into macrophages, whereas in classical and intermediate monocytes GLUT expression was downregulated. Alternatively activated M2 macrophages consumed more glucose compared with classically activated M1 macrophages. The GLUT4 mRNA was only detectable in unstimulated macrophages. Neither monocytes nor macrophages were insulin responsive. In the peripartum period, monocyte GLUT1 and GLUT3 expression and the GLUT3/GLUT1 ratio were negatively correlated with lactose production. The high-affinity GLUT3 transporter appears to be the predominant glucose transporter on bovine monocytes and macrophages, especially in the peripartal period when blood glucose levels decline. Glucose transporter expression in monocytes is downregulated as a function of lactose production, which

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

    PubMed Central

    Di Simone, Nicoletta; Di Nicuolo, Fiorella; Marzioni, Daniela; Castellucci, Mario; Sanguinetti, Maurizio; D’lppolito, Silvia; Caruso, Alessandro

    2009-01-01

    Abstract 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. PMID:18410529

  7. The regulation of glucose transport in the heart of control and diabetic rats: With special emphasis on the glucose transporter

    SciTech Connect

    Pleta, M. de Leoz.

    1989-01-01

    Glucose transport regulation with insulin and high perfusion pressure in the perfused rat hearts from control and diabetic rat hearts was investigated. ({sup 3}H)-cytochalasin B binding assay was used to study the distribution of glucose transporters within the subcellular membranes fractionated by linear sucrose density gradient centrifugation. In the present study, insulin increased glucose uptake in the perfused heart of control and diabetic animals. This coincided with an increase of glucose transporters on the plasma membrane. The increase in glucose transporters on the plasma membrane could not be accounted for by a decrease of glucose transporters from the microsomal membranes. High perfusion pressure did not change the number of glucose transporters on the plasma membrane compared to basal in the control and diabetic animals, though it increased glucose uptake above that observed for insulin in the control. Instead, high perfusion pressure altered the distribution of glucose transporters within the subcellular membranes in reverse to that with insulin, increasing an intermediate membrane pool believed to reside between the plasma membrane and microsomal membranes as well as the intracellular membrane pool.

  8. Antipsychotics inhibit glucose transport: Determination of olanzapine binding site in Staphylococcus epidermidis glucose/H+ symporter

    PubMed Central

    Babkin, Petr; George Thompson, Alayna M.; Iancu, Cristina V.; Walters, D. Eric; Choe, Jun-yong

    2015-01-01

    The antipsychotic drug olanzapine is widely prescribed to treat schizophrenia and other psychotic disorders. However, it often causes unwanted side effects, including diabetes, due to disruption of insulin-dependant glucose metabolism through a mechanism yet to be elucidated. To determine if olanzapine can affect the first step in glucose metabolism – glucose transport inside cells – we investigated the effect of this drug on the transport activity of a model glucose transporter. The glucose transporter from Staphylococcus epidermidis (GlcPSe) is specific for glucose, inhibited by various human glucose transporter (GLUT) inhibitors, has high sequence and structure homology to GLUTs, and is readily amenable to transport assay, mutagenesis, and computational modeling. We found that olanzapine inhibits glucose transport of GlcPSe with an IC50 0.9 ± 0.1 mM. Computational docking of olanzapine to the GlcPSe structure revealed potential binding sites that were further examined through mutagenesis and transport assay to identify residues important for olanzapine inhibition. These investigations suggest that olanzapine binds in a polar region of the cytosolic part of the transporter, and interacts with residues R129, strictly conserved in all GLUTs, and N136, conserved in only a few GLUTs, including the insulin-responsive GLUT4. We propose that olanzapine inhibits GlcPSe by impeding the alternating opening and closing of the substrate cavity necessary for glucose transport. It accomplishes this by disrupting a key salt bridge formed by conserved residues R129 and E362, that stabilizes the outward-facing conformation of the transporter. PMID:25941630

  9. Antipsychotics inhibit glucose transport: Determination of olanzapine binding site in Staphylococcus epidermidis glucose/H(+) symporter.

    PubMed

    Babkin, Petr; George Thompson, Alayna M; Iancu, Cristina V; Walters, D Eric; Choe, Jun-Yong

    2015-01-01

    The antipsychotic drug olanzapine is widely prescribed to treat schizophrenia and other psychotic disorders. However, it often causes unwanted side effects, including diabetes, due to disruption of insulin-dependant glucose metabolism through a mechanism yet to be elucidated. To determine if olanzapine can affect the first step in glucose metabolism - glucose transport inside cells - we investigated the effect of this drug on the transport activity of a model glucose transporter. The glucose transporter from Staphylococcus epidermidis (GlcPSe) is specific for glucose, inhibited by various human glucose transporter (GLUT) inhibitors, has high sequence and structure homology to GLUTs, and is readily amenable to transport assay, mutagenesis, and computational modeling. We found that olanzapine inhibits glucose transport of GlcPSe with an IC50 0.9 ± 0.1 mM. Computational docking of olanzapine to the GlcPSe structure revealed potential binding sites that were further examined through mutagenesis and transport assay to identify residues important for olanzapine inhibition. These investigations suggest that olanzapine binds in a polar region of the cytosolic part of the transporter, and interacts with residues R129, strictly conserved in all GLUTs, and N136, conserved in only a few GLUTs, including the insulin-responsive GLUT4. We propose that olanzapine inhibits GlcPSe by impeding the alternating opening and closing of the substrate cavity necessary for glucose transport. It accomplishes this by disrupting a key salt bridge formed by conserved residues R129 and E362, that stabilizes the outward-facing conformation of the transporter. PMID:25941630

  10. Energetics of low affinity amino acid transport into brain slices.

    PubMed

    Banay-Schwartz, M; Teller, D N; Lajtha, A

    1976-01-01

    It appears possible to dissect and study some of the potential energy sources for amino acid transport in brain slices despite the apparent complexity of the tissue in comparison to that of isolated bacterial vesicles23. The uptake capability of the tissue may be inadvertently damaged in some experimental protocols so that very special controls must be used to ensure that the treatment did not somehow inactivate the very mechanism that thereafter will be tested. We have presented some evidence that brain slice amino acid transport may not be obligatorily linked to glycolysis, ATP levels, Na+, K+-ATPase activity, K+ levels or direction of flux, or to Na+ flux. However, the energy source linkage for different amino acids appears to be rather specific, so that further generalizations are difficult to sustain. For instance, the incubation media and conditions we describe here were experimentally adjusted to maximize uptake of D-glu or alpha-AIB in the absence of glucose, or in lowered K+ or Na+. Therefore, these procedures, the results of which directly challenge some common assumptions regarding the energy basis for active transport in brain slices, probably will not be universally extensible to all other actively transported amino acids. PMID:782193

  11. Regulation of Intestinal Glucose Absorption by Ion Channels and Transporters

    PubMed Central

    Chen, Lihong; Tuo, Biguang; Dong, Hui

    2016-01-01

    The absorption of glucose is electrogenic in the small intestinal epithelium. The major route for the transport of dietary glucose from intestinal lumen into enterocytes is the Na+/glucose cotransporter (SGLT1), although glucose transporter type 2 (GLUT2) may also play a role. The membrane potential of small intestinal epithelial cells (IEC) is important to regulate the activity of SGLT1. The maintenance of membrane potential mainly depends on the activities of cation channels and transporters. While the importance of SGLT1 in glucose absorption has been systemically studied in detail, little is currently known about the regulation of SGLT1 activity by cation channels and transporters. A growing line of evidence suggests that cytosolic calcium ([Ca2+]cyt) can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca2+ in IEC are regulated by cation channels and transporters, such as Ca2+ channels, K+ channels, Na+/Ca2+ exchangers, and Na+/H+ exchangers. In this review, we consider the involvement of these cation channels and transporters in the regulation of glucose uptake in the small intestine. Modulation of them may be a potential strategy for the management of obesity and diabetes. PMID:26784222

  12. Radiotracers for Cardiac Sympathetic Innervation: Transport Kinetics and Binding Affinities for the Human Norepinephrine Transporter

    PubMed Central

    Raffel, David M.; Chen, Wei; Jung, Yong-Woon; Jang, Keun Sam; Gu, Guie; Cozzi, Nicholas V.

    2013-01-01

    Introduction Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [11C]-(−)-meta-hydroxyephedrine, [11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-labeled biogenic amines were also determined. Methods Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [3H]mazindol provided estimates of binding affinities (KI) for NET. Results Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for ‘tracer concentrations’ of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r2 = 0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. Conclusion The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density. PMID:23306137

  13. Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters.

    PubMed

    Gonçalves, Davi L; Matsushika, Akinori; de Sales, Belisa B; Goshima, Tetsuya; Bon, Elba P S; Stambuk, Boris U

    2014-09-01

    Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams. PMID:25039054

  14. Gastrointestinal transport of calcium and glucose in lactating ewes.

    PubMed

    Klinger, Stefanie; Schröder, Bernd; Gemmer, Anja; Reimers, Julia; Breves, Gerhard; Herrmann, Jens; Wilkens, Mirja R

    2016-06-01

    During lactation, mineral and nutrient requirements increase dramatically, particularly those for Ca and glucose. In contrast to monogastric species, in ruminants, it is rather unclear to which extend this physiological change due to increased demand for milk production is accompanied by functional adaptations of the gastrointestinal tract (GIT). Therefore, we investigated potential modulations of Ca and glucose transport mechanisms in the GIT of lactating and dried-off sheep. Ussing-chamber technique was applied to determine the ruminal and jejunal Ca flux rates. In the jejunum, electrophysiological properties in response to glucose were recorded. Jejunal brush-border membrane vesicles (BBMV) served to characterize glucose uptake via sodium-linked glucose transporter 1 (SGLT1), and RNA and protein expression levels of Ca and glucose transporting systems were determined. Ruminal Ca flux rate data showed a trend for higher absorption in lactating sheep. In the jejunum, small Ca absorption could only be observed in lactating ewes. From the results, it may be assumed that lactating ewes compensate for the Ca loss by increasing bone mobilization rather than by increasing supply through absorption from the GIT Presence of SGLT1 in the jejunum of both groups was shown by RNA and protein identification, but glucose uptake into BBMV could only be detected in lactating sheep. This, however, could not be attributed to electrogenic glucose absorption in lactating sheep under Ussing-chamber conditions, providing evidence that changes in jejunal glucose uptake may include additional factors, that is, posttranslational modifications such as phosphorylation. PMID:27273883

  15. Kinetics of glucose transport by the perfused mid-gut of the freshwater prawn Macrobrachium rosenberg ii.

    PubMed Central

    Ahearn, G A; Maginniss, L A

    1977-01-01

    1. Mucosal influx of [3H]glucose was examined in the mid-gut of a freshwater prawn, Macrobrachium rosenbergii, using an in vitro perfusion technique. 2. [3H]glucose transfer across the apical cell membrane of the epithelium exhibited Michaelis-Menten kinetics (Jmax.in = 0-15 mumole glucose equiv/g. min, Kt = 0-17 mM). Under Na-free conditions, glucose influx was significantly reduced and a linear function of substrate concentration, indicative of either slow cellular diffusion (KD = 7-6 X 10(3) mumole glucose equiv/g. min. mM) or a facilitated process with a low carrier affinity for the sugar. 3. Phlorizin was a potent competitive inhibitor of glucose influx (K1 = 3-6 X10(-3) mM), galactose and 3-O-methylglucose (3-O-MG) were weak inhibitors, and fructose had no evident effect on glucose uptake. Azide, but not iodoacetate (IAA), significantly depressed influx. 4. Absorbed [3H]glucose was rapidly metabolized by the mid-gut. The majority of accumulated activity within the tissue was in the form of phosphorylated compounds and tritiated water (THO), while only 0-3% was recovered as a free-glucose. 5. Preliminary studies examining transmural [3-H]glucose transport, however, demonstrated a significant net mucosal to serosal free-glucose flux across the prawn mid-gut which was Na-dependent and IAA- and phlorizin-sensitive. Two alternative interpretations of the data are advanced as possible mechanisms for transepithelial glucose transport: (1) group translocation, or (2) the operation of an energized, high affinity, baso-lateral sugar transport carrier. Images Plate 1 PMID:925985

  16. Cyanine-based 1-amino-1-deoxyglucose as fluorescent probes for glucose transporter mediated bioimaging.

    PubMed

    Xu, Hu; Liu, Xinyu; Yang, Jinna; Liu, Ran; Li, Taoli; Shi, Yunli; Zhao, Hongxia; Gao, Qingzhi

    2016-05-27

    Two novel cyanine-based 1-amino-1-deoxy-β-glucose conjugates (Glu-1N-Cy3 and Glu-1N-Cy5) were designed, synthesized and their fluorescence characteristics were studied. Both Glu-1N-Cy3 and Glu-1N-Cy5 accumulate in living HT29 human colon cancer cells, which overexpress glucose transporters (GLUTs). The cellular uptake of the bioprobes was inhibited by natural GLUT substrate d-glucose and 2-deoxy-d-glucose. The GLUT specificity of the probes was validated with quercetin, which is both a permeant substrate via GLUTs and a high-affinity inhibitor of GLUT-mediated glucose transport. Competitive fluorometric assay for GLUT substrate cell uptake revealed that Glu-1N-Cy3 and Glu-1N-Cy5 are 5 and 10 times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. This study provides fundamental data supporting the potential of these two conjugates as new powerful tools for GLUT-mediated theranostics, in vitro and in vivo molecular bioimaging and drug R&D. PMID:27033602

  17. Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes.

    PubMed Central

    Fischer, Y; Böttcher, U; Eblenkamp, M; Thomas, J; Jüngling, E; Rösen, P; Kammermeier, H

    1997-01-01

    Alternative substrates of energy metabolism are thought to contribute to the impairment of heart and muscle glucose utilization in insulin-resistant states. We have investigated the acute effects of substrates in isolated rat cardiomyocytes. Exposure to lactate, pyruvate, propionate, acetate, palmitate, beta-hydroxybutyrate or alpha-oxoglutarate led to the depression of glucose transport by up to 50%, with lactate, pyruvate and propionate being the most potent agents. The percentage inhibition was greater in cardiomyocytes in which glucose transport was stimulated with the alpha-adrenergic agonist phenylephrine or with a submaximal insulin concentration than in basal or fully insulin-stimulated cells. Cardiomyocytes from fasted or diabetic rats displayed a similar sensitivity to substrates as did cells from control animals. On the other hand, the amination product of pyruvate (alanine), as well as valine and the aminotransferase inhibitors cycloserine and amino-oxyacetate, stimulated glucose transport about 2-fold. In addition, the effect of pyruvate was counteracted by cycloserine. Since reversible transamination reactions are known to affect the pool size of the citrate cycle, the influence of substrates, amino acids and aminotransferase inhibitors on citrate, malate and glutamate content was examined. A significant negative correlation was found between alterations in glucose transport and the levels of citrate (P < 0.01) or malate (P < 0.01), and there was a positive correlation between glucose transport and glutamate levels (P < 0.05). In contrast, there was no correlation with changes in [1-(14)C]pyruvate oxidation or in glucose-6-phosphate levels. Finally, pyruvate decreased the abundance of GLUT4 glucose transporters at the surface of phenylephrine- or insulin-stimulated cells by 34% and 27 % respectively, as determined by using the selective photoaffinity label [3H]ATB-BMPA [[3H]2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-man nos-4-yloxy

  18. Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin-resistant glucose transport in adipose cells without restoring glucose transporter gene expression.

    PubMed Central

    Kahn, B B; Shulman, G I; DeFronzo, R A; Cushman, S W; Rossetti, L

    1991-01-01

    Evidence is emerging for a direct role of glucose, independent of changes in insulin, in the regulation of cellular glucose transport and glucose utilization in vivo. In this study we investigate potential cellular and molecular mechanisms for this regulatory effect of glucose by determining how normalization of glycemia without insulin therapy in diabetic rats influences 3-O-methylglucose transport and the expression and translocation of two genetically distinct species of glucose transporters (GTs) in adipose cells. These results are compared with alterations in glucose disposal in vivo measured by euglycemic clamp. In rats rendered diabetic by 90% pancreatectomy, insulin-stimulated glucose transport in adipose cells is decreased 50% in parallel with reduced insulin-mediated glucose disposal in vivo. Levels of adipose/muscle GTs measured by immunoblotting are decreased in adipose cell subcellular membrane fractions, as are the corresponding mRNA levels assessed by Northern blotting of total adipose cell RNA. Normalization of blood glucose in diabetic rats with phlorizin, which impairs renal tubular glucose reabsorption and thus enhances glucose excretion, restores insulin-stimulated glucose transport in adipose cells and insulin-mediated glucose disposal in vivo. Importantly, levels of the adipose/muscle GT protein remain 43% reduced in the low-density microsomes in the basal state and 46% reduced in the plasma membranes in the insulin-stimulated state. Adipose/muscle GT mRNA levels remain approximately 50% depressed. Levels of the HepG2/brain GT protein and mRNA are unaltered by diabetes or phlorizin treatment. Thus, changes in ambient glucose independent of changes in ambient insulin can regulate the glucose transport response to insulin in isolated adipose cells and changes in responsiveness parallel alterations in glucose uptake in vivo. Since this effect can occur without alteration in the expression of the two species of glucose transporters present in

  19. Regulation of. beta. -cell glucose transporter gene expression

    SciTech Connect

    Chen, Ling; Alam, Tausif; Johnson, J.H.; Unger, R.H. Department of Veterans Affairs Medical Center, Dallas, TX ); Hughes, S.; Newgard, C.B. )

    1990-06-01

    It has been postulated that a glucose transporter of {beta} cells (GLUT-2) may be important in glucose-stimulated insulin secretion. To determine whether this transporter is constitutively expressed or regulated, the authors subjected conscious unrestrained Wistar rats to perturbations in glucose homeostasis and quantitated {beta}-cell GLUT-2 mRNA by in situ hybridization. After 3 hr of hypoglycemia, GLUT-2 and proinsulin mRNA signal densities were reduced by 25% of the level in control rats. After 4 days, GLUT-2 and proinsulin mRNA densities were reduced by 85% and 65%, respectively. After 12 days of hypoglycemia, the K{sub m} for 3-O-methyl-D-glucose transport in isolated rat islets, normally 18-20 mM, was 2.5 mM. This provides functional evidence of a profound reduction of high K{sub m} glucose transporter in {beta} cells. In contrast, GLUT-2 was only slightly reduced by hypoglycemia in liver. To determine the effect of prolonged hyperglycemia, they also infused animals with 50% (wt/vol) glucose for 5 days. Hyperglycemic clamping increased GLUT-2 mRNA by 46% whereas proinsulin mRNA doubled. They conclude that GLUT-2 expression in {beta} cells, but not liver, is subject to regulation by certain perturbations in blood glucose homeostasis.

  20. Carbohydrate affinity for the glucose-galactose binding protein is regulated by allosteric domain motions.

    PubMed

    Ortega, Gabriel; Castaño, David; Diercks, Tammo; Millet, Oscar

    2012-12-01

    Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding. PMID:23148479

  1. Sodium coupled glucose co-transporters contribute to hypothalamic glucose-sensing

    PubMed Central

    O'Malley, Dervla; Reimann, Frank; Simpson, Anna K; Gribble, Fiona M

    2007-01-01

    Specialised neurons within the hypothalamus have the ability to sense and respond to changes in ambient glucose concentrations. We investigated the mechanisms underlying glucose-triggered activity in glucose-excited (GE) neurons, using primary cultures of rat hypothalamic neurons monitored by fluorescence calcium imaging. 35% (738/2139) of neurons were excited by increasing glucose from 3 to 15mM, but only 9% (6/64) of these GE neurons were activated by tolbutamide, suggesting the involvement of a KATP channel-independent mechanism. α-Methylglucopyranoside (αMDG, 12mM), a non-metabolisable substrate of sodium glucose co-transporters (SGLTs), mimicked the effect of high glucose in 67% of GE neurons, and both glucose and αMDG-triggered excitation were blocked by Na+ removal or by the SGLT inhibitor, phloridzin (100nM). In the presence of 0.5mM glucose and tolbutamide, responses could also be triggered by 3.5mM αMDG, supporting a role for an SGLT-associated mechanism at low as well as high substrate concentrations. By RT-PCR, we detected SGLT1, SGLT3a, SGLT3b in both cultured neurons and adult rat hypothalamus. Our findings suggest a novel role for SGLTs in glucose-sensing by hypothalamic GE neurons. PMID:17130483

  2. Regulation and biological function of a flagellar glucose transporter in Leishmania mexicana: a potential glucose sensor.

    PubMed

    Rodriguez-Contreras, Dayana; Aslan, Hamide; Feng, Xiuhong; Tran, Khoa; Yates, Phillip A; Kamhawi, Shaden; Landfear, Scott M

    2015-01-01

    In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ∼20-fold by increasing cell density but is up-regulated ∼50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a ∆lmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations. PMID:25300620

  3. Functional expression of sodium-glucose transporters in cancer

    PubMed Central

    Scafoglio, Claudio; Hirayama, Bruce A.; Kepe, Vladimir; Liu, Jie; Ghezzi, Chiara; Satyamurthy, Nagichettiar; Moatamed, Neda A.; Huang, Jiaoti; Koepsell, Hermann; Barrio, Jorge R.; Wright, Ernest M.

    2015-01-01

    Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl-4-deoxy-4-[18F]fluoro-d-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy. PMID:26170283

  4. Functional expression of sodium-glucose transporters in cancer.

    PubMed

    Scafoglio, Claudio; Hirayama, Bruce A; Kepe, Vladimir; Liu, Jie; Ghezzi, Chiara; Satyamurthy, Nagichettiar; Moatamed, Neda A; Huang, Jiaoti; Koepsell, Hermann; Barrio, Jorge R; Wright, Ernest M

    2015-07-28

    Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy. PMID:26170283

  5. Diabetic ketoacidosis, sodium glucose transporter-2 inhibitors and the kidney.

    PubMed

    Palmer, Biff F; Clegg, Deborah J; Taylor, Simeon I; Weir, Matthew R

    2016-08-01

    Diabetic ketoacidosis is a serious metabolic condition that may occur in patients with either Type 1 or Type 2 diabetes. The accumulation of ketoacids in the serum is a consequence of insulin deficiency and glucagon excess. Sodium Glucose Transporter 2 (SGLT2) inhibitors are novel therapeutic treatments for improving glucose homeostasis in patients with diabetes. Through reductions in glucose reabsorption by the kidney, they lower serum glucose in patients with Type 2 diabetes and they improve glucose control whether used alone or in combination with other therapies. Mechanistically, these drugs increase serum ketoacids and increase glucagon production, which in some individuals, can lead to formation of diabetic ketoacidosis. This review will first focus in how the kidney normally handles ketoacids, and second will discuss how the SGLT2 inhibitors affect the kidney in such a way so as to enhance the risk for development of ketoacidosis in susceptible individuals. PMID:27240541

  6. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  7. Genetic characterization of glucose transporter function in Leishmania mexicana

    PubMed Central

    Burchmore, Richard J. S.; Rodriguez-Contreras, Dayana; McBride, Kathleen; Barrett, Michael P.; Modi, Govind; Sacks, David; Landfear, Scott M.

    2003-01-01

    Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This Δlmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the Δlmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. Δlmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in Δlmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in Δlmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite. PMID:12651954

  8. Glucose and galactose transport in Bifidobacterium bifidum DSM 20082.

    PubMed

    Krzewinski, F; Brassart, C; Gavini, F; Bouquelet, S

    1997-09-01

    Sugar uptake was measured with 3H-galactose and 14C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters KM and Vmax were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K+ ions. PMID:9236301

  9. Increased glucose transport in response to phorbol ester growth factors, and insulin: relationship to phosphorylation of the glucose transporter

    SciTech Connect

    Allard, W.J.; Gibbs, E.M.; Witters, L.A.; Lienhard, G.E.

    1986-05-01

    The authors have examined the relationship between the increase in glucose transport induced by phorbol myristate acetate (PMA), EGF, PDGF, and insulin and the phosphorylation state of the glucose transporter in human fibroblasts. To assay transport, cells were cultured in medium with 10% serum for 5 days and then for 2 days in phosphate-free medium with 5% serum. Exposure to each agonist stimulated transport, as measured by the uptake of /sup 3/H-2-deoxyglucose over a 2 min period. Values for maximal percent stimulation, time needed to reach maximal stimulation, and concentration required to achieve half-maximal stimulation were as follows: PMA, 80%, 30 min, 2 nM; EGF, 30%, 10 min, 0.2 nM; Insulin, 45%, 10 min, 17 nM. In the case of PDGF, uptake was stimulated 65% by treatment with 0.7 or 1.4 nM for 20 min. Phosphorylation of the glucose transporter was measured in cells cultured for 5-7 days in medium with 10% serum and exposed to 670 ..mu..Ci/ml /sup 32/P/sub i/ for 100 min. The agonist was then added at a saturating dose for 20 min, and the glucose transporter was immunoprecipitated from cell lysates using a monoclonal antibody. Under these conditions, no basal phosphorylation of the transporter was detected, and only phorbol ester stimulated significant incorporation of phosphate into the transport protein. Experiments are currently in progress to quantitate transporter phosphorylation under conditions identical to those used for the assay of transport. These results suggest that while the transporter is a substrate for protein kinase C in vivo, phosphorylation of the transporter is not required for increased transport in response to growth factors and insulin.

  10. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  11. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  12. Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean

    PubMed Central

    Muñoz-Marín, María del Carmen; Luque, Ignacio; Zubkov, Mikhail V.; Hill, Polly G.; Diez, Jesús; García-Fernández, José Manuel

    2013-01-01

    Prochlorococcus is responsible for a significant part of CO2 fixation in the ocean. Although it was long considered an autotrophic cyanobacterium, the uptake of organic compounds has been reported, assuming they were sources of limited biogenic elements. We have shown in laboratory experiments that Prochlorococcus can take up glucose. However, the mechanisms of glucose uptake and its occurrence in the ocean have not been shown. Here, we report that the gene Pro1404 confers capability for glucose uptake in Prochlorococcus marinus SS120. We used a cyanobacterium unable to take up glucose to engineer strains that express the Pro1404 gene. These recombinant strains were capable of specific glucose uptake over a wide range of glucose concentrations, showing multiphasic transport kinetics. The Ks constant of the high affinity phase was in the nanomolar range, consistent with the average concentration of glucose in the ocean. Furthermore, we were able to observe glucose uptake by Prochlorococcus in the central Atlantic Ocean, where glucose concentrations were 0.5–2.7 nM. Our results suggest that Prochlorococcus are primary producers capable of tuning their metabolism to energetically benefit from environmental conditions, taking up not only organic compounds with key limiting elements in the ocean, but also molecules devoid of such elements, like glucose. PMID:23569224

  13. Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean.

    PubMed

    Muñoz-Marín, María del Carmen; Luque, Ignacio; Zubkov, Mikhail V; Hill, Polly G; Diez, Jesús; García-Fernández, José Manuel

    2013-05-21

    Prochlorococcus is responsible for a significant part of CO2 fixation in the ocean. Although it was long considered an autotrophic cyanobacterium, the uptake of organic compounds has been reported, assuming they were sources of limited biogenic elements. We have shown in laboratory experiments that Prochlorococcus can take up glucose. However, the mechanisms of glucose uptake and its occurrence in the ocean have not been shown. Here, we report that the gene Pro1404 confers capability for glucose uptake in Prochlorococcus marinus SS120. We used a cyanobacterium unable to take up glucose to engineer strains that express the Pro1404 gene. These recombinant strains were capable of specific glucose uptake over a wide range of glucose concentrations, showing multiphasic transport kinetics. The Ks constant of the high affinity phase was in the nanomolar range, consistent with the average concentration of glucose in the ocean. Furthermore, we were able to observe glucose uptake by Prochlorococcus in the central Atlantic Ocean, where glucose concentrations were 0.5-2.7 nM. Our results suggest that Prochlorococcus are primary producers capable of tuning their metabolism to energetically benefit from environmental conditions, taking up not only organic compounds with key limiting elements in the ocean, but also molecules devoid of such elements, like glucose. PMID:23569224

  14. 77 FR 28411 - Adrenalina, Affinity Technology Group, Inc., Braintech, Inc., Builders Transport, Incorporated...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-14

    ... From the Federal Register Online via the Government Publishing Office SECURITIES AND EXCHANGE COMMISSION Adrenalina, Affinity Technology Group, Inc., Braintech, Inc., Builders Transport, Incorporated... Adrenalina because it has not filed any periodic reports since the period ended September 30, 2008....

  15. Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin.

    PubMed Central

    Katagiri, H; Asano, T; Ishihara, H; Lin, J L; Inukai, K; Shanahan, M F; Tsukuda, K; Kikuchi, M; Yazaki, Y; Oka, Y

    1993-01-01

    GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity. Images Figure 1 Figure 2 PMID:8489512

  16. The Tuberous Sclerosis Complex Regulates Trafficking of Glucose Transporters and Glucose Uptake

    PubMed Central

    Jiang, Xiuyun; Kenerson, Heidi; Aicher, Lauri; Miyaoka, Robert; Eary, Janet; Bissler, John; Yeung, Raymond S.

    2008-01-01

    Human cancers often display an avidity for glucose, a feature that is exploited in clinical staging and response monitoring by using 18F-fluoro-deoxyglucose (FDG) positron emission tomography. Determinants of FDG accumulation include tumor blood flow, glucose transport, and glycolytic rate, but the underlying molecular mechanisms are incompletely understood. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin complex (mTORC) 1 pathway has been implicated in this process via the hypoxia-inducible factor alpha-dependent expression of vascular endothelial growth factor and glycolytic enzymes. Thus, we predicted that tumors with elevated mTORC1 activity would be accompanied by high FDG uptake. We tested this hypothesis in eight renal angiomyolipomas in which the loss of tuberous sclerosis complex (TSC) 1/2 function gave rise to constitutive mTORC1 activation. Surprisingly, these tumors displayed low FDG uptake on positron emission tomography. Exploring the underlying mechanisms in vitro revealed that Tsc2 regulates the membrane localization of the glucose transporter proteins (Glut)1, Glut2, and Glut4, and, therefore, glucose uptake. Down-regulation of cytoplasmic linker protein 170, an mTOR effector, rescued Glut4 trafficking in Tsc2−/− cells, whereas up-regulation of Akt activity in these cells was insufficient to redistribute Glut4 to the plasma membrane. The effect of mTORC1 on glucose uptake was confirmed using a liver-specific Tsc1- deletion mouse model in which FDG uptake was reduced in the livers of mutant mice compared with wild-type controls. Together, these data show that mTORC1 activity is insufficient for increased glycolysis in tumors and that constitutive mTOR activity negatively regulates glucose transporter trafficking. PMID:18511518

  17. Glucose transporter expression in human skeletal muscle fibers.

    PubMed

    Gaster, M; Handberg, A; Beck-Nielsen, H; Schroder, H D

    2000-09-01

    The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels. PMID:10950819

  18. Molecular Mechanism Underlying the Plant NRT1.1 Dual-Affinity Nitrate Transporter

    PubMed Central

    Sun, Ji; Zheng, Ning

    2015-01-01

    Nitrate (NO3−) is one of the most important sources of mineral nitrogen, which also serves as a key signaling molecule for plant growth and development. To cope with nitrate fluctuation in soil that varies by up to four orders of magnitude, plants have evolved high- and low-affinity nitrate transporter systems, consisting of distinct families of transporters. Interestingly, the first cloned nitrate transporter in Arabidopsis, NRT1.1 functions as a dual-affinity transporter, which can change its affinity for nitrate in response to substrate availability. Phosphorylation of a threonine residue, Thr101, switches NRT1.1 from low- to high-affinity state. Recent structural studies have unveiled that the unmodified NRT1.1 transporter works as homodimers with Thr101 located in close proximity to the dimer interface. Modification on the Thr101 residue is shown to not only decouple the dimer configuration, but also increase structural flexibility, thereby, altering the substrate affinity of NRT1.1. The structure of NRT1.1 helps establish a novel paradigm in which protein oligomerzation and posttranslational modification can synergistically expand the functional capacity of the major facilitator superfamily (MFS) transporters. PMID:26733879

  19. Peritoneal transport characteristics with glucose polymer based dialysate.

    PubMed

    Ho-dac-Pannekeet, M M; Schouten, N; Langendijk, M J; Hiralall, J K; de Waart, D R; Struijk, D G; Krediet, R T

    1996-09-01

    it remained unchanged during the dwell. In contrast, D/PNa+ of 1.36% glucose increased during the dwell, whereas D/PNa+ decreased with 3.86% glucose until 60 minutes, followed by a subsequent increase. The ultrafiltration coefficient (UFC) of the total peritoneal membrane was assessed using 3.86% glucose (0.18 +/- 0.04 ml/min/mm Hg), and the UFC of the small pores was assessed using icodextrin (0.06 +/- 0.008 ml/min/mm Hg). The difference between these represented the UFC through the transcellular pores, which averaged 50.5% of the total UFC, but with a very wide range (0 to 85%). An inverse relation existed between the duration of CAPD treatment and the total ultrafiltration coefficient (r = -0.68, P < 0.04), which could be attributed to a lower UFC of the transcellular pores in long-term patients (r = -0.66, P < 0.05), but not to the UFC of the small pores (r = -0.48, NS). The TCUFRo-60 min through the transcellular pores correlated with the sodium gradient, corrected for diffusion, in the first hour of the dwell (r = 0.69, P < 0.04), indicating that both parameters indeed measure transcellular water transport. It can be concluded that the glucose polymer solution induced sustained ultrafiltration and had no effect on peritoneal membrane characteristics. In addition, the results of the present study support the hypothesis that the glucose polymer solutions exerts its osmotic pressure across intercellular pores with radii of about 40 A. This leads to increased clearances of low molecular weight proteins such as beta 2m that are transported through these pores without sieving of Na+. The latter, as found during 3.86% glucose dialysate, is probably caused by transcellular water transport. The transcellular water transport accounted for 50% of the total ultrafiltration with glucose based dialysis solutions. It was lower in long-term CAPD patients. PMID:8872974

  20. Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

    NASA Astrophysics Data System (ADS)

    Sun, Ji; Bankston, John R.; Payandeh, Jian; Hinds, Thomas R.; Zagotta, William N.; Zheng, Ning

    2014-03-01

    Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101 however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

  1. Mystery solved: Trehalose kickstarts autophagy by blocking glucose transport.

    PubMed

    Mardones, Pablo; Rubinsztein, David C; Hetz, Claudio

    2016-01-01

    Although vertebrates cannot synthesize the natural disaccharide trehalose, exogenous administration of trehalose to mammalian cells may be beneficial for protein misfolding disorders. In this issue, DeBosch et al. show that trehalose may also be useful in treating nonalcoholic fatty liver disease and identify inhibition of cellular glucose import through SLC2A (also known as GLUT) transporters as a mechanism by which trehalose stimulates autophagy through the adenosine monophosphate-activated protein kinase (AMPK). PMID:26905424

  2. Crystal structure of the human glucose transporter GLUT1

    NASA Astrophysics Data System (ADS)

    Deng, Dong; Xu, Chao; Sun, Pengcheng; Wu, Jianping; Yan, Chuangye; Hu, Mingxu; Yan, Nieng

    2014-06-01

    The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2 Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters.

  3. AtKuP1: a dual-affinity K+ transporter from Arabidopsis.

    PubMed Central

    Fu, H H; Luan, S

    1998-01-01

    Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil. PMID:9477572

  4. Glucose transport by epithelia prepared from harvested enterocytes.

    PubMed

    Kimura, Yasuhiro; van der Merwe, Marie; Bering, Stine B; Penmatsa, Himabindu; Conoley, Veronica G; Sangild, Per T; Naren, Anjaparavanda P; Buddington, Randal K

    2015-01-01

    Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli. PMID:24166597

  5. Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport*

    PubMed Central

    Long, Wentong; Panwar, Pankaj; Witkowska, Kate; Wong, Kenneth; O'Neill, Debbie; Chen, Xing-Zhen; Lemieux, M. Joanne; Cheeseman, Chris I.

    2015-01-01

    High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes. PMID:25922070

  6. Molecular Dynamics Simulations of the Human Glucose Transporter GLUT1

    PubMed Central

    Park, Min-Sun

    2015-01-01

    Glucose transporters (GLUTs) provide a pathway for glucose transport across membranes. Human GLUTs are implicated in devastating diseases such as heart disease, hyper- and hypo-glycemia, type 2 diabetes and caner. The human GLUT1 has been recently crystalized in the inward-facing open conformation. However, there is no other structural information for other conformations. The X-ray structures of E. coli Xylose permease (XylE), a glucose transporter homolog, are available in multiple conformations with and without the substrates D-xylose and D-glucose. XylE has high sequence homology to human GLUT1 and key residues in the sugar-binding pocket are conserved. Here we construct a homology model for human GLUT1 based on the available XylE crystal structure in the partially occluded outward-facing conformation. A long unbiased all atom molecular dynamics simulation starting from the model can capture a new fully opened outward-facing conformation. Our investigation of molecular interactions at the interface between the transmembrane (TM) domains and the intracellular helices (ICH) domain in the outward- and inward-facing conformation supports that the ICH domain likely stabilizes the outward-facing conformation in GLUT1. Furthermore, inducing a conformational transition, our simulations manifest a global asymmetric rocker switch motion and detailed molecular interactions between the substrate and residues through the water-filled selective pore along a pathway from the extracellular to the intracellular side. The results presented here are consistent with previously published biochemical, mutagenesis and functional studies. Together, this study shed light on the structure and functional relationships of GLUT1 in multiple conformational states. PMID:25919356

  7. Adipocyte glucose transport regulation by eicosanoid precursors and inhibitors

    SciTech Connect

    Lee, H.C.C.

    1987-01-01

    Glucose uptake and free fatty acid release by adipocytes are increased by catecholamines. The mechanism of the stimulatory action of catecholamines on glucose uptake may be via eicosanoid production from release fatty acids. Rats were fed iso-nutrient diets with high or low safflower oil. After one month, 5 rats per diet group were fed diets with aspirin or without aspirin for 2 days. Isolated adipocytes from epididymal fat pads were incubated at 37/sup 0/C, gassed with 95% O/sub 2/-5% CO/sub 2/ in KRB buffer with 3% bovine serum albumin and with or without eicosanoid modifiers; a stimulator (10/sup -5/ M norepinephrine, N), or inhibitors (167 ..mu..l of antiserum to prostaglandin E (AntiE) per 1600 ..mu..l or 23mM Asp), or combinations of these. At 2-, 5-, and 10-min incubation, samples of incubation mixtures were taken to measure 2-deoxy glucose transport using /sup 3/H-2-deoxy glucose, /sup 14/C-inulin, and liquid scintillation counter.

  8. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity.

    PubMed Central

    Kim, E J; Kwak, J M; Uozumi, N; Schroeder, J I

    1998-01-01

    Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types. PMID:9477571

  9. Protective Role of Sodium-Glucose Co-Transporter 2 Inhibition Against Vascular Complications in Diabetes.

    PubMed

    Yamagishi, Sho-Ichi; Matsui, Takanori

    2016-04-01

    Diabetic micro- and macroangiopathy are devastating vascular complications that could account for disabilities and high mortality rate in patients with diabetes. Indeed, diabetic nephropathy and retinopathy are the leading causes of end-stage renal failure and acquired blindness, respectively, and atherosclerotic cardiovascular diseases (CVD) accounts for about 60% of death in diabetic subjects. As a result, the average life span of diabetic patients is about 10-15 years shorter than that of non-diabetic subjects. Furthermore, tight blood glucose control might have no more than a marginal impact on CVD in general and on all-cause mortality in particular in diabetes. Therefore, therapeutic strategies that target vascular complications in diabetes need to be developed. Recently, selective inhibition of sodium-glucose co-transporter 2 (SGLT2) has been proposed as a potential therapeutic target for the treatment of patients with diabetes because of low risk of hypoglycemia and no weight gain. Because 90% of glucose filtered by the glomerulus is reabsorbed by a low-affinity/high-capacity SGLT2 expressed in the S1 and S2 segments of the proximal tubule, blockade of SGLT2 promotes urinary glucose excretion and as a result improves hyperglycemia in an insulin-independent manner. Moreover, we have shown that SGLT2-mediated glucose overload to tubular cells could elicit inflammatory and pro-apoptotic reactions in this cell, being directly involved in diabetic nephropathy. In addition, several clinical studies have also shown that SGLT2 inhibitors could reduce blood pressure, body weight, and serum uric acid levels and ameliorate cardiovascular risk in patients with diabetes. This review summarizes the pathophysiological role of SGLT2 in vascular complications in diabetes and its potential therapeutic interventions. PMID:26228073

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

    PubMed

    Deng, Dong; Yan, Nieng

    2016-03-01

    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. PMID:26650681

  11. Ethanolic extract of Allium cepa stimulates glucose transporter typ 4-mediated glucose uptake by the activation of insulin signaling.

    PubMed

    Gautam, Sudeep; Pal, Savita; Maurya, Rakesh; Srivastava, Arvind K

    2015-02-01

    The present work was undertaken to investigate the effects and the molecular mechanism of the standardized ethanolic extract of Allium cepa (onion) on the glucose transport for controlling diabetes mellitus. A. cepa stimulates glucose uptake by the rat skeletal muscle cells (L6 myotubes) in both time- and dose-dependent manners. This effect was shown to be mediated by the increased translocation of glucose transporter typ 4 protein from the cytoplasm to the plasma membrane as well as the synthesis of glucose transporter typ 4 protein. The effect of A. cepa extract on glucose transport was stymied by wortmannin, genistein, and AI½. In vitro phosphorylation analysis revealed that, like insulin, A. cepa extract also enhances the tyrosine phosphorylation of the insulin receptor-β, insulin receptor substrate-1, and the serine phosphorylation of Akt under both basal and insulin-stimulated conditions without affecting the total amount of these proteins. Furthermore, it is also shown that the activation of Akt is indispensable for the A. cepa-induced glucose uptake in L6 myotubes. Taken together, these findings provide ample evidence that the ethanolic extract of A. cepa stimulates glucose transporter typ 4 translocation-mediated glucose uptake by the activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt dependent pathway. PMID:25654406

  12. Radiation inactivation target size of rat adipocyte glucose transporter

    SciTech Connect

    Jung, C.Y.; Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.

    1987-05-01

    In situ assembly states of rat adipocyte glucose transport protein in plasma membrane (PM) and in microsomal pool (MM) were assessed by measuring target size (TS) of D glucose-sensitive, cytochalasin B binding activity. High energy radiation inactivated the binding in both PM and MM by reducing the total capacity of the binding (B/sub T/) without affecting the dissociation constant (K/sub D/). The reduction in B/sub T/ as a function of radiation dose was analyzed based on classical target theory, from which TS was calculated. TS in the PM of insulin-treated adipocytes was 58 KDa. TS in the MM of noninsulin-treated and insulin-treated adipocytes were 112 and 109 KDa, respectively. With MM, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses showing a shoulder in the semilog plots, which may be due to an interaction with a radiation sensitive inhibitor. With these results, they propose the following model: Adipocyte glucose transporter, while exists as a monomer (T) in PM, occurs in MM either as a homodimer (T/sub 2/) or as a heterodimer (TX) with a protein X of a similar size. These dimers (T/sub 2/ or TX) in MM, furthermore, may form a multi-molecular assembly with another, large (300-400 KDa) protein Y, and insulin increases this assembly formation. These putative, transporter-associated proteins X and Y may play an important role in control of transporter distribution between PM and MM, particularly in response to insulin.

  13. Assessing Glucose Uptake through the Yeast Hexose Transporter 1 (Hxt1)

    PubMed Central

    Roy, Adhiraj; Dement, Angela D.; Cho, Kyu Hong; Kim, Jeong-Ho

    2015-01-01

    The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1N370A and HXT1W473A are resistant to such degradation. Hxt1N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1. PMID:25816250

  14. Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

    PubMed

    Adewoye, L O; Worobec, E A

    2000-08-01

    The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins. PMID:10940570

  15. Is it time to think about the sodium glucose co-transporter 2 sympathetically?

    PubMed

    Elliott, Rosemary H; Matthews, Vance B; Rudnicka, Caroline; Schlaich, Markus P

    2016-04-01

    Disturbances in glucose homeostasis are a key feature of the metabolic syndrome and type 2 diabetes. Renal glucose reabsorption is an important factor in glycaemic control. Glucose reabsorption in the proximal tubules is mediated by the sodium glucose co-transporter 2. The capacity for glucose reabsorption is increased in type 2 diabetes and contributes significantly to hyperglycaemia and impaired glucose control. Understanding the mechanisms underpinning the regulation of the sodium glucose co-transporter 2 is therefore of high clinical relevance. However, despite recent advances in the field and the availability of pharmacological inhibitors of this glucose transporter for the treatment of type 2 diabetes, the mechanisms that regulate sodium glucose co-transporter 2 expression are not fully understood. The sympathetic nervous system is an important modulator of glucose homeostasis, and sympathetic hyperactivity is a characteristic feature of obesity, the metabolic syndrome and type 2 diabetes. Sympathetic inhibition either achieved pharmacologically or by renal sympathetic denervation has been associated with improved glucose control. Importantly, sympathetic nerves innervate the proximal tubules of the kidney where they have been shown to regulate the expression of other transporters such as the sodium hydrogen exchanger 3. This review aims to explore the evidence for the regulation of sodium glucose co-transporter 2-mediated glucose reabsorption by the sympathetic nervous system. PMID:26369359

  16. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast

    PubMed Central

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D.; Hong Cho, Kyu; Kim, Jeong-Ho

    2016-01-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor. PMID:26764094

  17. Translocation of two glucose transporters in heart: effects of rotenone, uncouplers, workload, palmitate, insulin and anoxia.

    PubMed

    Wheeler, T J; Fell, R D; Hauck, M A

    1994-12-30

    Our previous studies on the acute regulation of glucose transport in perfused rat hearts were extended to explore further the mechanism of regulation by anoxia; to test the effects of palmitate, a transport inhibitor; and to compare the translocation of two glucose transporter isoforms (GLUT1 and GLUT4). Following heart perfusions under various conditions, glucose transporters in intracellular membranes were quantitated by reconstitution of transport activity and by Western blotting. Rotenone stimulated glucose uptake and decreased the intracellular contents of glucose transporters. This indicates that it activates glucose transport via net outward translocation, similarly to anoxia. However, two uncouplers of oxidative phosphorylation produced little or no effect. Increased workload (which stimulates glucose transport) reduced the intracellular contents of transporters, while palmitate increased the contents, indicating that these factors cause net translocation from or to the intracellular pool, respectively. Relative changes in GLUT1 were similar to those in GLUT4 for most factors tested. A plot of changes in total intracellular transporter content vs. changes in glucose uptake was roughly linear, with a slope of -0.18. This indicates that translocation accounts for most of the changes in glucose transport, and the basal pool of intracellular transporters is five times as large as the plasma membrane pool. PMID:7841183

  18. Steviol Glycosides Modulate Glucose Transport in Different Cell Types

    PubMed Central

    Rizzo, Benedetta; Zambonin, Laura; Leoncini, Emanuela; Vieceli Dalla Sega, Francesco; Prata, Cecilia; Fiorentini, Diana; Hrelia, Silvana

    2013-01-01

    Extracts from Stevia rebaudiana Bertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently, Stevia extracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies on Stevia and steviol glycosides in vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercial Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt. Furthermore, Stevia extracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis that Stevia extracts could mimic insulin effects modulating PI3K/Akt pathway. PMID:24327825

  19. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    PubMed

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  20. Improvement of glucose uptake rate and production of target chemicals by overexpressing hexose transporters and transcriptional activator Gcr1 in Saccharomyces cerevisiae.

    PubMed

    Kim, Daehee; Song, Ji-Yoon; Hahn, Ji-Sook

    2015-12-01

    Metabolic engineering to increase the glucose uptake rate might be beneficial to improve microbial production of various fuels and chemicals. In this study, we enhanced the glucose uptake rate in Saccharomyces cerevisiae by overexpressing hexose transporters (HXTs). Among the 5 tested HXTs (Hxt1, Hxt2, Hxt3, Hxt4, and Hxt7), overexpression of high-affinity transporter Hxt7 was the most effective in increasing the glucose uptake rate, followed by moderate-affinity transporters Hxt2 and Hxt4. Deletion of STD1 and MTH1, encoding corepressors of HXT genes, exerted differential effects on the glucose uptake rate, depending on the culture conditions. In addition, improved cell growth and glucose uptake rates could be achieved by overexpression of GCR1, which led to increased transcription levels of HXT1 and ribosomal protein genes. All genetic modifications enhancing the glucose uptake rate also increased the ethanol production rate in wild-type S. cerevisiae. Furthermore, the growth-promoting effect of GCR1 overexpression was successfully applied to lactic acid production in an engineered lactic acid-producing strain, resulting in a significant improvement of productivity and titers of lactic acid production under acidic fermentation conditions. PMID:26431967

  1. Diabetic Hyperglycemia: Link to Impaired Glucose Transport in Pancreatic β Cells

    NASA Astrophysics Data System (ADS)

    Unger, Roger H.

    1991-03-01

    Glucose uptake into pancreatic β cells by means of the glucose transporter GLUT-2, which has a high Michaelis constant, is essential for the normal insulin secretory response to hyperglycemia. In both autoimmune and nonautoimmune diabetes, this glucose transport is reduced as a consequence of down-regulation of the normal β-cell transporter. In autoimmune diabetes, circulating immunoglobulins can further impair this glucose transport by inhibiting functionally intact transporters. Insights into mechanisms of the unresponsiveness of β cells to hyperglycemia may improve the management and prevention of diabetes.

  2. Overexpression of GLUT3 placental glucose transporter in diabetic rats.

    PubMed Central

    Boileau, P; Mrejen, C; Girard, J; Hauguel-de Mouzon, S

    1995-01-01

    The localization of the two major placental glucose transporter isoforms, GLUT1 and GLUT3 was studied in 20-d pregnant rats. Immunocytochemical studies revealed that GLUT1 protein is expressed ubiquitously in the junctional zone (maternal side) and the labyrinthine zone (fetal side) of the placenta. In contrast, expression of GLUT3 protein is restricted to the labyrinthine zone, specialized in nutrient transfer. After 19-d maternal insulinopenic diabetes (streptozotocin), placental GLUT3 mRNA and protein levels were increased four-to-fivefold compared to nondiabetic rats, whereas GLUT1 mRNA and protein levels remained unmodified. Placental 2-deoxyglucose uptake and glycogen concentration were also increased fivefold in diabetic rats. These data suggest that GLUT3 plays a major role in placental glucose uptake and metabolism. The role of hyperglycemia in the regulation of GLUT3 expression was assessed by lowering the glycemia of diabetic pregnant rats. After a 5-d phlorizin infusion to pregnant diabetic rats, placental GLUT3 mRNA and protein levels returned to levels similar to those observed in nondiabetic rats. Furthermore, a short-term hyperglycemia (12 h), achieved by performing hyperglycemic clamps induced a fourfold increase in placental GLUT3 mRNA and protein with no concomitant change in GLUT1 expression. This study provides the first evidence that placental GLUT3 mRNA and protein expression can be stimulated in vivo under hyperglycemic conditions. Thus, GLUT3 transporter isoform appears to be highly sensitive to ambient glucose levels and may play a pivotal role in the severe alterations of placental function observed in diabetic pregnancies. Images PMID:7615800

  3. Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose–H+ symporter

    PubMed Central

    Leandro, Maria José; Gonçalves, Paula; Spencer-Martins, Isabel

    2006-01-01

    Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose–proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose–H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide–H+ symporters encompassing proteins from several yeasts and filamentous fungi. PMID:16402921

  4. Substrate specificity and mapping of residues critical for transport in the high-affinity glutathione transporter Hgt1p.

    PubMed

    Zulkifli, Mohammad; Yadav, Shambhu; Thakur, Anil; Singla, Shiffalli; Sharma, Monika; Bachhawat, Anand Kumar

    2016-08-01

    The high-affinity glutathione transporter Hgt1p of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter (OPT) family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher affinity for reduced glutathione (GSH), whereas it transported oxidized glutathione (GSSG) and other glutathione conjugates with lower affinity. To identify the residues of Hgt1p critical for substrate binding and translocation, all amino acid residues of the 13 predicted transmembrane domains (TMDs) have been subjected to mutagenesis. Functional evaluation of these 269 mutants by growth and biochemical assay followed by kinetic analysis of the severely defective mutants including previous mutagenic studies on this transporter have led to the identification of N124 (TMD1), V185 (TMD3), Q222, G225 and Y226 (TMD4), P292 (TMD5), Y374 (TMD6), L429 (TMD7) and F523 and Q526 (TMD9) as critical for substrate binding with at least 3-fold increase in Km upon mutagenesis to alanine. In addition residues Y226 and Y374 appeared to be important for differential substrate specificity. An ab initio model of Hgt1p was built and refined using these mutagenic data that yielded a helical arrangement that includes TMD3, TMD4, TMD5, TMD6, TMD7, TMD9 and TMD13 as pore-lining helices with the functionally important residues in a channel-facing orientation. Taken together the results of this study provides the first mechanistic insights into glutathione transport by a eukaryotic high-affinity glutathione transporter. PMID:27252386

  5. Gene expression of epithelial glucose transporters: the role of diabetes mellitus.

    PubMed

    Dominguez, J H; Song, B; Maianu, L; Garvey, W T; Qulali, M

    1994-11-01

    The functions of absorption of dietary glucose by the small intestine and reabsorption of filtered glucose by the renal proximal tubule are strikingly similar in their organization and in the way they adapt to uncontrolled diabetes mellitus. In both cases, transepithelial glucose and Na+ fluxes are augmented. The epithelial adaptations to hyperglycemia of uncontrolled diabetes are accomplished by increasing the glucose transport surface area and the number of the efflux glucose transporter GLUT2 located in the basolateral membrane. The signals that modify the size of the epithelium and the overexpression of basolateral GLUT2 are not known. It was speculated that high glucose levels and enhanced Na+ flux may be important factors in the signaling event that culminates in a renal and intestinal epithelium that is modified to transport higher rates of glucose against a higher extracellular level of glucose. PMID:7873742

  6. Glucose transporter 1 deficiency syndrome and hemiplegic migraines as a dominant presenting clinical feature.

    PubMed

    Mohammad, Shekeeb S; Coman, David; Calvert, Sophie

    2014-12-01

    Glucose transporter 1 deficiency syndrome (OMIM 606777) is a treatable epileptic encephalopathy caused by mutations in the SLC2A1 gene (OMIM 138140) causing impaired glucose transport into the brain. The classical phenotype is associated with seizures, developmental delay, ataxia and spasticity; however, milder phenotypes are emerging. We describe an 8-year-old boy with glucose transporter 1 deficiency syndrome whose clinical presentation was dominated by hemiplegic migraines that resolved with institution of a modified Atkins diet. PMID:25440161

  7. Glucose transporters and maximal transport are increased in endurance-trained rat soleus

    NASA Technical Reports Server (NTRS)

    Slentz, C. A.; Gulve, E. A.; Rodnick, K. J.; Henriksen, E. J.; Youn, J. H.; Holloszy, J. O.

    1992-01-01

    Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS).

  8. Isoform-selective Inhibition of Facilitative Glucose Transporters

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

    2014-01-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  9. Simultaneous measurement of glucose transport and utilization in the human brain

    PubMed Central

    Shestov, Alexander A.; Emir, Uzay E.; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R.

    2011-01-01

    Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, KMt and Vmaxt, in humans have so far been obtained by measuring steady-state brain glucose levels by proton (1H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMRglc) obtained from other tracer studies, such as 13C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state 1H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMRglc, this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain. PMID:21791622

  10. A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood.

    PubMed

    Palmer, Clovis S; Anzinger, Joshua J; Butterfield, Tiffany R; McCune, Joseph M; Crowe, Suzanne M

    2016-01-01

    Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood. PMID:27584036

  11. Dysregulated hepatic expression of glucose transporters in chronic disease: contribution of semicarbazide-sensitive amine oxidase to hepatic glucose uptake

    PubMed Central

    Karim, Sumera; Liaskou, Evaggelia; Fear, Janine; Garg, Abhilok; Reynolds, Gary; Claridge, Lee; Adams, David H.; Newsome, Philip N.

    2014-01-01

    Insulin resistance is common in patients with chronic liver disease (CLD). Serum levels of soluble vascular adhesion protein-1 (VAP-1) are also increased in these patients. The amine oxidase activity of VAP-1 stimulates glucose uptake via translocation of transporters to the cell membrane in adipocytes and smooth muscle cells. We aimed to document human hepatocellular expression of glucose transporters (GLUTs) and to determine if VAP-1 activity influences receptor expression and hepatic glucose uptake. Quantitative PCR and immunocytochemistry were used to study human liver tissue and cultured cells. We also used tissue slices from humans and VAP-1-deficient mice to assay glucose uptake and measure hepatocellular responses to stimulation. We report upregulation of GLUT1, -3, -5, -6, -7, -8, -9, -10, -11, -12, and -13 in CLD. VAP-1 expression and enzyme activity increased in disease, and provision of substrate to hepatic VAP-1 drives hepatic glucose uptake. This effect was sensitive to inhibition of VAP-1 and could be recapitulated by H2O2. VAP-1 activity also altered expression and subcellular localization of GLUT2, -4, -9, -10, and -13. Therefore, we show, for the first time, alterations in hepatocellular expression of glucose and fructose transporters in CLD and provide evidence that the semicarbazide-sensitive amine oxidase activity of VAP-1 modifies hepatic glucose homeostasis and may contribute to patterns of GLUT expression in chronic disease. PMID:25342050

  12. Dysregulated hepatic expression of glucose transporters in chronic disease: contribution of semicarbazide-sensitive amine oxidase to hepatic glucose uptake.

    PubMed

    Karim, Sumera; Liaskou, Evaggelia; Fear, Janine; Garg, Abhilok; Reynolds, Gary; Claridge, Lee; Adams, David H; Newsome, Philip N; Lalor, Patricia F

    2014-12-15

    Insulin resistance is common in patients with chronic liver disease (CLD). Serum levels of soluble vascular adhesion protein-1 (VAP-1) are also increased in these patients. The amine oxidase activity of VAP-1 stimulates glucose uptake via translocation of transporters to the cell membrane in adipocytes and smooth muscle cells. We aimed to document human hepatocellular expression of glucose transporters (GLUTs) and to determine if VAP-1 activity influences receptor expression and hepatic glucose uptake. Quantitative PCR and immunocytochemistry were used to study human liver tissue and cultured cells. We also used tissue slices from humans and VAP-1-deficient mice to assay glucose uptake and measure hepatocellular responses to stimulation. We report upregulation of GLUT1, -3, -5, -6, -7, -8, -9, -10, -11, -12, and -13 in CLD. VAP-1 expression and enzyme activity increased in disease, and provision of substrate to hepatic VAP-1 drives hepatic glucose uptake. This effect was sensitive to inhibition of VAP-1 and could be recapitulated by H2O2. VAP-1 activity also altered expression and subcellular localization of GLUT2, -4, -9, -10, and -13. Therefore, we show, for the first time, alterations in hepatocellular expression of glucose and fructose transporters in CLD and provide evidence that the semicarbazide-sensitive amine oxidase activity of VAP-1 modifies hepatic glucose homeostasis and may contribute to patterns of GLUT expression in chronic disease. PMID:25342050

  13. Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I.

    PubMed

    Zoidis, E; Ghirlanda-Keller, C; Schmid, C

    2011-02-01

    Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts. PMID:21076856

  14. Role of reactive oxygen species in regulation of glucose transport in skeletal muscle during exercise.

    PubMed

    Katz, Abram

    2016-06-01

    Glucose derived from extracellular sources serves as an energy source in virtually all eukaryotic cells, including skeletal muscle. Its contribution to energy turnover increases with exercise intensity up to moderately heavy workloads. However, at very high workloads, the contribution of extracellular glucose to energy turnover is negligible, despite the high rate of glucose transport. Reactive oxygen species (ROS) are involved in the stimulation of glucose transport in isolated skeletal muscle preparations during intense repeated contractions. Consistent with this observation, heavy exercise is associated with significant production of ROS. However, during more mild to moderate stimulation or exercise conditions (in vitro, in situ and in vivo) antioxidants do not affect glucose transport. It is noteworthy that the production of ROS is limited or not observed under these conditions and that the concentration of the antioxidant used was extremely low. The results to date suggest that ROS involvement in activation of glucose transport occurs primarily during intense short-term exercise and that other mechanisms are involved during mild to moderate exercise. What remains puzzling is why ROS-mediated activation of glucose transport would occur under conditions where glucose transport is highest and utilization (i.e. phosphorylation of glucose by hexokinase) is low. Possibly ROS production is involved in priming glucose transport during heavy exercise to accelerate glycogen biogenesis during the initial recovery period after exercise, as well as altering other aspects of intracellular metabolism. PMID:26791627

  15. Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.

    PubMed

    Heilig, C W; Concepcion, L A; Riser, B L; Freytag, S O; Zhu, M; Cortes, P

    1995-10-01

    An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT1 gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial beta-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis. PMID:7560072

  16. PET measurement of glucose membrane transport using labeled analogs: Distinction of transport from metabolic processes

    SciTech Connect

    Holden, J.E.; Koeppe, R.A.; Gatley, S.J.

    1984-01-01

    Carrier mediated glucose transport rates across brain capillary and myocardial cell membranes are many times higher than those expected for simple diffusion, and transport regulation can be an important determinant of tissue metabolic status. The authors have investigated the use of glucose analogs and dynamic positron tomography for the non-invasive measurement of unidirectional membrane transport rates. If analog extraction is sufficiently low, transport rates can be inferred directly from fitted kinetic rate constants. Fitting calculations were seen to be sensitive to the difficult to measure rapid components of the arterial input curves, to contributions from blood-borne label in the early data points, and to interference from other chemical forms in cases of significant phosphorylation. This last uncertainty was studied using serial scans of normal brain after venous injection of the well-transported but poorly phosphorylated analog 3-deoxy-3-fluoroglucose. Transport rate constants derived from 4-parameter fits of three hours of data were compared to those derived from 2-parameter fits of the first 12-20 minutes of data. Errors due to trapped label were absorbed primarily into the apparent distribution volume, allowing accurate estimation of transport rate constants from a brief data acquisition period. The study of the distinction of transport from phosphorylation also bears on the important question of the significance of the individual rate constants in the four-parameter fitting of brief dynamic scan sequences in studies of metabolic rate using 2-deoxy-2-fluoroglucose.

  17. Characterization of the fetal glucose transporter in rabbit kidney. Comparison with the adult brush border electrogenic Na+-glucose symporter.

    PubMed Central

    Beck, J C; Lipkowitz, M S; Abramson, R G

    1988-01-01

    Glucose transport was characterized in rabbit renal brush border membrane vesicles (BBMV) of the fetus late in gestation. Highly purified, osmotically reactive fetal BBMV contained a glucose transporter that was qualitatively indistinguishable from that in the adult: both are concentrative, Na+ dependent, electrogenic, stereospecific, and sensitive to phlorizin. Although the apparent Km for glucose is similar in the fetus and adult, the Vmax is significantly higher in the adult. When the membrane potential was clamped with a protonophore, this difference diminished; however, Vmax remained significantly higher in adult BBMV. This postnatal increase in Vmax was paralleled by a similar increase in the number of phlorizin binding sites. These findings indicate that the maturational increase in glucose transport is, in part, consequent to a more favorable electrical potential for Na+-dependent glucose transport and, in part, the result of the insertion of new transporters. The homogenate activity of several brush border enzymes also demonstrated significant maturational increases. The magnitude of these changes was variable and enzyme dependent. These combined observations suggest that mature expression of membrane proteins (transporters and enzymes) occurs at different stages of development of renal proximal tubule cells. PMID:3403709

  18. Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

    SciTech Connect

    Beck, J.T.; Ullman, B. )

    1989-08-22

    An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 {mu}M concentration of either activated ({sup 3}H)folate or activated ({sup 3}H)methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms.

  19. Endocytosis, recycling, and regulated exocytosis of glucose transporter 4.

    PubMed

    Foley, Kevin; Boguslavsky, Shlomit; Klip, Amira

    2011-04-19

    Glucose transporter 4 (GLUT4) is responsible for the uptake of glucose into muscle and adipose tissues. Under resting conditions, GLUT4 is dynamically retained through idle cycling among selective intracellular compartments, from whence it undergoes slow recycling to the plasma membrane (PM). This dynamic retention can be released by command from intracellular signals elicited by insulin and other stimuli, which result in 2-10-fold increases in the surface level of GLUT4. Insulin-derived signals promote translocation of GLUT4 to the PM from a specialized compartment termed GLUT4 storage vesicles (GSV). Much effort has been devoted to the characterization of the intracellular compartments and dynamics of GLUT4 cycling and to the signals by which GLUT4 is sorted into, and recruited from, GSV. This review summarizes our understanding of intracellular GLUT4 traffic during its internalization from the membrane, its slow, constitutive recycling, and its regulated exocytosis in response to insulin. In spite of specific differences in GLUT4 dynamic behavior in adipose and muscle cells, the generalities of its endocytic and exocytic itineraries are consistent and an array of regulatory proteins that regulate each vesicular traffic event emerges from these cell systems. PMID:21405107

  20. Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts

    SciTech Connect

    Whitesell, R.R.; Regen, D.M.; Pelletier, D.; Abumrad, N.A. )

    1990-10-01

    Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of ({sup 14}C)methylglucose and D-({sup 14}C)glucose with (1) L-({sup 3}H)glucose as an extracellular marker together with the ({sup 14}C)glucose or ({sup 14}C)methylglucose in the substrate mixture, (2) sampling times as short as 2 s, (3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and (4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.

  1. Importin {beta}-type nuclear transport receptors have distinct binding affinities for Ran-GTP

    SciTech Connect

    Hahn, Silvia; Schlenstedt, Gabriel

    2011-03-18

    Highlights: {yields} Determination of binding properties of nuclear transport receptor/Ran-GTP complexes. {yields} Biosensor measurements provide constants for dissociation, on-rates, and off-rates. {yields} The affinity of receptors for Ran-GTP is widely divergent. {yields} Dissociation constants differ for three orders of magnitude. {yields} The cellular concentration of yeast Ran is not limiting. -- Abstract: Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin {beta} family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of {beta}-receptors and of other Ran-binding proteins was determined. We found that the number of {beta}-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

  2. Apolipoprotein A-I glycation by Glucose and Reactive Aldehydes Alters Phospholipid Affinity but Not Cholesterol Export from Lipid-Laden Macrophages

    PubMed Central

    Brown, Bronwyn E.; Nobecourt, Estelle; Zeng, Jingmin; Jenkins, Alicia J.; Rye, Kerry-Anne; Davies, Michael J.

    2013-01-01

    Increased protein glycation in people with diabetes may promote atherosclerosis. This study examined the effects of non-enzymatic glycation on the association of lipid-free apolipoproteinA-I (apoA-I) with phospholipid, and cholesterol efflux from lipid-loaded macrophages to lipid-free and lipid-associated apoA-I. Glycation of lipid-free apoA-I by methylglyoxal and glycolaldehyde resulted in Arg, Lys and Trp loss, advanced glycation end-product formation and protein cross-linking. The association of apoA-I glycated by glucose, methylglyoxal or glycolaldehyde with phospholipid multilamellar vesicles was impaired in a glycating agent dose-dependent manner, with exposure of apoA-I to both 30 mM glucose (42% decrease in kslow) and 3 mM glycolaldehyde (50% decrease in kfast, 60% decrease in kslow) resulting is significantly reduced affinity. Cholesterol efflux to control or glycated lipid-free apoA-I, or discoidal reconstituted HDL containing glycated apoA-I (drHDL), was examined using cholesterol-loaded murine (J774A.1) macrophages treated to increase expression of ATP binding cassette transporters A1 (ABCA1) or G1 (ABCG1). Cholesterol efflux from J774A.1 macrophages to glycated lipid-free apoA-I via ABCA1 or glycated drHDL via an ABCG1-dependent mechanism was unaltered, as was efflux to minimally modified apoA-I from people with Type 1 diabetes, or controls. Changes to protein structure and function were prevented by the reactive carbonyl scavenger aminoguanidine. Overall these studies demonstrate that glycation of lipid-free apoA-I, particularly late glycation, modifies its structure, its capacity to bind phospholipids and but not ABCA1- or ABCG1-dependent cholesterol efflux from macrophages. PMID:23741493

  3. Glycogen supercompensation masks the effect of a traininginduced increase in GLUT-4 on muscle glucose transport.

    PubMed

    Host, H H; Hansen, P A; Nolte, L A; Chen, M M; Holloszy, J O

    1998-07-01

    Endurance exercise training induces a rapid increase in the GLUT-4 isoform of the glucose transporter in muscle. In fasted rats, insulin-stimulated muscle glucose transport is increased in proportion to the increase in GLUT-4. There is evidence that high muscle glycogen may decrease insulin-stimulated glucose transport. This study was undertaken to determine whether glycogen supercompensation interferes with the increase in glucose transport associated with an exercise-induced increase in GLUT-4. Rats were trained by means of swimming for 6 h/day for 2 days. Rats fasted overnight after the last exercise bout had an approximately twofold increase in epitrochlearis muscle GLUT-4 and an associated approximately twofold increase in maximally insulin-stimulated glucose transport activity. Epitrochlearis muscles of rats fed rodent chow after exercise were glycogen supercompensated (86.4 +/- 4.8 micromol/g wet wt) and showed no significant increase in maximally insulin-stimulated glucose transport above the sedentary control value despite an approximately twofold increase in GLUT-4. Fasting resulted in higher basal muscle glucose transport rates in both sedentary and trained rats but did not significantly increase maximally insulin-stimulated transport in the sedentary group. We conclude that carbohydrate feeding that results in muscle glycogen supercompensation prevents the increase in maximally insulin-stimulated glucose transport associated with an exercise training-induced increase in muscle GLUT-4. PMID:9655766

  4. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter.

    PubMed

    Samsudin, Firdaus; Parker, Joanne L; Sansom, Mark S P; Newstead, Simon; Fowler, Philip W

    2016-02-18

    Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the ?-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepTSt, and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theoretically exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homology model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families. PMID:27028887

  5. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter

    PubMed Central

    Samsudin, Firdaus; Parker, Joanne L.; Sansom, Mark S.P.; Newstead, Simon; Fowler, Philip W.

    2016-01-01

    Summary Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the β-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepTSt, and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theoretically exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homology model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families. PMID:27028887

  6. The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes.

    PubMed

    Kaddai, V; Gonzalez, T; Bolla, M; Le Marchand-Brustel, Y; Cormont, M

    2008-07-01

    NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor kappaB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes. PMID:18492771

  7. Glucose Transporter Type 1 Deficiency Syndrome with Carbohydrate-Responsive Symptoms but without Epilepsy

    ERIC Educational Resources Information Center

    Koy, Anne; Assmann, Birgit; Klepper, Joerg; Mayatepek, Ertan

    2011-01-01

    Glucose transporter type 1 deficiency syndrome (GLUT1-DS) is caused by a defect in glucose transport across the blood-brain barrier. The main symptoms are epilepsy, developmental delay, movement disorders, and deceleration of head circumference. A ketogenic diet has been shown to be effective in controlling epilepsy in GLUT1-DS. We report a female…

  8. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    PubMed

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply. PMID:25164101

  9. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    PubMed

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid. PMID:24484197

  10. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin.

    PubMed

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  11. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  12. Low and high affinity dopamine transporter inhibitors block dopamine uptake within 5 sec of intravenous injection

    PubMed Central

    Yorgason, Jordan T.; Jones, Sara R.; España, Rodrigo A.

    2011-01-01

    Extensive evidence suggests that the reinforcing effects of cocaine involve inhibition of dopamine transporters (DAT) and subsequent increases in dopamine (DA) levels in the striatum. We have previously reported that cocaine inhibits the DAT within 4–5 sec of intravenous injection, matching the temporal profile of the behavioral and subjective effects of cocaine. Intravenous injection of GBR-12909, a high affinity, long-acting DAT inhibitor, also inhibits DA uptake within 5 sec. Given that high affinity, long-acting drugs are considered to have relatively low abuse potential, we found it intriguing that GBR-12909 had an onset profile similar to that of cocaine. To further explore the onset kinetics of both low and high affinity DAT inhibitors, we examined the effects of intravenous cocaine (1.5 mg/kg), methylphenidate (1.5 mg/kg), nomifensine (1.5 mg/kg), GBR-12909 (1.5 mg/kg), PTT (0.5 mg/kg), and WF23 (0.5 mg/kg) on electrically-evoked DA release and uptake in the nucleus accumbens core. Results indicate that all of the DAT inhibitors significantly inhibited DA uptake within 5 sec of injection. However, the timing of peak uptake inhibition varied greatly between the low and high affinity uptake inhibitors. Uptake inhibition following cocaine, methylphenidate, and nomifensine peaked 30 sec following injection. In contrast, peak effects for GBR-12909, PTT, and WF23 occurred between 20 and 60 min following injection. These observations suggest that the initial onset for intravenous DAT inhibitors is extremely rapid and does not appear to be dictated by a drug’s affinity. PMID:21402130

  13. Pretranslational Suppression of an Insulin-Responsive Glucose Transporter in Rats with Diabetes Mellitus

    NASA Astrophysics Data System (ADS)

    Garvey, W. Timothy; Huecksteadt, Thomas P.; Birnbaum, Morris J.

    1989-07-01

    A prominent feature of diabetes mellitus is the inability of insulin to appropriately increase the transport of glucose into target tissues. The contributions of different glucose transport proteins to insulin resistance in rats with streptozotocin-induced diabetes was evaluated. A glucose transporter messenger RNA and its cognate protein that are exclusively expressed in muscle and adipose tissue were specifically depleted in diabetic animals, and these effects were reversed after insulin therapy; a different glucose transporter and its messenger RNA that exhibit a less restricted tissue distribution were not specifically modulated in this way. Depletion of the muscle- and adipose-specific glucose transporter species correlates with and may account for the major portion of cellular insulin resistance in diabetes in these animals.

  14. Structural state of the Na+/D-glucose cotransporter in calf kidney brush-border membranes. Target size analysis of Na+-dependent phlorizin binding and Na+-dependent D-glucose transport.

    PubMed

    Lin, J T; Szwarc, K; Kinne, R; Jung, C Y

    1984-11-01

    Target sizes of the renal sodium-D-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at -50 degrees C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na+-dependent D-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4-4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na+-dependent phlorizin binding unit was estimated to be 230 000 +/- 38 000. From the tracer exchange experiments a molecular weight of 345 000 +/- 24 500 was calculated for the D-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and gamma-glutamyltransferase, whose target sizes were found to be 68 570 +/- 2670 and 73 500 +/- 2270, respectively. These findings provide further evidence for the assumption that the sodium-D-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and D-glucose translocation. PMID:6148966

  15. Glycaemia regulates the glucose transporter number in the plasma membrane of rat skeletal muscle.

    PubMed Central

    Dimitrakoudis, D; Ramlal, T; Rastogi, S; Vranic, M; Klip, A

    1992-01-01

    The number of glucose transporters was measured in isolated membranes from diabetic-rat skeletal muscle to determine the role of circulating blood glucose levels in the control of glucose uptake into skeletal muscle. Three experimental groups of animals were investigated in the post-absorptive state: normoglycaemic/normoinsulinaemic, hyperglycaemic/normoinsulinaemic and hyperglycaemic/normoinsulinaemic made normoglycaemic/normoinsulinaemic by phlorizin treatment. Hyperglycaemia caused a reversible decrease in total transporter number, as measured by cytochalasin B binding, in both plasma membranes and internal membranes of skeletal muscle. Changes in GLUT4 glucose transporter protein mirrored changes in cytochalasin B binding in plasma membranes. However, there was no recovery of GLUT4 levels in intracellular membranes with correction of glycaemia. GLUT4 mRNA levels decreased with hyperglycaemia and recovered only partially with correction of glycaemia. Conversely, GLUT1 glucose transporters were only detectable in the plasma membranes; the levels of this protein varied directly with glycaemia, i.e. in the opposite direction to GLUT4 glucose transporters. This study demonstrates that hyperglycaemia, in the absence of hypoinsulinaemia, is capable of down-regulating the glucose transport system in skeletal muscle, the major site of peripheral resistance to insulin-stimulated glucose transport in diabetes. Furthermore, correction of hyperglycaemia causes a complete restoration of the transport system in the basal state (determined by the transporter number in the plasma membrane), but possibly only an incomplete recovery of the transport system's ability to respond to insulin (since there is no recovery of GLUT4 levels in the intracellular membrane insulin-responsive transporter pool). Finally, the effect of hyperglycaemia is specific for glucose transporter isoforms, with GLUT1 and GLUT4 proteins varying respectively in parallel and opposite directions to levels of

  16. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

    PubMed Central

    Tomatsu, Hajime; Takano, Junpei; Takahashi, Hideki; Watanabe-Takahashi, Akiko; Shibagaki, Nakako; Fujiwara, Toru

    2007-01-01

    Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the Km value of MOT1 for molybdate is ≈20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil. PMID:18003916

  17. Downregulation of mouse intestinal Na(+)-coupled glucose transporter SGLT1 by gum arabic (Acacia Senegal).

    PubMed

    Nasir, Omaima; Artunc, Ferruh; Wang, Kan; Rexhepaj, Rexhep; Föller, Michael; Ebrahim, Ammar; Kempe, Daniela S; Biswas, Raja; Bhandaru, Madhuri; Walter, Michael; Mohebbi, Nilufar; Wagner, Carsten A; Saeed, Amal M; Lang, Florian

    2010-01-01

    Intestinal Na(+)-coupled glucose transporter SGLT1 determines the rate of glucose transport, which in turn influences glucose-induced insulin release and development of obesity. The present study explored effects of Gum Arabic (GA), a dietary polysaccharide from dried exudates of Acacia Senegal, on intestinal glucose transport and body weight in wild-type C57Bl/6 mice. Treatment with GA (100 g/l) in drinking water for four weeks did not affect intestinal SGLT1 transcript levels but decreased SGLT1 protein abundance in jejunal brush border membrane vesicles. Glucose-induced jejunal short-circuit currents revealed that GA treatment decreased electrogenic glucose transport. Drinking a 20% glucose solution for four weeks significantly increased body weight and fasting plasma glucose concentrations, effects significantly blunted by simultaneous treatment with GA. GA further significantly blunted the increase in body weight, fasting plasma glucose and fasting insulin concentrations during high fat diet. In conclusion, the present observations disclose a completely novel effect of gum arabic, i.e. its ability to decrease intestinal SGLT1 expression and activity and thus to counteract glucose-induced obesity. PMID:20110681

  18. The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

    SciTech Connect

    Galperin, M.Y.; Noll, K.M.; Romano, A.H.

    1996-08-01

    The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external D-glucose. This active transport of 2-DOG was dependent upon the presence of sodium ion and an external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T.neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation. 33 refs., 3 figs., 1 tab.

  19. HIV Protease Inhibitors Act as Competitive Inhibitors of the Cytoplasmic Glucose Binding Site of GLUTs with Differing Affinities for GLUT1 and GLUT4

    PubMed Central

    Hresko, Richard C.; Hruz, Paul W.

    2011-01-01

    The clinical use of several first generation HIV protease inhibitors (PIs) is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a KI of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity. PMID:21966466

  20. Glucose Transporters are Abundant in Cells with "Occluding" Junctions at the Blood-Eye Barriers

    NASA Astrophysics Data System (ADS)

    Harik, Sami I.; Kalaria, Rajesh N.; Whitney, Paul M.; Andersson, Lars; Lundahl, Per; Ledbetter, Steven R.; Perry, George

    1990-06-01

    We studied the distribution of the "erythroid/brain" glucose transporter protein in the human and rat eye by immunocytochemistry with monoclonal and polyclonal antibodies to the C terminus of the human erythrocyte glucose transporter. We found intense immunocytochemical staining in the endothelium of microvessels of the retina, optic nerve, and iris but not in microvessels of the choroid, ciliary body, sclera, and other retro-orbital tissues. In addition, we found marked immunocytochemical staining of retinal pigment epithelium, ciliary body epithelium, and posterior epithelium of the iris. The common feature of all those endothelial and epithelial cells that stained intensely for the glucose transporter is the presence of "occluding" intercellular junctions, which constitute the anatomical bases of the blood-eye barriers. We propose that a high density of the glucose transporter is a biochemical concomitant of epithelial and endothelial cells with barrier characteristics, at least in tissues that have a high metabolic requirement for glucose.

  1. Glucose transporters are abundant in cells with "occluding" junctions at the blood-eye barriers.

    PubMed Central

    Harik, S I; Kalaria, R N; Whitney, P M; Andersson, L; Lundahl, P; Ledbetter, S R; Perry, G

    1990-01-01

    We studied the distribution of the "erythroid/brain" glucose transporter protein in the human and rat eye by immunocytochemistry with monoclonal and polyclonal antibodies to the C terminus of the human erythrocyte glucose transporter. We found intense immunocytochemical staining in the endothelium of microvessels of the retina, optic nerve, and iris but not in microvessels of the choroid, ciliary body, sclera, and other retro-orbital tissues. In addition, we found marked immunocytochemical staining of retinal pigment epithelium, ciliary body epithelium, and posterior epithelium of the iris. The common feature of all those endothelial and epithelial cells that stained intensely for the glucose transporter is the presence of "occluding" intercellular junctions, which constitute the anatomical bases of the blood-eye barriers. We propose that a high density of the glucose transporter is a biochemical concomitant of epithelial and endothelial cells with barrier characteristics, at least in tissues that have a high metabolic requirement for glucose. Images PMID:2190218

  2. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

    PubMed

    Orlowski, S; Champeil, P

    1991-01-15

    We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium

  3. Rapid high-affinity transport of a chemotherapeutic amino acid across the blood-brain barrier.

    PubMed

    Takada, Y; Vistica, D T; Greig, N H; Purdon, D; Rapoport, S I; Smith, Q R

    1992-04-15

    The therapeutic efficacy of many anticancer drugs against intracerebral tumors is limited by poor uptake into the central nervous system. One way to enhance brain delivery is to design agents that are transported into the brain by the saturable nutrient carriers of the blood-brain barrier. In this paper, we describe a nitrogen mustard amino acid, DL-2-amino-7-bis[(2-chloroethyl)amino/bd-1,2,3,4-tetrahydro-2-napthoi c acid, that is taken up into brain with high affinity by the large neutral amino acid carrier of the blood-brain barrier. Brain transport of DL-2-amino-7-bis[(2-chloroethyl)aminol-1,2,3,4-tetrahydro-2-naphth oic acid in the rat was found to be rapid (cerebrovascular permeability-surface area product approximately 2 x 10(-2) ml/s/g), saturable and inhibitable by large neutral amino acids. Maximal influx rate (Vmax) and half-saturation (Km) constants equaled 0.26 nmol/min/g and 0.19 microM, respectively, in the parietal cortex. Regional brain uptake of acid exceeded that of the clinical analogue, melphalan, by greater than 20-fold. The results demonstrate that drug modification to produce high-affinity ligands for the cerebrovascular nutrient carriers is a viable means to enhance drug delivery to brain for the treatment of brain tumors and other central nervous system disorders. PMID:1559223

  4. Glucose Transport into Everted Sacks of Intestine of Mice: A Model for the Study of Active Transport.

    ERIC Educational Resources Information Center

    Deyrup-Olsen, Ingrith; Linder, Alison R.

    1979-01-01

    Described is a laboratory procedure which uses the small intestines of mice as models for the transport of glucose and other solutes. Demonstrations are suitable for either introductory or advanced physiology courses. (RE)

  5. Differentiation of the insulin-sensitive glucose transporter in 3T3-L1 adipocytes

    SciTech Connect

    Frost, S.C.; Baly, D.L.; Cushman, S.W.; Lane, M.D.; Simpson, I.A.

    1986-05-01

    3T3-L1 fibroblasts differentiate in culture to resemble adipocytes both morphologically and biochemically. Insulin-sensitive glucose transport, as measured by 2-deoxy-(1-/sup 14/C)- glucose uptake in the undifferentiated cell is small (2X). In contrast, the rate of glucose transport in fully differentiated cells is elevated 15-fold over basal in the presence of insulin. To determine if this is due to an increase in the number of transporters/cell or accessibility to the transporters, the number of transporters was measured in subcellular fractions over differentiation using a /sup 3/H-cytochalasin B binding assay. The increase in the rate of insulin-sensitive glucose transport directly parallels an increase in the number of transporters which reside in an insulin-responsive intracellular compartment. This observation was confirmed by identifying the transporters by immunoblotting using an antibody generated against the human erythrocyte transporter. The molecular weight of this transporter increases over differentiation from a single band of 40kDa to a heterogeneous triplet of 40, 44 and 48kDa. These data suggest that the transporter undergoes differential processing and that the functional, insulin-responsive transporter may be different from the insulin-insensitive (basal) transporter.

  6. Glucose Transport into Everted Sacs of the Small Intestine of Mice

    ERIC Educational Resources Information Center

    Hamilton, Kirk L.; Butt, A. Grant

    2013-01-01

    The Na[superscript +]-glucose cotransporter is a key transport protein that is responsible for absorbing Na[superscript +] and glucose from the luminal contents of the small intestine and reabsorption by the proximal straight tubule of the nephron. Robert K. Crane originally described the cellular model of absorption of Na[superscript +] and…

  7. Melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer.

    PubMed

    Hevia, David; González-Menéndez, Pedro; Quiros-González, Isabel; Miar, Ana; Rodríguez-García, Aida; Tan, Dun-Xian; Reiter, Russel J; Mayo, Juan C; Sainz, Rosa M

    2015-03-01

    Melatonin is present in a multitude of taxa and it has a broad range of biological functions, from synchronizing circadian rhythms to detoxifying free radicals. Some functions of melatonin are mediated by its membrane receptors but others are receptor-independent. For the latter, melatonin must enter into the cell. Melatonin is a derivative of the amino acid tryptophan and reportedly easily crosses biological membranes due to its amphipathic nature. However, the mechanism by which melatonin enters into cells remains unknown. Changes in redox state, endocytosis pathways, multidrug resistance, glycoproteins or a variety of strategies have no effect on melatonin uptake. Herein, it is demonstrated that members of the SLC2/GLUT family glucose transporters have a central role in melatonin uptake. When studied by docking simulation, it is found that melatonin interacts at the same location in GLUT1 where glucose does. Furthermore, glucose concentration and the presence of competitive ligands of GLUT1 affect the concentration of melatonin into cells. As a regulatory mechanism, melatonin reduces the uptake of glucose and modifies the expression of GLUT1 transporter in prostate cancer cells. More importantly, glucose supplementation promotes prostate cancer progression in TRAMP mice, while melatonin attenuated glucose-induced tumor progression and prolonged the lifespan of tumor-bearing mice. This is the first time that a facilitated transport of melatonin is suggested. In fact, the important role of glucose transporters and glucose metabolism in cell fate might explain some of the diverse functions described for melatonin. PMID:25612238

  8. A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii.

    PubMed

    Montanini, Barbara; Moretto, Nadia; Soragni, Elisabetta; Percudani, Riccardo; Ottonello, Simone

    2002-06-01

    An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia. PMID:12051892

  9. Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes.

    PubMed Central

    Le Quesne, S A; Fairlamb, A H

    1996-01-01

    Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027]. PMID:8687391

  10. Sodium-Glucose Cotransporter Inhibitors: Effects on Renal and Intestinal Glucose Transport: From Bench to Bedside.

    PubMed

    Mudaliar, Sunder; Polidori, David; Zambrowicz, Brian; Henry, Robert R

    2015-12-01

    Type 2 diabetes is a chronic disease with disabling micro- and macrovascular complications that lead to excessive morbidity and premature mortality. It affects hundreds of millions of people and imposes an undue economic burden on populations across the world. Although insulin resistance and insulin secretory defects play a major role in the pathogenesis of hyperglycemia, several other metabolic defects contribute to the initiation/worsening of the diabetic state. Prominent among these is increased renal glucose reabsorption, which is maladaptive in patients with diabetes. Instead of an increase in renal glucose excretion, which could ameliorate hyperglycemia, there is an increase in renal glucose reabsorption, which helps sustain hyperglycemia in patients with diabetes. The sodium-glucose cotransporter (SGLT) 2 inhibitors are novel antidiabetes agents that inhibit renal glucose reabsorption and promote glucosuria, thereby leading to reductions in plasma glucose concentrations. In this article, we review the long journey from the discovery of the glucosuric agent phlorizin in the bark of the apple tree through the animal and human studies that led to the development of the current generation of SGLT2 inhibitors. PMID:26604280

  11. Rco-3, a Gene Involved in Glucose Transport and Conidiation in Neurospora Crassa

    PubMed Central

    Madi, L.; McBride, S. A.; Bailey, L. A.; Ebbole, D. J.

    1997-01-01

    Macroconidiation in Neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. Conidia formation is normally observed when the fungus is exposed to air. However, carbon limitation can induce conidiation in mycelia submerged in an aerated liquid medium. A mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been cloned. RCO3 exhibits sequence similarity to members of the sugar transporter gene superfamily, with greatest similarity to glucose transporters of yeast. Consistent with this structural similarity, we find that glucose transport activity is altered in the mutant. However, growth of the mutant in media containing alternate carbon sources does not suppress conidiation in submerged culture. The properties of the mutant suggest that RCO3 is required for expression of glucose transport activity, glucose regulation of gene expression, and general carbon repression of development. PMID:9178001

  12. A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

    PubMed Central

    Larsen, Mads B.; Fontana, Andréia C. K.; Magalhães, Lizandra G.; Rodrigues, Vanderlei; Mortensen, Ole V.

    2011-01-01

    The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from Schistosoma mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercaria stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. PMID:21251927

  13. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

    PubMed

    Fischer, Marcus; Hopkins, Adam P; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G; Hubbard, Roderick E; Thomas, Gavin H

    2015-11-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  14. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

    PubMed Central

    Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

    2015-01-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  15. Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters.

    PubMed

    Bucci, Diego; Galeati, Giovanna; Giaretta, Elisa; Tamanini, Carlo; Spinaci, Marcella

    2013-12-01

    Sex-sorting damages spermatozoa function, shortening their lifespan and fertility. This study used an immunofluorescence technique to investigate the effect of sex-sorting on the localization of glucose transporters (GLUTs) in boar spermatozoa. GLUTs are trans-membrane proteins responsible for glucose transport within cells. Distribution of GLUTs on sperm cells was similar in unsorted and sex-sorted semen, suggesting that the flow cytometric sex-sorting process did not affect the sperm energy apparatus. PMID:24287043

  16. Effect of insulin and glucocorticoids on glucose transporters in rat adipocytes

    SciTech Connect

    Carter-Su, C.; Okamoto, K.

    1987-04-01

    The ability of glucocorticoids to modify the effect of insulin on glucose (L-1-/sup 3/H(N))glucose and D-(/sup 14/C-U)glucose) transport was investigated in both intact isolated rat adipocytes and in membranes isolated from hormone-treated adipocytes. In intact adipocytes, dexamethasone, a potent synthetic glucocorticoid, inhibited insulin-stimulated 3-O-methylglucose transport at all concentrations of insulin tested. Insulin sensitivity, as well as the maximal response to insulin, was decreased by dexamethasone in the absence of a change in /sup 125/I insulin binding. The inhibition was observed regardless of which hormone acted first, was blocked by actinomycin D, and resulted from a decrease in V/sub max/ rather than an increase in K/sub t/ of transport. In plasma membranes isolated from insulin-treated adipocytes, glucose transport activity and the amount of glucose transporter covalently labeled with (/sup 3/H)cytochalasin B were increased in parallel in a dose-dependent fashion. The amount of labeled transporter in a low-density microsomal fraction (LDMF) was decreased in a reciprocal fashion. In contrast, addition of dexamethasone to insulin-stimulated cells caused decreases in both transport activity and amount of labeled transporter in the plasma membranes. This was accompanied by a small increase in the amount of (/sup 3/H)cytochalasin B incorporated into the glucose transporter in the LDMF. These results are consistent with both insulin and glucocorticoids altering the distribution of glucose transporters between the plasma membrane and LDMF, in opposite directions.

  17. Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione.

    PubMed Central

    Paulusma, C C; van Geer, M A; Evers, R; Heijn, M; Ottenhoff, R; Borst, P; Oude Elferink, R P

    1999-01-01

    The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114+/-24 versus 181+/-20 nmol/min per kg body weight). Madin-Darby canine kidney (MDCK) II cells stably expressing the human cMOAT protein displayed >10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141+/-6.1 pmol/min per mg of protein versus 13.2+/-1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki approximately 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5-10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover. PMID:10024515

  18. [Relationships of glucose transporter 4 with cognitive changes induced by high fat diet and glucose metabolism in hippocampus].

    PubMed

    Zhang, Yun-Li; Wang, Lin

    2016-06-25

    The hippocampus not only plays a role in appetite and energy balance, but also is particularly important in learning and memory. Figuring out the relationships of hippocampal glucose transporter 4 (GLUT4) with hippocampal glucose metabolism and hippocampus-dependent cognitive function is very important to clearly understand the pathophysiological basis of nutritional obesity and diabetes-related diseases, and treat obesity and cognitive dysfunction. Therefore, this study reviewed recent researches conducted on hippocampal GLUT4, hippocampal glucose metabolism, and hippocampus-dependent cognitive function. In this review, we mainly discussed: (1) The structure of GLUT4 and the distribution and function of GLUT4 in the hippocampus; (2) The translocation of GLUT4 in the hippocampus; (3) The relationships of the PI3K-Akt-GLUT4 signaling pathway with the high fat diet-induced changes of cognitive function and the glucose metabolism in the hippocampus; (4) The associations of the PI3K-Akt-GLUT4 signaling pathway with the diabetes-related cognitive dysfunction in the hippocampus; (5) The potential mechanisms of cognitive dysfunction induced by glucose metabolic disorder. PMID:27350206

  19. Early alterations in soleus GLUT-4, glucose transport, and glycogen in voluntary running rats

    NASA Technical Reports Server (NTRS)

    Henriksen, Erik J.; Halseth, Amy E.

    1994-01-01

    Voluntary wheel running (WR) by juvenile female rats was used as a noninterventional model of soleus muscle functional overload to study the regulation of insulin-stimulated glucose transport activity by the glucose transporter (GLUT-4 isoform) protein level and glycogen concentration. Soleus total protein content was significantly greater (+18%;P greater than 0.05) than in age-matched controls after 1 wk of WR, and this hypertrophic response continued in weeks 2-4 (+24-32%). GLUT-4 protein was 39% greater than in controls in 1-wk WR soleus, and this adaptation was accompanied by a similar increase in in vitro insulin-stimulated glucose transport activity(+29%). After 2 and 4 wk of WR, however, insulin-stimulated glucose transport activity had returned to control levels, despite a continued elevation (+25-28%) of GLUT-4 protein. At these two time points, glycogen concentration was significantly enhanced in WR soleus (+21-42%), which coincided with significant reductions in glycogen synthase activity ratios (-23 to-41%). These results indicate that, in this model of soleus muscle functional overload, the GLUT-4 protein level may initially regulate insulin-stimulated glucose transport activity in the absence of changes in other modifying factors. However,this regulation of glucose transport activity by GLUT-4 protein may be subsequently overridden by elevated glycogen concentration.

  20. Impaired stimulation of glucose transport in cardiac myocytes exposed to very low-density lipoproteins.

    PubMed

    Papageorgiou, I; Viglino, C; Brulhart-Meynet, M-C; James, R W; Lerch, R; Montessuit, C

    2016-07-01

    We recently observed that free fatty acids impair the stimulation of glucose transport into cardiomyocytes in response to either insulin or metabolic stress. In vivo, fatty acids for the myocardium are mostly obtained from triglyceride-rich lipoproteins (chylomicrons and Very Low-Density Lipoproteins). We therefore determined whether exposure of cardiac myocytes to VLDL resulted in impaired basal and stimulated glucose transport. Primary adult rat cardiac myocytes were chronically exposed to VLDL before glucose uptake was measured in response to insulin or metabolic stress, provoked by the mitochondrial ATP synthase inhibitor oligomycin. Exposure of cardiac myocytes to VLDL reduced both insulin-and oligomycin-stimulated glucose uptake. The reduction of glucose uptake was associated with a moderately reduced tyrosine phosphorylation of the insulin receptor. No reduction of the phosphorylation of the downstream effectors of insulin signaling Akt and AS160 was however observed. Similarly only a modest reduction of the activating phosphorylation of the AMP-activated kinase (AMPK) was observed in response to oligomycin. Similar to our previous observations with free fatty acids, inhibition of fatty acid oxidation restored oligomycin-stimulated glucose uptake. In conclusions, VLDL-derived fatty acids impair stimulated glucose transport in cardiac myocytes by a mechanism that seems to be mediated by a fatty acid oxidation intermediate. Thus, in the clinical context of the metabolic syndrome high VLDL may contribute to enhancement of ischemic injury by reduction of metabolic stress-stimulated glucose uptake. PMID:27052924

  1. Monte Carlo charge transport and photoemission from negative electron affinity GaAs photocathodes

    NASA Astrophysics Data System (ADS)

    Karkare, Siddharth; Dimitrov, Dimitre; Schaff, William; Cultrera, Luca; Bartnik, Adam; Liu, Xianghong; Sawyer, Eric; Esposito, Teresa; Bazarov, Ivan

    2013-03-01

    High quantum yield, low transverse energy spread, and prompt response time make GaAs activated to negative electron affinity an ideal candidate for a photocathode in high brightness photoinjectors. Even after decades of investigation, the exact mechanism of electron emission from GaAs is not well understood. Here, photoemission from such photocathodes is modeled using detailed Monte Carlo electron transport simulations. Simulations show a quantitative agreement with the experimental results for quantum efficiency, energy distributions of emitted electrons, and response time without the assumption of any ad hoc parameters. This agreement between simulation and experiment sheds light on the mechanism of electron emission and provides an opportunity to design novel semiconductor photocathodes with optimized performance.

  2. Effect of pycnogenol on glucose transport in mature 3T3-L1 adipocytes.

    PubMed

    Lee, Hee-Hyun; Kim, Kui-Jin; Lee, Ok-Hwan; Lee, Boo-Yong

    2010-08-01

    Pycnogenol, a procyanidins-enriched extract of Pinus maritima bark, possesses antidiabetic properties, which improves the altered parameters of glucose metabolism that are associated with type 2 diabetes mellitus (T2DM). Since the insulin-stimulated antidiabetic activities of natural bioactive compounds are mediated by GLUT4 via the phosphatidylinositol-3-kinase (PI3K) and/or p38 mitogen activated protein kinase (p38-MAPK) pathway, the effects of pycnogenol were examined on the molecular mechanism of glucose uptake by the glucose transport system. 3T3-L1 adipocytes were treated with various concentrations of pycnogenol, and glucose uptake was examined using a non-radioisotope enzymatic assay and by molecular events associated with the glucose transport system using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results show that pycnogenol increased glucose uptake in fully differentiated 3T3-L1 adipocytes and increased the relative abundance of both GLUT4 and Akt mRNAs through the PI3K pathway in a dose dependent manner. Furthermore, pycnogenol restored the PI3K antagonist-induced inhibition of glucose uptake in the presence of wartmannin, an inhibitor of the PI3K. Overall, these results indicate that pycnogenol may stimulate glucose uptake via the PI3K dependent tyrosine kinase pathways involving Akt. Further the results suggest that pycnogenol might be useful in maintaining blood glucose control. PMID:20658573

  3. Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum.

    PubMed Central

    Cohen, N R; Knecht, D A; Lodish, H F

    1996-01-01

    To facilitate expression of the rat GLUT 1 glucose transporter cDNA in Dictyostelium discoideum, we mutated the 5' end of the coding sequence such that the codons for the first ten amino acids conformed to preferred Dictyostelium codon usage. As determined by Western-blot analysis, a population of Dictyostelium transformed with the mutated cDNA expressed nonglycosylated GLUT 1 protein. Cell lines expressing GLUT 1 transport radiolabelled 2-deoxy-D-glucose at a rate 6-10 times that of cell lines transformed with vector alone. The initial rate of inward transport of 2-deoxy-D-glucose was stimulated several-fold by the presence of unlabelled glucose in the Dictyostelium cytoplasm, exemplifying the trans-activation of GLUT 1 transport characteristic of GLUT 1 present in erythrocyte membranes. The K(m) and Ki values for 2-deoxy-D-glucose, D-glucose, D-mannose and D-galactose were 3.7 mM, 2.6 mM, 11 mM and 30 mM respectively, similar to the values for GLUT 1 expressed in mammalian cells. L-Glucose and L-galactose, which are not transported by GLUT 1, do not inhibit uptake of 2-deoxy-D-glucose in Dictyostelium expressing GLUT 1. Thus, even though GLUT 1 expressed in Dictyostelium is not N-glycosylated, it transports hexoses normally; this is the first example of functional expression of a mammalian transport protein in this lower eukaryote. PMID:8645185

  4. Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport.

    PubMed

    Xia, Xiudong; Fan, Xiaorong; Wei, Jia; Feng, Huimin; Qu, Hongye; Xie, Dan; Miller, Anthony J; Xu, Guohua

    2015-01-01

    Plant proteins belonging to the NPF (formerly NRT1/PTR) family are well represented in every genome and function in transporting a wide variety of substrates. In this study, we showed that rice OsNPF2.4 is located in the plasma membrane and is expressed mainly in the epidermis, xylem parenchyma, and phloem companion cells. Functional analysis in oocytes showed that OsNPF2.4 is a pH-dependent, low-affinity NO₃⁻ transporter. Short-term (¹⁵NO₃⁻) influx rate, long-term NO₃⁻ acquisition by root, and upward transfer from root to shoot were decreased by disruption of OsNPF2.4 and increased by OsNPF2.4 overexpression under high NO₃⁻ supply. Moreover, the redistribution of NO₃⁻ in the mutants in comparison with the wild type from the oldest leaf to other organs, particularly to N-starved roots, was dramatically changed. Knockout of OsNPF2.4 decreased rice growth and potassium (K) concentration in xylem sap, root, culm, and sheath, but increased the shoot:root ratio of tissue K under higher NO₃⁻. We conclude that OsNPF2.4 functions in acquisition and long-distance transport of NO₃⁻ , and that altering its expression has an indirect effect on K recycling between the root and shoot. PMID:25332358

  5. Glucose transport and microvillus membrane physical properties along the crypt-villus axis of the rabbit.

    PubMed Central

    Meddings, J B; DeSouza, D; Goel, M; Thiesen, S

    1990-01-01

    Both transport function and microvillus membrane physical properties evolve as the enterocyte matures and migrates up the crypt-villus axis. We isolated enriched fractions of villus tip, mid-villus, and crypt enterocytes from which microvillus membrane vesicles were prepared. Using this material we characterized the alterations that occur in microvillus membrane fluidity as the rabbit enterocyte matures and correlated these with kinetic studies of glucose transport. With increasing maturity the microvillus membrane becomes more rigid due to both an increase in the cholesterol/phospholipid ratio and alterations in individual phospholipid subclasses. Maximal rates of glucose transport were greatest in microvillus membrane vesicles prepared from mature cells. However, the glucose concentration producing half-maximal rates of transport (Km) was significantly lower in crypt microvillus membrane vesicles, suggesting that a distinct glucose transporter existed in crypt enterocytes. This distinction disappeared when differences between membrane lipid environments were removed. By fluidizing villus-tip microvillus membrane vesicles, in vitro, to levels seen in the crypt microvillus membrane, we observed a reduction in the Km of this transport system. These data suggest that the kinetic characteristics of the sodium-dependent glucose transporter are dependent upon its local membrane environment. Images PMID:2318967

  6. Stability, metabolism and transport of D-Asp(OBzl)-Ala--a model prodrug with affinity for the oligopeptide transporter.

    PubMed

    Steffansen, B; Lepist, E I; Taub, M E; Larsen, B D; Frokjaer, S; Lennernäs, H

    1999-04-01

    The model prodrug D-Asp(OBzl)-Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of D-Asp(OBzl)-Ala and its in vitro metabolism in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of D-Asp(OBzl)-Ala in situ using the single-pass rat perfusion model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of D-Asp(OBzl)-Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids, the half-lives were >3 h and 2.3+/-0. 03 h, respectively. Using the rat single-pass perfusion technique, the effective jejunal permeability (Peff) of D-Asp(OBzl)-Ala was determined to be high (1.29+/-0.5.10-4 cm/s). The 32 times higher Peff value found in the perfusion model compared to Caco-2 cells is most likely due to a higher functional expression of the oligopeptide transporter. Rat jejuna Peff was reduced by approximately 50% in the presence of well known oligopeptide transporter substrates, such as Gly-Sar and cephalexin. It may be that D-Asp(OBzl)-Ala is primarily absorbed intact by the rat jejunal oligopeptide transporter, since the stability in the intestinal homogenate and fluids was rather high (t1/2>2.3 h). PMID:10072480

  7. Study on glucose transport in muscle cells by extracts from Mitragyna speciosa (Korth) and mitragynine.

    PubMed

    Purintrapiban, Juntipa; Keawpradub, Niwat; Kansenalak, Supaporn; Chittrakarn, Somsmorn; Janchawee, Benjamas; Sawangjaroen, Kitja

    2011-09-01

    The leaves of Mitragyna speciosa Korth (Rubiaceae) have been used in folk medicine for its unique medicinal properties. This study examined the water, methanolic and crude alkaloidal extracts from M. speciosa leaves and its major constituent mitragynine for the enhancement of glucose transport. Cellular uptake of radioactive 2-deoxyglucose was determined in rat L8 myotubes. Involving signalling pathway was determined with the specific inhibitors. Cell cytotoxicity was monitored by lactate dehydrogenase assay. Protein levels of glucose transporters (GLUTs) were measured by Western blotting. The results show that test samples significantly increased the rate of glucose uptake. The uptake was associated with increase in GLUT1 protein content. Co-incubation with insulin had no additional effect, but the cellular uptake was decreased by wortmannin and SB 203580, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK), respectively. It is concluded that the increased glucose transport activity of M. speciosa is associated with increases in activities of the key enzymes dependent to the insulin-stimulated glucose transport for its acute action, and increases in the GLUT1 content for its long-term effect. This study demonstrated the effect of M. speciosa in stimulating glucose transport in muscle cells, implicating the folkloric use of M. speciosa leaves for treating diabetes. PMID:18846471

  8. Impaired insulin-stimulated glucose transport in ATM-deficient mouse skeletal muscle.

    PubMed

    Ching, James Kain; Spears, Larry D; Armon, Jennifer L; Renth, Allyson L; Andrisse, Stanley; Collins, Roy L; Fisher, Jonathan S

    2013-06-01

    There are reports that ataxia telangiectasia mutated (ATM) plays a role in insulin-stimulated Akt phosphorylation, although this is not the case in some cell types. Because Akt plays a key role in insulin signaling, which leads to glucose transport in skeletal muscle, the predominant tissue in insulin-stimulated glucose disposal, we examined whether insulin-stimulated Akt phosphorylation and (or) glucose transport would be decreased in skeletal muscle of mice lacking functional ATM, compared with muscle from wild-type mice. We found that in vitro insulin-stimulated Akt phosphorylation was normal in soleus muscle from mice with 1 nonfunctional allele of ATM (ATM+/-) and from mice with 2 nonfunctional alleles (ATM-/-). However, insulin did not stimulate glucose transport or the phosphorylation of AS160 in ATM-/- soleus. ATM protein level was markedly higher in wild-type extensor digitorum longus (EDL) than in wild-type soleus. In EDL from ATM-/- mice, insulin did not stimulate glucose transport. However, in contrast to findings for soleus, insulin-stimulated Akt phosphorylation was blunted in ATM-/- EDL, concomitant with a tendency for insulin-stimulated phosphatidylinositol 3-kinase activity to be decreased. Together, the findings suggest that ATM plays a role in insulin-stimulated glucose transport at the level of AS160 in muscle comprised of slow and fast oxidative-glycolytic fibers (soleus) and at the level of Akt in muscle containing fast glycolytic fibers (EDL). PMID:23724874

  9. Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle.

    PubMed

    Henriksen, E J; Stump, C S; Trinh, T H; Beaty, S D

    1996-05-01

    Hindlimb weight bearing after a 3-day period of hindlimb suspension (reweighting) of juvenile rats results in a marked transient elevation in soleus glycogen concentration that cannot be explained on the basis of the activities of glycogen synthase and phosphorylase. We have hypothesized that enhanced glucose transport activity could underlie this response. We directly tested this hypothesis by assessing the response of insulin-dependent and insulin-independent glucose transport activity (in vitro 2-[1,2-3H]deoxy-D-glucose uptake) as well as glucose transporter (GLUT-4) protein levels during a 48-h reweighting period. After a net glycogen loss (from 29 +/- 2 to 16 +/- 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmol.mg-1.h-1 up to 18 h, reaching an apex of 38 +/- 1 nmol/mg. During this same reweighting period, insulin-independent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weight-bearing control values) and was associated with an elevated level of GLUT-4 protein and the specific activity of total hexokinase. The specific activity of citrate synthase was also increased. By 24 h of reweighting, although insulin-independent glucose transport activity and GLUT-4 protein remained elevated, glycogen accumulation had ceased, likely due to enhanced phosphorylase activity at this time point. These results are consistent with the interpretation that the glycogen supercompensation seen during reweighting of the rat soleus may be regulated in part by an enhanced glucose flux arising from an increase in insulin-independent glucose transport activity and hexokinase activity. PMID:8727537

  10. Enhanced Glucose Transport, but not Phosphorylation Capacity, Ameliorates Lipopolysaccharide-Induced Impairments in Insulin-Stimulated Muscle Glucose Uptake.

    PubMed

    Otero, Yolanda F; Mulligan, Kimberly X; Barnes, Tammy M; Ford, Eric A; Malabanan, Carlo M; Zong, Haihong; Pessin, Jeffrey E; Wasserman, David H; McGuinness, Owen P

    2016-06-01

    Lipopolysaccharide (LPS) is known to impair insulin-stimulated muscle glucose uptake (MGU). We determined if increased glucose transport (GLUT4) or phosphorylation capacity (hexokinase II; HKII) could overcome the impairment in MGU. We used mice that overexpressed GLUT4 (GLUT4) or HKII (HK) in skeletal muscle. Studies were performed in conscious, chronically catheterized (carotid artery and jugular vein) mice. Mice received an intravenous bolus of either LPS (10 μg/g body weight) or vehicle (VEH). After 5 h, a hyperinsulinemic-euglycemic clamp was performed. As MGU is also dependent on cardiovascular function that is negatively affected by LPS, cardiac function was assessed using echocardiography. LPS decreased whole body glucose disposal and MGU in wild-type (WT) and HK mice. In contrast, the decrease was attenuated in GLUT4 mice. Although membrane-associated GLUT4 was increased in VEH-treated GLUT4 mice, LPS impaired membrane-associated GLUT4 in GLUT4 mice to the same level as LPS-treated WT mice. This suggested that overexpression of GLUT4 had further benefits beyond preserving transport activity. In fact, GLUT4 overexpression attenuated the LPS-induced decrease in cardiac function. The maintenance of MGU in GLUT4 mice following LPS was accompanied by sustained anaerobic glycolytic flux as suggested by increased muscle Pdk4 expression, and elevated lactate availability. Thus, enhanced glucose transport, but not phosphorylation capacity, ameliorates LPS-induced impairments in MGU. This benefit is mediated by long-term adaptations to the overexpression of GLUT4 that sustain muscle anaerobic glycolytic flux and cardiac function in response to LPS. PMID:26682946

  11. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-01-01

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins. PMID:27525850

  12. Enhancement of binding kinetics on affinity substrates by laser point heating induced transport.

    PubMed

    Wang, Bu; Cheng, Xuanhong

    2016-03-01

    Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used the COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates is quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces the reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predictable binding patterns. This optothermal strategy holds promise to improve the time-response and sensitivity of biosensors and microarrays. PMID:26898559

  13. Glucose transport in human skeletal muscle cells in culture. Stimulation by insulin and metformin.

    PubMed Central

    Sarabia, V; Lam, L; Burdett, E; Leiter, L A; Klip, A

    1992-01-01

    Primary human muscle cell cultures were established and the regulation of glucose transport was investigated. Primary cultures were allowed to proceed to the stage of myotubes through fusion of myoblasts or were used for clonal selection based on fusion potential. In clonally selected cultures, hexose (2-deoxy-glucose) uptake into myotubes was linear within the time of study and inhibitable by cytochalasin B (IC50 = 400 nM). Cytochalasin B photolabeled a protein(s) of 45,000-50,000 D in a D-glucose-protectable manner, suggesting identity with the glucose transporters. In the myotube stage, the cells expressed both the GLUT1 and GLUT4 glucose transporter protein isoforms at an average molar ratio of 7:1. Preincubation in media of increasing glucose concentrations (range 5-25 mM) progressively decreased the rate of 2-deoxyglucose uptake. Insulin elevated 2-deoxyglucose uptake in a dose-dependent manner, with half maximal stimulation achieved at 3.5 nM. Insulin also stimulated the transport of the nonmetabolizable hexose 3-O-methylglucose, as well as the activity of glycogen synthase, responsible for nonoxidative glucose metabolism. The oral antihyperglycemic drug metformin stimulated the cytochalasin B-sensitive component of both 2-deoxyglucose and 3-O-methylglucose uptake. Maximal stimulation was observed at 8 h of exposure to 50 microM metformin, and this effect was not prevented by incubation with the protein-synthesis inhibitor cycloheximide. The relative effect of metformin was higher in cells incubated in 25 mM glucose than in 5 mM glucose, consistent with its selective action in hyperglycemic conditions in vivo. Metformin (50 microM for 24 h) was more effective than insulin (1 microM for 1 h) in stimulating hexose uptake and the hormone was effective on top of the stimulation caused by the biguanide, suggesting independent mechanisms of action. Images PMID:1401073

  14. Transport of galactose, glucose and their molecular analogues by Escherichia coli K12.

    PubMed Central

    Henderson, P J; Giddens, R A; Jones-Mortimer, M C

    1977-01-01

    1. Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM. 2. Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside. 3. Galactose, glucose and to a lesser extent fucose are substrates for both transport systems. 4. 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system. 5. The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity. Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter. Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism. 6. Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977), 161,1. PMID:15558

  15. Oat β-glucan depresses SGLT1- and GLUT2-mediated glucose transport in intestinal epithelial cells (IEC-6).

    PubMed

    Abbasi, Nazanin N; Purslow, Peter P; Tosh, Susan M; Bakovic, Marica

    2016-06-01

    Oat β-glucan consumption is linked to reduced risk factors associated with diabetes and obesity by lowering glycemic response and serum level of low-density lipoproteins. The purpose of this study was to identify the mechanism of action of oat β-glucan at the interface between the gut wall and the lumen responsible for attenuating glucose levels. We proposed that viscous oat β-glucan acts as a physical barrier to glucose uptake in normally absorptive gut epithelial cells IEC-6 by affecting the expression of intestinal glucose transporters. Concentration and time-dependent changes in glucose uptake were established by using a nonmetabolizable glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose. The effectiveness of nutrient transport in IEC-6 cells was shown by significant differences in glucose uptake and corresponding transporter expression. The expressions of glucose transporters sodium-glucose-linked transport protein 1 (SGLT1) and glucose transporter 2 (GLUT2) increased with time (0-60 minutes) and glucose levels (5-25 mmol/L). The suppression of glucose uptake and SGLT1 and GLUT2 expression by increasing concentrations (4-8 mg/mL) of oat β-glucan demonstrated a direct effect of the physical properties of oat β-glucan on glucose transport. These results affirmed oat β-glucan as a dietary agent for minimizing postprandial glucose and showed that modulating the activity of the key intestinal glucose transporters with oat β-glucan could be an effective way of lowering blood glucose levels in patients with diabetes. PMID:27188900

  16. Glucose metabolism transporters and epilepsy: only GLUT1 has an established role.

    PubMed

    Hildebrand, Michael S; Damiano, John A; Mullen, Saul A; Bellows, Susannah T; Oliver, Karen L; Dahl, Hans-Henrik M; Scheffer, Ingrid E; Berkovic, Samuel F

    2014-02-01

    The availability of glucose, and its glycolytic product lactate, for cerebral energy metabolism is regulated by specific brain transporters. Inadequate energy delivery leads to neurologic impairment. Haploinsufficiency of the glucose transporter GLUT1 causes a characteristic early onset encephalopathy, and has recently emerged as an important cause of a variety of childhood or later-onset generalized epilepsies and paroxysmal exercise-induced dyskinesia. We explored whether mutations in the genes encoding the other major glucose (GLUT3) or lactate (MCT1/2/3/4) transporters involved in cerebral energy metabolism also cause generalized epilepsies. A cohort of 119 cases with myoclonic astatic epilepsy or early onset absence epilepsy was screened for nucleotide variants in these five candidate genes. No epilepsy-causing mutations were identified, indicating that of the major energetic fuel transporters in the brain, only GLUT1 is clearly associated with generalized epilepsy. PMID:24483274

  17. Rapid kinetics of liver microsomal glucose-6-phosphatase. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity

    SciTech Connect

    Berteloot, A.; Vidal, H.; van de Werve, G. )

    1991-03-25

    Rapid kinetics of both glucose-6-P uptake and hydrolysis in fasted rat liver microsomes were investigated with a recently developed fast-sampling, rapid-filtration apparatus. Experiments were confronted with both the substrate transport and conformational models currently proposed for the glucose-6-phosphatase system. Accumulation in microsomes of 14C products from (U-14C)glucose-6-P followed biexponential kinetics. From the inside to outside product concentrations, it could be inferred that mostly glucose should accumulate inside the vesicles. While biexponential kinetics are compatible with the mathematical predictions of a simplified substrate transport model, the latter fails in explaining the burst in total glucose production over a similar time scale to that used for the uptake measurements. Since the initial rate of the burst phase in untreated microsomes exactly matched the steady-state rate of glucose production in detergent-treated vesicles, it can be definitely concluded that the substrate transport model does not describe adequately our results. While the conformational model accounts for both the burst of glucose production and the kinetics of glucose accumulation into the vesicles, it cannot explain the burst in 32Pi production from (32P)glucose-6-P measured under the same conditions. Since the amplitude of the observed bursts is not compatible with a presteady state in enzyme activity, we propose that a hysteretic transition best explains our results in both untreated and permeabilized microsomes, thus providing a new rationale to understand the molecular mechanism of the glucose-6-phosphatase system.

  18. Arabidopsis thaliana High-Affinity Phosphate Transporters Exhibit Multiple Levels of Posttranslational Regulation[C][W

    PubMed Central

    Bayle, Vincent; Arrighi, Jean-François; Creff, Audrey; Nespoulous, Claude; Vialaret, Jérôme; Rossignol, Michel; Gonzalez, Esperanza; Paz-Ares, Javier; Nussaume, Laurent

    2011-01-01

    In Arabidopsis thaliana, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high-affinity phosphate transporters. They are transcriptionally induced by phosphate starvation and require PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1) to exit the endoplasmic reticulum (ER), indicating intracellular traffic as an additional level of regulation of PHT1 activity. Our study revealed that PHF1 acts on PHT1, upstream of vesicle coat protein COPII formation, and that additional regulatory events occur during PHT1 trafficking and determine its ER exit and plasma membrane stability. Phosphoproteomic and mutagenesis analyses revealed modulation of PHT1;1 ER export by Ser-514 phosphorylation status. Confocal microscopy analysis of root tip cells showed that PHT1;1 is localized to the plasma membrane and is present in intracellular endocytic compartments. More precisely, PHT1;1 was localized to sorting endosomes associated with prevacuolar compartments. Kinetic analysis of PHT1;1 stability and targeting suggested a modulation of PHT1 internalization from the plasma membrane to the endosomes, followed by either subsequent recycling (in low Pi) or vacuolar degradation (in high Pi). For the latter condition, we identified a rapid mechanism that reduces the pool of PHT1 proteins present at the plasma membrane. This mechanism is regulated by the Pi concentration in the medium and appears to be independent of degradation mechanisms potentially regulated by the PHO2 ubiquitin conjugase. We propose a model for differential trafficking of PHT1 to the plasma membrane or vacuole as a function of phosphate concentration. PMID:21521698

  19. Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

    PubMed Central

    Daram, P; Brunner, S; Rausch, C; Steiner, C; Amrhein, N; Bucher, M

    1999-01-01

    An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function. PMID:10559441

  20. A mutation Ser213/Asn in the hexokinase 1 from Schizosaccharomyces pombe increases its affinity for glucose.

    PubMed

    Petit, T; Herrero, P; Gancedo, C

    1998-10-29

    Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed that Schizosaccharomyces pombe hexokinase 1 had a Ser residue in a place where all other kinases had an Asn. We changed an AGT codon to AAT to place an Asn in the Ser213 position. This mutation decreased Km for glucose from 9.4 mM to 1.6 mM and the ratio Vmax (Fructose)/Vmax (Glucose) from 5 to 2.5. Also the Km for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM. A mutation in the similar position of S. pombe hexokinase 2 (Asn196/Ser) increased the Km for glucose from 0.16 mM to 0.56 mM. Fermentation of glucose is not detectable in a S. pombe mutant with only hexokinase 1 activity but expression of the hxk1(S213/N) gene conferred ability to ferment the sugar. While the mutated hexokinase 1 partially mimicked S. cerevisiae hexokinase II in catabolite repression of invertase, the wild type one could not substitute for it. PMID:9790975

  1. Derivativation of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

    SciTech Connect

    Wadzinski, B.; Shanahan, M.; Ruoho, A.

    1987-05-01

    An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin (IAPS-Fsk), has been synthesized, purified, and characterized. The K/sub i/ for inhibition of 3-0-methylglucose transport by TAPS-Fsk in human erythrocytes was found to be 0.1 uM. The carrier-free radioiodinated label has been shown to be a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes with 1-10 nM (I-125)IAPS-Fsk and analysis by SDS-PAGE showed specific derivatization of a broad band with an apparent molecular weight of 40-70 kDa. Photoincorporation using 2 nM (I-125)IAPS-Fsk was protected with D-glucose, cytochalasin B, and forskolin. No protection was observed with L-glucose. Endo-B-galactosidase digestion and trypsinization of (I-125)IAPS-Fsk labelled erythrocytes reduced the specifically radiolabelled transporter to 40 kDa and 18 kDa respectively. (I-125)-IAPS-Fsk will be used to study the structural aspects of the glucose transporter.

  2. Functional characterization of the human facilitative glucose transporter 12 (GLUT12) by electrophysiological methods.

    PubMed

    Pujol-Giménez, Jonai; Pérez, Alejandra; Reyes, Alejandro M; Loo, Donald D F; Lostao, Maria Pilar

    2015-06-15

    GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results showed that GLUT12 is a facilitative sugar transporter with substrate selectivity: d-glucose ≥ α-methyl-d-glucopyranoside (α-MG) > 2-deoxy-d-glucose(2-DOG) > d-fructose = d-galactose. α-MG is a characteristic substrate of the Na(+)/glucose (SGLT) family and has not been shown to be a substrate of any of the GLUTs. In the absence of sugar, (22)Na(+) was transported through GLUT12 at a higher rate (40%) than noninjected oocytes, indicating that there is a Na(+) leak through GLUT12. Genistein, an inhibitor of GLUT1, also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) but not by the PKC activator phorbol-12-myristate-13-acetate (PMA). In high K(+) concentrations, glucose uptake was blocked. Addition of glucose to the external solution induced an inward current with a reversal potential of approximately -15 mV and was blocked by Cl(-) channel blockers, indicating the current was carried by Cl(-) ions. The sugar-activated Cl(-) currents were unaffected by genistein. In high external K(+) concentrations, sugar-activated Cl(-) currents were also blocked, indicating that GLUT12 activity is voltage dependent. Furthermore, glucose-induced current was increased by the PKA activator 8-Br-cAMP but not by the PKC activator PMA. These new features of GLUT12 are very different from those described for other GLUTs, indicating that GLUT12 must have a specific physiological role within glucose homeostasis, still to be discovered. PMID:25855082

  3. The effect of gonadotropin on glucose transport and apoptosis in rat ovary.

    PubMed

    Zhang, Cheng; Niu, Wanbao; Wang, Zhengpin; Wang, Xiaoxia; Xia, Guoliang

    2012-01-01

    Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1-4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake. PMID:22870326

  4. The Effect of Gonadotropin on Glucose Transport and Apoptosis in Rat Ovary

    PubMed Central

    Zhang, Cheng; Niu, Wanbao; Wang, Zhengpin; Wang, Xiaoxia; Xia, Guoliang

    2012-01-01

    Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1–4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake. PMID:22870326

  5. Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation.

    PubMed

    Mark, R J; Pang, Z; Geddes, J W; Uchida, K; Mattson, M P

    1997-02-01

    A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD. PMID:8994059

  6. Estimation of kinetic parameters for glucose transport in human brain cortex

    SciTech Connect

    Vyska, K.; Machulla, H.J.; Mehdorn, H.M.; Notohamiprodjo, G.; Knapp, W.H.; Feinendegen, L.E.

    1985-05-01

    3-O-C-11-methyl-D-glucose (CMG), F-18-3-deoxy-3-fluoro D-glucose (3FDG), and dynamic positron-emission-tomography (dPET) were used to measure the rate constants for glucose transport across the blood brain barrier (BBB) in human cortex. The assay takes advantage of CMG or 3FDG being practically not metabolized in brain and being transported back from the tissue into the circulation. The simultaneous registration of tracer concentration in blood and tissue by dPET at 1 min intervals for 40 min yields time activity curves, which permit the in vivo determination of the rate constants for CMG or 3FDG transport across the BBB. In the present study, 4 healthy volunteers and 10 patients suffering from a single-sided ischemic brain disease were examined. In all cases the CMG/3FDG measurements were carried out at two different glucose plasma concentrations i.e. at normoglycemia and hyperglycemia after i.v. application of 10 g glucose. The determination of glucose plasma concentration was performed just before and immediately after the CMG/3FDG study. Using these data and a new mathematical model the Michaelis-Menten constant (K/sub M/) and maximal velocity (V/sub M/) for CMG, 3FDG and glucose transport across the BBB in normal and non-affected human cortex were determined. K/sub M CMG/ was 7.21 ..mu..mol/g; K/sub M 3FDG/ was 3.93 ..mu..mol/g and K/sub M gluc/ was 6.31 ..mu..mol/g. V/sub M/ was found in all cases to be 2.1 ..mu..mol/min g. The data obtained suggest that the CMG/3FDG method might provide a powerful tool for studying the mechanisms involved in the pathological alterations of glucose carrier system.

  7. Molecular physiology of cellular glucose transport - a potential area for clinical studies in diabetes mellitus.

    PubMed

    Tatoń, Jan; Piatkiewicz, Paweł; Czech, Anna

    2010-01-01

    The normalization of cellular glucose assimilation is the basic aim of metabolic therapy in type 2 diabetes mellitus (T2DM). It requires parallel changes in the process of cellular glucose transport (CGT). This review presents the pathophysiological and clinical outlines of CGT. Sequentially, the advances in the mechanisms and classification of CGT and their physiological and molecular base are described. The role of CGT pathogenetic significance in diabetes mellitus is stressed. Finally, the opinion is expressed that the CGT study is a potentially important approach to clinical interpretation of glucose metabolism disturbances and their pharmacotherapy. PMID:20602306

  8. Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose disposal

    PubMed Central

    Jarmin, S.; Eilers, W.; Elashry, M.; Andersen, D. K.; Dickson, G.; Foster, K.

    2014-01-01

    Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes. PMID:24473441

  9. Cholinergic activation of the murine trachealis muscle via non-vesicular acetylcholine release involving low-affinity choline transporters.

    PubMed

    Nassenstein, Christina; Wiegand, Silke; Lips, Katrin S; Li, Guanfeng; Klein, Jochen; Kummer, Wolfgang

    2015-11-01

    In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release. Classical enzyme histochemistry demonstrated acetylcholinesterase (AChE) activity in nerve fibers in the muscle and butyrylcholinesterase (BChE) activity in the smooth muscle cells. Acute inhibition of both esterases by eserine significantly raised tracheal tone which was fully sensitive to atropine. This effect was reduced, but not abolished, in AChE, but not in BChE gene-deficient mice. The eserine-induced increase in tracheal tone was unaffected by vesamicol (10(-5)M), an inhibitor of the vesicular acetylcholine transporter, and by corticosterone (10(-4)M), an inhibitor of organic cation transporters. Hemicholinium-3, in low concentrations an inhibitor of the high-affinity choline transporter-1 (CHT1), completely abrogated the eserine effects when applied in high concentrations (10(-4)M) pointing towards an involvement of low-affinity choline transporters. To evaluate the cellular sources of non-quantal ACh release in the trachea, expression of low-affinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR analysis. Even though these transporters were largely abundant in the epithelium, denudation of airway epithelial cells had no effect on eserine-induced tracheal contraction, indicating a non-quantal release of ACh from non-epithelial sources in the airways. These data provide evidence for an epithelium-independent non-vesicular, non-quantal ACh release in the mouse trachea involving low-affinity choline transporters. PMID:26278668

  10. Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

    PubMed Central

    Klip, A; Ramlal, T; Douen, A G; Bilan, P J; Skorecki, K L

    1988-01-01

    The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate

  11. Evidence for a Novel Affinity Mechanism of Motor-assisted Transport Along MicrotubulesV⃞

    PubMed Central

    Wada, Yuuko; Hamasaki, Toshikazu; Satir, Peter

    2000-01-01

    In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (−) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that β tubulin is exposed at the (+) end of the MT. PMID:10637299

  12. Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.

    PubMed Central

    Bringaud, F; Baltz, T

    1993-01-01

    A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2. Images PMID:8423781

  13. Directed evolution of xylose specific transporters to facilitate glucose-xylose co-utilization.

    PubMed

    Wang, Meng; Yu, Chenzhao; Zhao, Huimin

    2016-03-01

    A highly active xylose specific transporter without glucose inhibition is highly desirable in cost-effective production of biofuels from lignocellulosic biomass. However, currently available xylose specific transporters suffer from low overall activity and most are inhibited by glucose. In this study, we applied a directed evolution strategy to engineer the xylose specific transporter AN25 from Neurospora crassa with improved xylose transportation capacity. After four rounds of directed evolution using two different strategies, we obtained an AN25 mutant AN25-R4.18 with 43-fold improvement in terms of xylose transportation capacity while maintaining its high xylose specificity. In addition, glucose inhibition was almost completely eliminated in the final evolved mutant. We demonstrated that improved xylose transportation of AN25 mutants in the exponential growth phase led to significant improvement of xylose consumption in high cell-density fermentation. Finally, we showed that AN25 mutant AN25-R4.18 can enable relatively efficient glucose-xylose co-utilization in high concentrations of mixed sugars. PMID:26284760

  14. Promotion of Streptococcus mutans glucose transport by human whole saliva and parotid fluid.

    PubMed Central

    Germaine, G R; Tellefson, L M

    1985-01-01

    Human saliva and parotid fluid have two effects on glucose uptake by Streptococcus mutans: a reduction in the overall rate of uptake, and the promotion of a biphasic mode of uptake. The former effect had been previously shown to result from lactoperoxidase-mediated inhibition of transport or metabolism or both. The objective of the present study was to uncover the basis of the second effect. Biphasic glucose uptake consisted of a rapid phase of low capacity and short duration (approximately 10 to 15 s) followed by a slower phase of high capacity and long duration (several minutes). The slow phase is typical of cells not exposed to the secretions (control cells). S. mutans BHT cells pretreated with as little as 10 microM glucose for 10 min at 37 degrees C, followed by its removal, subsequently exhibit biphasic glucose uptake typical of saliva- or parotid fluid-treated cells. Since pretreatment of the organism with glucose, whole saliva supernatant, or parotid fluid supported subsequent transport of the nonmetabolized glucose analog, 2-deoxyglucose, we concluded that pretreatments established a relatively stable pool of glycolytic intermediates (i.e., a phosphoenolpyruvate potential). Thin-layer chromatographic analysis of extracts from [14C]glucose-pretreated cells confirmed the presence of a stable pool of triose phosphates. Dialysis experiments indicated that high-molecular-weight substrates in the secretions were readily utilized by the organism to establish a phosphoenolpyruvate potential, especially when the lactoperoxidase system was rendered inactive. A survey of several carbohydrate constituents of salivary glycoproteins revealed that mannose, galactose, and N-acetylglucosamine, in addition to glucose, established phosphoenolpyruvate potentials in the organisms. Inactive substances included, among others, N-acetylgalactosamine and N-acetylneuraminic acid. In a survey of selected amino acids, arginine alone promoted 2-deoxyglucose accumulation by the organism

  15. Promotion of Streptococcus mutans glucose transport by human whole saliva and parotid fluid.

    PubMed

    Germaine, G R; Tellefson, L M

    1985-04-01

    Human saliva and parotid fluid have two effects on glucose uptake by Streptococcus mutans: a reduction in the overall rate of uptake, and the promotion of a biphasic mode of uptake. The former effect had been previously shown to result from lactoperoxidase-mediated inhibition of transport or metabolism or both. The objective of the present study was to uncover the basis of the second effect. Biphasic glucose uptake consisted of a rapid phase of low capacity and short duration (approximately 10 to 15 s) followed by a slower phase of high capacity and long duration (several minutes). The slow phase is typical of cells not exposed to the secretions (control cells). S. mutans BHT cells pretreated with as little as 10 microM glucose for 10 min at 37 degrees C, followed by its removal, subsequently exhibit biphasic glucose uptake typical of saliva- or parotid fluid-treated cells. Since pretreatment of the organism with glucose, whole saliva supernatant, or parotid fluid supported subsequent transport of the nonmetabolized glucose analog, 2-deoxyglucose, we concluded that pretreatments established a relatively stable pool of glycolytic intermediates (i.e., a phosphoenolpyruvate potential). Thin-layer chromatographic analysis of extracts from [14C]glucose-pretreated cells confirmed the presence of a stable pool of triose phosphates. Dialysis experiments indicated that high-molecular-weight substrates in the secretions were readily utilized by the organism to establish a phosphoenolpyruvate potential, especially when the lactoperoxidase system was rendered inactive. A survey of several carbohydrate constituents of salivary glycoproteins revealed that mannose, galactose, and N-acetylglucosamine, in addition to glucose, established phosphoenolpyruvate potentials in the organisms. Inactive substances included, among others, N-acetylgalactosamine and N-acetylneuraminic acid. In a survey of selected amino acids, arginine alone promoted 2-deoxyglucose accumulation by the organism

  16. Effect of insulin-like factors on glucose transport activity in unweighted rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Henriksen, Erik J.; Ritter, Leslie S.

    1993-01-01

    The effect of 3 or 6 days of unweighting on glucose transport activity, as assessed by 2-deoxyglucose uptake, in soleus strips stimulated by maximally effective concentrations of insulin, IGF-I, vanadate, or phospholipase C (PLC) is examined. Progressively increased responses to maximally effective doses of insulin or insulin-like growth factor were observed after 3 and 6 days of unweighting compared with weight matched control strips. Enhanced maximal responses to vanadate (6 days only) and PLC (3 and 6 days) were also observed. The data provide support for the existance of postreceptor binding mechanisms for the increased action of insulin on the glucose transport system in unweighted rat skeletal muscle.

  17. Glucose transporter type 1 deficiency syndrome effectively treated with modified Atkins diet.

    PubMed

    Haberlandt, Edda; Karall, Daniela; Jud, Veronika; Baumgartner, Sara Sigl; Zotter, Sibylle; Rostasy, Kevin; Baumann, Matthias; Scholl-Buergi, Sabine

    2014-04-01

    This is a report on the successful treatment of a 6-year-old girl with genetically proven glucose transporter type 1 deficiency syndrome (GLUT1-DS) with modified Atkins diet (MAD). GLUT1-DS is an inborn disorder of glucose transport across the blood-brain barrier, which leads to energy deficiency of the brain with a broad spectrum of neurological symptoms including therapy-resistant epilepsy. Usually classical ketogenic diet (KD) is the standard treatment for patients with GLUT1-DS. Treatment with MAD, a variant of KD, for an observation period of 17 months resulted in improvement of seizures, alertness, cognitive abilities, and electroencephalography in this patient. PMID:23888468

  18. Evolutionary ancestry and novel functions of the mammalian glucose transporter (GLUT) family

    PubMed Central

    2010-01-01

    Background In general, sugar porters function by proton-coupled symport or facilitative transport modes. Symporters, coupled to electrochemical energy, transport nutrients against a substrate gradient. Facilitative carriers transport sugars along a concentration gradient, thus transport is dependent upon extracellular nutrient levels. Across bacteria, fungi, unicellular non-vertebrates and plants, proton-coupled hexose symport is a crucial process supplying energy under conditions of nutrient flux. In mammals it has been assumed that evolution of whole body regulatory mechanisms would eliminate this need. To determine whether any isoforms bearing this function might be conserved in mammals, we investigated the relationship between the transporters of animals and the proton-coupled hexose symporters found in other species. Results We took a comparative genomic approach and have performed the first comprehensive and statistically supported phylogenetic analysis of all mammalian glucose transporter (GLUT) isoforms. Our data reveals the mammalian GLUT proteins segregate into five distinct classes. This evolutionary ancestry gives insight to structure, function and transport mechanisms within the groups. Combined with biological assays, we present novel evidence that, in response to changing nutrient availability and environmental pH, proton-coupled, active glucose symport function is maintained in mammalian cells. Conclusions The analyses show the ancestry, evolutionary conservation and biological importance of the GLUT classes. These findings significantly extend our understanding of the evolution of mammalian glucose transport systems. They also reveal that mammals may have conserved an adaptive response to nutrient demand that would have important physiological implications to cell survival and growth. PMID:20487568

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

    PubMed

    Kikuta, Shingo; Nakamura, Yuki; Hattori, Makoto; Sato, Ryoichi; Kikawada, Takahiro; Noda, Hiroaki

    2015-09-01

    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. PMID:26226652

  20. Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

    NASA Astrophysics Data System (ADS)

    Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

    1990-09-01

    Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

  1. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    PubMed

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake. PMID:18258590

  2. SGLT1 sugar transporter/sensor is required for post-oral glucose appetition.

    PubMed

    Sclafani, Anthony; Koepsell, Hermann; Ackroff, Karen

    2016-04-01

    Recent findings suggest that the intestinal sodium-glucose transporter 1 (SGLT1) glucose transporter and sensor mediates, in part, the appetite-stimulation actions of intragastric (IG) glucose and nonmetabolizable α-methyl-d-glucopyranoside (MDG) infusions in mice. Here, we investigated the role of SGLT1 in sugar conditioning using SGLT1 knockout (KO) and C57BL/6J wild-type (WT) mice. An initial experiment revealed that both KO and WT mice maintained on a very low-carbohydrate diet display normal preferences for saccharin, which was used in the flavored conditioned stimulus (CS) solutions. In experiment 2, mice were trained to drink one flavored solution (CS+) paired with an IG MDG infusion and a different flavored solution (CS-) paired with IG water infusion. In contrast to WT mice, KO mice decreased rather than increased the intake of the CS+ during training and failed to prefer the CS+ over the CS- in a choice test. In experiment 3, the KO mice also decreased their intake of a CS+ paired with IG glucose and avoided the CS+ in a choice test, unlike WT mice, which preferred the CS+ to CS-. In experiment 4, KO mice, like WT mice preferred a glucose + saccharin solution to a saccharin solution. These findings support the involvement of SGLT1 in post-oral glucose and MDG conditioning. The results also indicate that sugar malabsorption in KO mice has inhibitory effects on sugar intake but does not block their natural preference for sweet taste. PMID:26791832

  3. Glucose Transport in Cultured Animal Cells: An Exercise for the Undergraduate Cell Biology Laboratory

    PubMed Central

    Ledbetter, Mary Lee S; Lippert, Malcolm J

    2002-01-01

    Membrane transport is a fundamental concept that undergraduate students of cell biology understand better with laboratory experience. Formal teaching exercises commonly used to illustrate this concept are unbiological, qualitative, or intricate and time consuming to prepare. We have developed an exercise that uses uptake of radiolabeled nutrient analogues by attachment-dependent animal cells cultured on multiwell trays. This system can readily be manipulated within a typical 3-h laboratory period to yield reproducible, biologically relevant, quantitative data regarding key aspects of membrane transport. Each 24-well tray of cultures allows a group of two to four students to compare eight conditions in triplicate. If different groups of students test different conditions or different types of cells, data can be shared for an even broader experience. The exercise is also readily adaptable for open-ended student projects. Here we illustrate the exercise measuring uptake of the nonmetabolizable glucose analogue [3H]-2-deoxy-d-glucose. Students successfully tested the effects of competing sugars, putative inhibitors of the GLUT1 transporter, and changes in cell physiology that might be expected to affect glucose transport in epithelial cells and fibroblasts. In this exercise students find the nutritional and medical implications of glucose transport and its regulation intriguing. They also learn to handle radioisotopes and cultured cells. PMID:12459793

  4. Sodium-glucose co-transporter 2 (SGLT2) inhibitors: a growing class of antidiabetic agents

    PubMed Central

    Vivian, Eva M

    2014-01-01

    Although several treatment options are available to reduce hyperglycemia, only about half of individuals with diagnosed diabetes mellitus (DM) achieve recommended glycemic targets. New agents that reduce blood glucose concentrations by novel mechanisms and have acceptable safety profiles are needed to improve glycemic control and reduce the complications associated with type 2 diabetes mellitus (T2DM). The renal sodium-glucose co-transporter 2 (SGLT2) is responsible for reabsorption of most of the glucose filtered by the kidney. Inhibitors of SGLT2 lower blood glucose independent of the secretion and action of insulin by inhibiting renal reabsorption of glucose, thereby promoting the increased urinary excretion of excess glucose. Canagliflozin, dapagliflozin, and empagliflozin are SGLT2 inhibitors approved as treatments for T2DM in the United States, Europe, and other countries. Canagliflozin, dapagliflozin, and empagliflozin increase renal excretion of glucose and improve glycemic parameters in patients with T2DM when used as monotherapy or in combination with other antihyperglycemic agents. Treatment with SGLT2 inhibitors is associated with weight reduction, lowered blood pressure, and a low intrinsic propensity to cause hypoglycemia. Overall, canagliflozin, dapagliflozin, and empagliflozin are well tolerated. Cases of genital infections and, in some studies, urinary tract infections have been more frequent in canagliflozin-, dapagliflozin-, and empagliflozin-treated patients compared with those receiving placebo. Evidence from clinical trials suggests that SGLT2 inhibitors are a promising new treatment option for T2DM. PMID:25598831

  5. Effect of polyphenols on glucose and lactate transport by breast cancer cells.

    PubMed

    Martel, F; Guedes, M; Keating, E

    2016-05-01

    One of the cancer molecular hallmarks is a deviant energetic metabolism, known as the Warburg effect, whereby the rate of glucose uptake is significantly increased and a high rate of glycolysis and lactic acid production occurs even when oxygen is present-"aerobic lactatogenesis". Accordingly, GLUT1 and MCT1, which are the main glucose and lactate transporters in cancer cells, respectively, have been proposed as oncogenes and are currently seen as potential therapeutic targets in cancer treatment. Polyphenols, commonly contained in fruits and vegetables, have long been associated with a protective role against cancer. Generally considered as nontoxic, dietary polyphenols are considered ideal chemopreventive and possibly chemotherapeutic agents. Several mechanisms of action of polyphenols in breast cancer cells have been proposed including modulation of intracellular signaling, induction of apoptosis through redox regulation or modulation of epigenetic alterations. Additionally, in vitro studies have shown that several polyphenols act as specific inhibitors of glucose transport in breast cancer cell lines and an association between their anticarcinogenic effect and inhibition of glucose cellular uptake has been described. Also, some polyphenols were found to inhibit lactate transport. Importantly, some polyphenols behave as inhibitors of both glucose and lactate cellular uptake by breast cancer cells and these compounds are thus very interesting in the context of a chemopreventive effect, because they deplete breast cancer cells of their two most important energy suppliers. So, the antimetabolic effect of polyphenols should be regarded as a mechanism of action contributing to their chemopreventive/chemotherapeutic potential in relation to breast cancer. PMID:27097608

  6. Isoproterenol stimulates phosphorylation of the insulin-regulatable glucose transporter in rat adipocytes

    SciTech Connect

    James, D.E.; Hiken, J.; Lawrence, J.C. Jr. )

    1989-11-01

    The authors have examined the acute effect of insulin and isoproterenol on the phosphorylation state of the insulin-regulatable glucose transporter (IRGT) in rat adipocytes. The IRGT was immunoprecipitated from either detergent-solubilized whole-cell homogenates or subcellular fraction of {sup 32}P-labeled fat cells and subjected to sodium dodecyl sulfate/polyarcylamide gel electrophoresis. The {sup 32}P-labeled IRGT was detected by autoradiography as a species of apparent M{sub r} 46,000. Insulin stimulated translocation of the IRGT from low-density microsomes to the plasma membrane but did not affect phosphorylation of the transporter in either fraction. Isoproterenol inhibited insulin-stimulated glucose transport by 40% but was without effect on the subcellar distribution of the transporter in either the presence or absence of insulin. Isoproterenol stimulated phosphorylation of the IRGT 2-fold. Incubating cells with dibutyryl-cAMP and 8-bromo-cAMP also stimulated phosphorylation 2-fold, and the transporter was phosphorylated in vitro when IRGT-enriched vesicles were incubated with cAMP-dependent protein kinase and ({gamma}-{sup 32}P)ATP. These results suggest that isoproterenol stimulates phosphorylation of the IRGT via a cAMP-dependent pathway and that phosphorylation of the transporter may modulate its ability to transport glucose.

  7. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

    PubMed

    Fishwick, Katherine J; Rylett, R Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  8. Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

    PubMed

    Bauché, Stéphanie; O'Regan, Seana; Azuma, Yoshiteru; Laffargue, Fanny; McMacken, Grace; Sternberg, Damien; Brochier, Guy; Buon, Céline; Bouzidi, Nassima; Topf, Ana; Lacène, Emmanuelle; Remerand, Ganaelle; Beaufrere, Anne-Marie; Pebrel-Richard, Céline; Thevenon, Julien; El Chehadeh-Djebbar, Salima; Faivre, Laurence; Duffourd, Yannis; Ricci, Federica; Mongini, Tiziana; Fiorillo, Chiara; Astrea, Guja; Burloiu, Carmen Magdalena; Butoianu, Niculina; Sandu, Carmen; Servais, Laurent; Bonne, Gisèle; Nelson, Isabelle; Desguerre, Isabelle; Nougues, Marie-Christine; Bœuf, Benoit; Romero, Norma; Laporte, Jocelyn; Boland, Anne; Lechner, Doris; Deleuze, Jean-François; Fontaine, Bertrand; Strochlic, Laure; Lochmuller, Hanns; Eymard, Bruno; Mayer, Michèle; Nicole, Sophie

    2016-09-01

    The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse. PMID:27569547

  9. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  10. Interaction of Maltose Transport with the Transport of Glucose and Galactosides

    PubMed Central

    McKinstry, Grenetta; Koch, Arthur L.

    1972-01-01

    In cells of Escherichia coli possessing both maltose and galactoside permease, fluxes via one permease are independent of the substrate for the other permease. However, both fluxes are partially inhibited by glucose or α-methyl glucoside at low concentrations in cells grown on glucose. Neither maltose nor galactosides have an inhibitory effect on glucose permease function. These observations are consistent with the hypothesis that the number of glucose permease systems on the cell surface of such cells is much larger than the number for maltose or galactosides. PMID:4550675

  11. The Role of Sodium-Dependent Glucose Transporter 1 and Glucose Transporter 2 in the Absorption of Cyanidin-3-O-β-Glucoside in Caco-2 Cells

    PubMed Central

    Zou, Tang-Bin; Feng, Dan; Song, Gang; Li, Hua-Wen; Tang, Huan-Wen; Ling, Wen-Hua

    2014-01-01

    Anthocyanins have multiple biological activities of benefit to human health. While a few studies have been conducted to evaluate the bioavailability of anthocyanins, the mechanisms of their absorption mechanism remain ill-defined. In the present study, we investigated the absorption mechanism of cyanidin-3-O-β-glucoside (Cy-3-G) in human intestinal epithelial (Caco-2) cells. Cy-3-G transport was assessed by measuring the absorptive and efflux direction. Inhibition studies were conducted using the pharmacological agents, phloridzin, an inhibitor of sodium-dependent glucose transporter 1 (SGLT1), or phloretin, an inhibitor of glucose transporter 2 (GLUT2). The results showed that phloridzin and phloretin significantly inhibited the absorption of Cy-3-G. In addition, Caco-2 cells transfected with small interfering RNA (siRNA) specific for SGLT1 or GLUT2 showed significantly decreased Cy-3-G absorption. These siRNA transfected cells also showed a significantly decreased rate of transport of Cy-3-G compared with the control group. These findings suggest that Cy-3-G absorption is dependent on the activities of SGLT1 and GLUT2 in the small intestine and that SGLT1 and GLUT2 could be a limiting step for the bioavailability of Cy-3-G. PMID:25314643

  12. The role of sodium-dependent glucose transporter 1 and glucose transporter 2 in the absorption of cyanidin-3-o-β-glucoside in Caco-2 cells.

    PubMed

    Zou, Tang-Bin; Feng, Dan; Song, Gang; Li, Hua-Wen; Tang, Huan-Wen; Ling, Wen-Hua

    2014-10-01

    Anthocyanins have multiple biological activities of benefit to human health. While a few studies have been conducted to evaluate the bioavailability of anthocyanins, the mechanisms of their absorption mechanism remain ill-defined. In the present study, we investigated the absorption mechanism of cyanidin-3-O-β-glucoside (Cy-3-G) in human intestinal epithelial (Caco-2) cells. Cy-3-G transport was assessed by measuring the absorptive and efflux direction. Inhibition studies were conducted using the pharmacological agents, phloridzin, an inhibitor of sodium-dependent glucose transporter 1 (SGLT1), or phloretin, an inhibitor of glucose transporter 2 (GLUT2). The results showed that phloridzin and phloretin significantly inhibited the absorption of Cy-3-G. In addition, Caco-2 cells transfected with small interfering RNA (siRNA) specific for SGLT1 or GLUT2 showed significantly decreased Cy-3-G absorption. These siRNA transfected cells also showed a significantly decreased rate of transport of Cy-3-G compared with the control group. These findings suggest that Cy-3-G absorption is dependent on the activities of SGLT1 and GLUT2 in the small intestine and that SGLT1 and GLUT2 could be a limiting step for the bioavailability of Cy-3-G. PMID:25314643

  13. Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

    PubMed

    Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

    2012-10-01

    The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function. PMID:22858636

  14. Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

    PubMed Central

    Baumann, Marc U.; Schneider, Henning; Malek, Antoine; Palta, Vidya; Surbek, Daniel V.; Sager, Ruth; Zamudio, Stacy; Illsley, Nicholas P.

    2014-01-01

    Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth. PMID:25157747

  15. Effect of denervation on the expression of two glucose transporter isoforms in rat hindlimb muscle.

    PubMed Central

    Block, N E; Menick, D R; Robinson, K A; Buse, M G

    1991-01-01

    Denervation rapidly (within 24 h) induces insulin resistance of several insulin-responsive pathways in skeletal muscle, including glucose transport; resistance is usually maximal by 3 d. We examined the effect of denervation on the expression of two glucose transporter isoforms (GLUT-1 and GLUT-4) in rat hindlimb muscle; GLUT-4 is the predominant species in muscle. 1 d postdenervation, GLUT-1 and GLUT-4 mRNA and protein concentrations were unchanged. 3 and 7 d postdenervation, GLUT-4 mRNA and protein (per microgram DNA) were decreased by 50%. The minor isoform, GLUT-1 mRNA increased by approximately 500 and approximately 100%, respectively, on days 3 and 7 while GLUT-1 protein increased by approximately 60 and approximately 100%. The data suggest that the insulin resistance of glucose transport early after denervation does not reflect a decrease in total glucose transporter number; however, decreased GLUT-4 expression may contribute to its increased severity after 3 d. Parallel decreases in GLUT-4 mRNA and GLUT-4 protein postdenervation are consistent with pretranslational regulation; GLUT-1 expression may be regulated pre- and posttranslationally. The cell type(s) which overexpress GLUT-1 postdenervation need to be identified. Nervous stimuli and/or contractile activity may modulate the expression of GLUT-1 and GLUT-4 in skeletal muscle tissue. Images PMID:1939643

  16. Pathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-function.

    PubMed

    Raja, Mobeen; Kinne, Rolf K H

    2015-01-01

    Two families of glucose transporter - the Na(+)-dependent glucose cotransporter-1 (SGLT family) and the facilitated diffusion glucose transporter family (GLUT family) - play a crucial role in the translocation of glucose across the epithelial cell membrane. How genetic mutations cause life-threatening diseases like GLUT1-deficiency syndrome (GLUT1-DS) is not well understood. In this review, we have combined previous functional data with our in silico analyses of the bacterial homologue of GLUT members, XylE (an outward-facing, partly occluded conformation) and previously proposed GLUT1 homology model (an inward-facing conformation). A variety of native and mutant side chain interactions were modeled to highlight the potential roles of mutations in destabilizing protein-protein interaction hence triggering structural and functional defects. This study sets the stage for future studies of the structural properties that mediate GLUT1 dysfunction and further suggests that both SGLT and GLUT families share conserved domains that stabilize the transporter structure/function via a similar mechanism. PMID:25863194

  17. Low-affinity transport of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.

    PubMed

    Tagawa, Maki; Yumoto, Ryoko; Oda, Keisuke; Nagai, Junya; Takano, Mikihisa

    2008-01-01

    FITC-albumin uptake by cultured alveolar type II epithelial cells, RLE-6TN, is mediated by high- and low-affinity transport systems. In this study, characteristics of the low-affinity transport system were evaluated. The uptake of FITC-albumin was time and temperature dependent and was inhibited by metabolic inhibitors and bafilomycin A1. Confocal laser scanning microscopic analysis showed punctate localization of the fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by caveolae-mediated endocytosis inhibitors such as nystatin, but was inhibited by clathrin-mediated endocytosis inhibitors such as phenylarsine oxide. The uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. In addition, macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride inhibited the uptake. These results indicate that the low-affinity transport of FITC-albumin in RLE-6TN cells is at least in part mediated by clathrin-mediated endocytosis, but not by caveolae-mediated endocytosis. Possible involvement of macropinocytosis was also suggested. PMID:18974609

  18. Dissociation of insulin receptor phosphorylation and stimulation of glucose transport in BC3H-1 myocytes

    SciTech Connect

    Mojsilovic, L.P.; Standaert, M.L.; Rosic, N.K.; Pollet, R.J.

    1986-05-01

    The authors have investigated insulin receptor phosphorylation in differentiated cultured BC3H-1 myocytes. As for other insulin-responsive cell systems in partially purified wheat germ agglutinin receptor preparations, insulin stimulates the phosphorylation of its own receptor (95K ..beta..-subunits) in a dose dependent manner (0-400 nM), as identified by immunoprecipitation with antiinsulin receptor antibodies and SDS-PAGE. In the same preparations they show that 12-0-tetradecanyl phorbol acetate (TPA), which in many respect ..beta..-subunits in the same dose dependent manner (0-5 ..mu..M). In addition, antiinsulin receptor antibodies (B-10) also induced phosphorylation of mimics insulin action, also induced phosphorylation of the insulin receptor and HPLC tryptic maps of the /sup 32/P-labeled ..beta..-subunit were identical to those for insulin-induced receptor phosphorylation. However, while insulin and TPA are potent stimulators of glucose transport in these muscle cells, the antireceptor antibodies alone failed to provoke glucose transport at any concentration. The specificity and activity of these antibodies were confirmed in their system by their ability to inhibit insulin binding and insulin-stimulated glucose transport in a concentration-dependent manner. Their results indicate that phosphorylation of insulin receptor is not a crucial event in mediating insulin action, at least with respect to glucose transport. While the effects of the B-10 antibody in the BC3H-1 myocyte differ from those in the adipocyte, their results provide independent confirmation of their essential conclusion that phosphorylation of the insulin receptor may not be necessary nor sufficient for its acute action in promoting glucose transport.

  19. Functional Characterization and Expression Analyses of the Glucose-Specific AtSTP9 Monosaccharide Transporter in Pollen of Arabidopsis1

    PubMed Central

    Schneidereit, Alexander; Scholz-Starke, Joachim; Büttner, Michael

    2003-01-01

    A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated. Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a Km for glucose in the micromolar range. In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose. Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte. AtSTP9 expression is not found in other floral organs or vegetative tissues. Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes. This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth. The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars. PMID:12970485

  20. Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

    SciTech Connect

    Tomioka, Shigemasa; Kaneko, Miyuki; Satomura, Kazuhito; Mikyu, Tomiko; Nakajo, Nobuyoshi

    2009-10-09

    We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-D-[1,2-{sup 3}H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 {mu}M) significantly increased V{sub max} but not K{sub m} of GLUT3 for 2-deoxy-D-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucose uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.

  1. Deoxyandrographolide promotes glucose uptake through glucose transporter-4 translocation to plasma membrane in L6 myotubes and exerts antihyperglycemic effect in vivo.

    PubMed

    Arha, Deepti; Pandeti, Sukanya; Mishra, Akansha; Srivastava, Swayam Prakash; Srivastava, Arvind Kumar; Narender, Tadigoppula; Tamrakar, Akhilesh Kumar

    2015-12-01

    Skeletal muscle is the principal site for postprandial glucose utilization and augmenting the rate of glucose utilization in this tissue may help to control hyperglycemia associated with diabetes mellitus. Here, we explored the effect of Deoxyandrographolide (DeoAn) isolated from the Andrographis paniculata Nees on glucose utilization in skeletal muscle and investigated its antihyperglycemic effect in vivo in streptozotocin-induced diabetic rats and genetically diabetic db/db mice. In L6 myotubes, DeoAn dose-dependently stimulated glucose uptake by enhancing the translocation of glucose transporter 4 (GLUT4) to cell surface, without affecting the total cellular GLUT4 and GLUT1 content. These effects of DeoAn were additive to insulin. Further analysis revealed that DeoAn activated PI-3-K- and AMPK-dependent signaling pathways, account for the augmented glucose transport in L6 myotubes. Furthermore, DeoAn lowered postprandial blood glucose levels in streptozotocin-induced diabetic rats and also suppressed the rises in the fasting blood glucose, serum insulin, triglycerides and LDL-Cholesterol levels of db/db mice. These findings suggest the therapeutic efficacy of the DeoAn for type 2 diabetes mellitus and can be potential phytochemical for its management. PMID:26528798

  2. The Escherichia coli glucose transporter enzyme IICBGlc recruits the global repressor Mlc

    PubMed Central

    Nam, Tae-Wook; Cho, Seung-Hyon; Shin, Dongwoo; Kim, Ja-Hee; Jeong, Jin-Young; Lee, Joon-Hee; Roe, Jung-Hye; Peterkofsky, Alan; Kang, Sa-Ouk; Ryu, Sangryeol; Seok, Yeong-Jae

    2001-01-01

    In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state-dependent interaction of a membrane-bound protein with a repressor. The dephospho-form of enzyme IICBGlc, but not its phospho-form, interacts directly with Mlc and induces transcription of Mlc-regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc-regulated genes is caused by dephosphorylation of the membrane-bound transporter enzyme IICBGlc, which directly recruits Mlc to derepress its regulon. PMID:11157755

  3. Ciglitazone enhances ovarian cancer cell death via inhibition of glucose transporter-1.

    PubMed

    Shin, So Jin; Kim, Jin Young; Kwon, Sun Young; Mun, Kyo-Cheol; Cho, Chi Heum; Ha, Eunyoung

    2014-11-15

    Ciglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist and improves insulin sensitivity. Apart from antidiabetic activity, ciglitazone elicits inhibitory effects on cancer cell growth. Recent studies indicate that glucose metabolism plays a key role in malignant diseases. Significant increase in glucose consumption is found under malignant conditions. The role of ciglitazone in cancer cell death in relation to glucose metabolism is unclear. Thus we designed this study to determine the effect of ciglitazone on glucose metabolism. First, we found ciglitazone inhibited glucose uptake in ovarian cancer cells but did not affect hexokinase activity. Ciglitazone decreased expression levels of glucose transporter-1 (GLUT-1). We also found that ciglitazone and siGLUT-1 treatments induced cell death in ovarian cancer cells. We identified that ciglitazone decreased expressions of specific protein 1 (Sp-1) and β-catenin while increased phosphorylation levels of AMP-activated protein kinase. In vivo study using NOD-scid IL2Rgamma(null) mice confirmed that ciglitazone significantly decreased ovarian cancer mass transplanted onto the back of the mice. Finally, we determined GLUT-1 expressions in patients with serous type ovarian cancer and found that GLUT-1 expression was markedly increased in cancer patients and expression level was proportional to the degree of cancer stages. These results suggest that ciglitazone induces apoptosis in ovarian cancer cells by the inhibition of GLUT-1 and provides a possible therapeutic effect of ciglitazone as an adjuvant drug in the treatment of ovarian cancer. PMID:25240713

  4. High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.

    PubMed

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-03-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  5. Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes.

    PubMed

    Bourzac, Jean-François; L'Ériger, Karine; Larrivée, Jean-François; Arguin, Guillaume; Bilodeau, Maude S; Stankova, Jana; Gendron, Fernand-Pierre

    2013-01-01

    With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two-thirds of villi. These data suggest a different role for this receptor in IECs. Using the non-cancerous IEC-6 cells and differentiated Caco-2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7-induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7-dependent Glut2 internalization involved the calcium-independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. PMID:22566162

  6. The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

    PubMed

    McCoy, Jason G; Ren, Zhenning; Stanevich, Vitali; Lee, Jumin; Mitra, Sharmistha; Levin, Elena J; Poget, Sebastien; Quick, Matthias; Im, Wonpil; Zhou, Ming

    2016-06-01

    The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters. PMID:27161976

  7. Sodium-glucose co-transporter 2 inhibitors: from apple tree to 'Sweet Pee'.

    PubMed

    Hardman, Timothy C; Rutherford, Peter; Dubrey, Simon W; Wierzbicki, Anthony S

    2010-01-01

    The sodium-glucose co-transporter 2 (SGLT2), located in the plasma membrane of cells lining the proximal tubule, facilitates the reabsorbtion of glucose in the kidney. Inhibition of SGLT2 has the potential to reduce blood glucose and represents an opportune target for managing blood glucose. By promoting the excretion of glucose, SGLT2 inhibitors are the first anti-diabetic treatment to target the removal rather than the metabolic redirection of glucose. Their mechanism of action is independent of that of endogenous insulin status and thus provides a means of managing plasma glucose irrespective of a patient's glycaemic status or treatments being used in combination. Several candidate SGLT2 inhibitors based on the core glucoside structure of phlorizin are currently being developed, of which, the metabolically more stable aromatic and heteroaromatic C-glucosides have demonstrated the most promising preclinical and clinical data. The inhibition of SGLT2 by messenger antisense technology is also being investigated. Current indications suggest that short-term benefits, in terms of HbA1(c) reductions, are modest and it remains to be seen whether encouraging exogenous glucose disposal will result in long term patient benefits in terms of returning metabolic balance or even weight loss. Indications are that clinical efficacy will be greater with molecules based on an O-glucoside structure. Concerns have been raised over the safety of these agents, particularly a possible predisposition to urinary tract infections, but these concerns have yet to be confirmed in clinical studies. Clinical development programs will need to establish those patients most likely to benefit from inhibition of SGLT2. PMID:21128897

  8. The Sodium Glucose Cotransporter SGLT1 Is an Extremely Efficient Facilitator of Passive Water Transport.

    PubMed

    Erokhova, Liudmila; Horner, Andreas; Ollinger, Nicole; Siligan, Christine; Pohl, Peter

    2016-04-29

    The small intestine is void of aquaporins adept at facilitating vectorial water transport, and yet it reabsorbs ∼8 liters of fluid daily. Implications of the sodium glucose cotransporter SGLT1 in either pumping water or passively channeling water contrast with its reported water transporting capacity, which lags behind that of aquaporin-1 by 3 orders of magnitude. Here we overexpressed SGLT1 in MDCK cell monolayers and reconstituted the purified transporter into proteoliposomes. We observed the rate of osmotic proteoliposome deflation by light scattering. Fluorescence correlation spectroscopy served to assess (i) SGLT1 abundance in both vesicles and plasma membranes and (ii) flow-mediated dilution of an aqueous dye adjacent to the cell monolayer. Calculation of the unitary water channel permeability, pf, yielded similar values for cell and proteoliposome experiments. Neither the absence of glucose or Na(+), nor the lack of membrane voltage in vesicles, nor the directionality of water flow grossly altered pf Such weak dependence on protein conformation indicates that a water-impermeable occluded state (glucose and Na(+) in their binding pockets) lasts for only a minor fraction of the transport cycle or, alternatively, that occlusion of the substrate does not render the transporter water-impermeable as was suggested by computational studies of the bacterial homologue vSGLT. Although the similarity between the pf values of SGLT1 and aquaporin-1 makes a transcellular pathway plausible, it renders water pumping physiologically negligible because the passive flux would be orders of magnitude larger. PMID:26945065

  9. Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids.

    PubMed

    Hresko, Richard C; Kraft, Thomas E; Quigley, Andrew; Carpenter, Elisabeth P; Hruz, Paul W

    2016-08-12

    The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. PMID:27302065

  10. Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids*

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Quigley, Andrew; Carpenter, Elisabeth P.; Hruz, Paul W.

    2016-01-01

    The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. PMID:27302065

  11. Combined phylogenetic and neighbourhood analysis of the hexose transporters and glucose sensors in yeasts.

    PubMed

    Palma, Margarida; Seret, Marie-Line; Baret, Philippe V

    2009-06-01

    The sugar porter family in yeasts encompasses a wide variety of transporters including the hexose transporters and glucose sensors. We analysed a total of 75 members from both groups in nine hemiascomycetous species, with complete and well-annotated genomes: Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces rouxii, Kluyveromyces thermotolerans, Saccharomyces kluyverii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii and Yarrowia lipolytica. We present a model for the evolution of the hexose transporters and glucose sensors, supported by two types of complementary evidences: phylogeny and neighbourhood analysis. Five lineages of evolution were identified and discussed according to different mechanisms of gene evolution: lineage A for HXT1, HXT3, HXT4, HXT5, HXT6 and HXT7; lineage B for HXT2 and HXT10; lineage C for HXT8; lineage D for HXT14; and lineage E for SNF3 and RGT2. PMID:19459981

  12. Structural basis of human erythrocyte glucose transporter function in proteoliposome vesicles: circular dichroism measurements.

    PubMed Central

    Chin, J J; Jung, E K; Chen, V; Jung, C Y

    1987-01-01

    The secondary structural compositions of the human erythrocyte glucose transporter in proteoliposome vesicles were assessed on the basis of circular dichroism (CD) spectra measured in the absence and in the presence of D-glucose or an inhibitor, cytochalasin B. We designed and used a scattered-light-collecting device, which corrects CD spectra for optical artifacts originating from light scattering. Relative contents of eight types of secondary structure were estimated by using basis spectra generated by the eigenvector method based on CD spectra of 15 proteins of known structure. Results indicate that the glucose transporter is composed of approximately 82% alpha-helices, 10% beta-turns, and 8% other random structure, with no beta-strands. In the presence of an excess of D-glucose, the alpha-helical content is reduced by more than 10% and there is a significant increase in the random structure content. Cytochalasin B does not appear to affect the secondary structural composition of the transporter to any significant degree. PMID:3473495

  13. Effect of the peroxisome proliferator perfluoro-n-decanoic acid on glucose transport in the isolated perfused rat liver.

    PubMed

    Goecke-Flora, C M; Wyman, J F; Jarnot, B M; Reo, N V

    1995-01-01

    The perfluorinated carboxylic acid, perfluoro-n-decanoic acid (PFDA), is a known peroxisome proliferator which displays toxicity in rodents. Using a paired-tracer first-pass extraction technique, the effect of PFDA on hepatic glucose transport was determined in the isolated perfused rat liver. In brief, livers isolated from PFDA-treated and control rats on day 5 posttreatment were administered the radiolabeled glucose analog, 3-O-[14C]methyl-D-glucose ([14C]3-O-MG) in addition to [fructose-1-3H(N)]sucrose ([3H]sucrose), which served as a measure of extracellular volume. Hepatic glucose transport was calculated from the change in the ratio [14C]3-O-MG/[3H]sucrose during passage through the liver. Data from this study indicate that PFDA inhibits hepatic glucose transport. Percent hepatic glucose extraction is 1.8-fold greater in controls than in PFDA-treated rats. No significant difference in lactate dehydrogenase levels was observed in the liver perfusate from PFDA-treated and control rats. This suggests that the difference in percent glucose extraction between PFDA-treated and control groups is specifically due to the PFDA treatment and is not attributed to differences in liver viability between groups. Although the exact mechanism for this inhibition in hepatic glucose transport is not known, it is hypothesized that PFDA may have a major impact on membrane structure/function which, in turn, may alter glucose transport. PMID:7703370

  14. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

    PubMed Central

    Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

    2015-01-01

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

  15. Skeletal muscle glucose transporter protein responses to antenatal glucocorticoids in the ovine fetus.

    PubMed

    Gray, Susan; Stonestreet, Barbara S; Thamotharan, Shanthie; Sadowska, Grazyna B; Daood, Molly; Watchko, Jon; Devaskar, Sherin U

    2006-05-01

    We investigated the effects of maternal antenatal dexamethasone (Dex) treatment given as a single course (4 doses) or multiple courses (20 doses) on fetal skeletal muscle glucose transporter (GLUT) protein concentrations at 70% of gestation (106 to 107 days with term being 145 to 150 days) in the ovine fetus. Antenatal corticosteroid administration was associated with a decrease in endogenous fetal plasma cortisol concentrations (P < 0.05), fetal hyperglycemia (P < 0.02) and hyperinsulinemia (P < 0.05). These metabolic/hormonal changes were associated with a decrease in fetal body weight (P < 0.05) in the multiple course Dex group compared with the multiple course placebo group. These perturbations were associated with an increase in fetal skeletal muscle GLUT 1 concentrations that mediate basal glucose transport in the extensor digitorum lateralis and extensor digitorum longus muscles (P < 0.05) 18 h after the last dose of Dex was given in the single course group. However, in the multiple course Dex group, a small increase in GLUT 1 was observed only in the biceps femoris. In contrast, both single and multiple courses of antenatal Dex were associated with an increase in the extensor digitorum lateralis and biceps femoris muscle GLUT 4 (insulin-responsive) concentrations (P < 0.05). We conclude that antenatal corticosteroids perturb fetal glucose/insulin homeostasis, which is associated with increases in fetal skeletal muscle glucose transporters to compensate for and attenuate the associated catabolic fetal state. These changes consist of an increase in proteins that mediate basal glucose transport (GLUT 1) to meet immediate energy requirements of the fetal skeletal muscle with an increase in basal insulin sensitivity (GLUT 4) to compensate for the Dex-induced catabolic state after exposure to multiple courses of Dex. PMID:16648290

  16. Heterologous expression of rab4 reduces glucose transport and GLUT4 abundance at the cell surface in oocytes.

    PubMed Central

    Mora, S; Monden, I; Zorzano, A; Keller, K

    1997-01-01

    To evaluate the role of the small rab GTP-binding proteins in glucose transporter trafficking, we have heterologously co-expressed rab4 or rab5 and GLUT4 or GLUT1 glucose transporters in Xenopus oocytes. Co-injection of rab4 and GLUT4 cRNAs resulted in a dose-dependent decrease in glucose transport; this effect was specific for rab4, since co-injection of an inactive rab4 mutant or rab5 cRNA did not have any effect on glucose transport. The effect of rab4 was selective for GLUT4, since no effect was detected in GLUT1-expressing oocytes. The inhibitory effect of rab4 on GLUT4-induced glucose transport was not the result of a change in overall cellular levels of GLUT4 glucose transporters. However, rab4 expression caused a marked decrease in the abundance of GLUT4 transporters present at the cell surface. Finally, rab4 and inhibitors of PtdIns 3-kinase showed additive effects in decreasing glucose transport in GLUT4-expressing oocytes. We conclude that rab4 plays an important role in the regulation of the intracellular GLUT4 trafficking pathway, by contributing to the intracellular retention of GLUT4 through a PtdIns 3-kinase-independent mechanism. PMID:9182703

  17. Identification of residues important for substrate uptake in a glucose transporter from the filamentous fungus Trichoderma reesei

    PubMed Central

    Zhang, Weixin; Cao, Yanli; Gong, Jing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

    2015-01-01

    The glucose transporter is an important player in cell metabolism that mediates the intracellular uptake of glucose. Here, we characterized the glucose transporter Stp1 from the filamentous fungus Trichoderma reesei. The individual substitution of several conserved residues for Ala in Stp1 corresponding to those interacting with D-glucose in the xylose/H+ symporter XylE inflicted contrasting effects on its ability to support the growth of an hxt-null yeast on glucose. The targeted change of Phe 50, proximal to the substrate-binding site, was also found to exert a profound effect on the activity of Stp1. In contrast with the charged residues, the substitution of Phe 50 with either the hydrophilic residues Asn and Gln or the small residues Gly and Ala significantly enhanced the transport of glucose and its fluorescent analogue, 2-NBDG. On the other hand, a variant with the three substitutions I115F, F199I and P214L displayed remarkably improved activity on glucose and 2-NBDG transport. Further analysis indicated that the combined mutations of Ile 115 and Pro 214, positioned on the lateral surface of the Stp1 N-domain, fully accounted for the enhanced transport activity. These results provide insight into the structural basis for glucose uptake in fungal sugar transporters. PMID:26345619

  18. Effects of stevioside on glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal muscle.

    PubMed

    Lailerd, Narissara; Saengsirisuwan, Vitoon; Sloniger, Julie A; Toskulkao, Chaivat; Henriksen, Erik J

    2004-01-01

    Stevioside (SVS), a natural sweetener extracted from Stevia rebaudiana, has been used as an antihyperglycemic agent. However, little is known regarding its potential action on skeletal muscle, the major site of glucose disposal. Therefore, the purpose of the present study was to determine the effect of SVS treatment on skeletal muscle glucose transport activity in both insulin-sensitive lean (Fa/-) and insulin-resistant obese (fa/fa) Zucker rats. SVS was administered (500 mg/kg body weight by gavage) 2 hours before an oral glucose tolerance test (OGTT). Whereas the glucose incremental area under the curve (IAUC(glucose)) was not affected by SVS in lean Zucker rats, the insulin incremental area under the curve (IAUC(insulin)) and the glucose-insulin index (product of glucose and insulin IAUCs and inversely related to whole-body insulin sensitivity) were decreased (P<.05) by 42% and 45%, respectively. Interestingly, in the obese Zucker rat, SVS also reduced the IAUC(insulin) by 44%, and significantly decreased the IAUC(glucose) (30%) and the glucose-insulin index (57%). Muscle glucose transport was assessed following in vitro SVS treatment. In lean Zucker rats, basal glucose transport in type I soleus and type IIb epitrochlearis muscles was not altered by 0.01 to 0.1 mmol/L SVS. In contrast, 0.1 mmol/L SVS enhanced insulin-stimulated (2 mU/mL) glucose transport in both epitrochlearis (15%) and soleus (48%). At 0.5 mmol/L or higher, the SVS effect was reversed. Similarly, basal glucose transport in soleus and epitrochlearis muscles in obese Zucker rats was not changed by lower doses of SVS (0.01 to 0.1 mmol/L). However, these lower doses of SVS significantly increased insulin-stimulated glucose transport in both obese epitrochlearis and soleus (15% to 20%). In conclusion, acute oral SVS increased whole-body insulin sensitivity, and low concentrations of SVS (0.01 to 0.1 mmol/L) modestly improved in vitro insulin action on skeletal muscle glucose transport in both lean

  19. Exogenous nitric oxide reduces glucose transporters translocation and lactate production in ischemic myocardium in vivo

    PubMed Central

    Lei, Biao; Matsuo, Ken; Labinskyy, Volodymyr; Sharma, Naveen; Chandler, Margaret P.; Ahn, Anna; Hintze, Thomas H.; Stanley, William C.; Recchia, Fabio A.

    2005-01-01

    Nitric oxide (NO) inhibits myocardial glucose transport and metabolism, although the underlying mechanism(s) and functional consequences of this effect are not clearly understood. We tested the hypothesis that NO inhibits the activation of AMP-activated protein kinase (AMPK) and translocation of cardiac glucose transporters (GLUTs; GLUT-4) and reduces lactate production. Ischemia was induced in open-chest dogs by a 66% flow reduction in the left anterior descending coronary artery (LAD). During ischemia, dogs were untreated (control) or treated by direct LAD infusion of (i) nitroglycerin (NTG) (0.5 μg·kg–1·min–1); (ii) 8-Br-cGMP (50 μg·kg–1·min–1); or (iii) NO synthase inhibitor l-nitro-argininemethylester (40 μg·kg–1·min–1; n = 9 per group). Cardiac substrate oxidation was measured with isotopic tracers. There were no differences in myocardial blood flow or oxygen delivery among groups; however, at 45 min of ischemia, the activation of AMPK was significantly less in NTG (77 ± 12% vs. nonischemic myocardium) and 8-Br-cGMP (104 ± 13%), compared with control (167 ± 17%). Similarly, GLUT-4 translocation was significantly reduced in NTG (74 ± 7%) and 8-Br-cGMP (120 ± 11%), compared with control (165 ± 17%). Glucose uptake and lactate output were 30% and 60% lower in NTG compared with control. Inhibition of NO synthesis stimulated glucose oxidation (67% increase compared with control) but did not affect AMPK phosphorylation, GLUT-4 translocation and glucose uptake. Contractile function in the ischemic region was significantly improved by NTG and l-nitro-argininemethylester. In conclusion, in ischemic myocardium an NO donor inhibits glucose uptake and lactate production via a reduction in AMPK stimulation of GLUT-4 translocation, revealing a mechanism of metabolic modulation and myocardial protection activated by NO donors. PMID:15870202

  20. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed Central

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-01-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  1. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-05-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  2. Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats

    PubMed Central

    Habold, Caroline; Foltzer-Jourdainne, Charlotte; Le Maho, Yvon; Lignot, Jean-Hervé; Oudart, Hugues

    2005-01-01

    Intestinal hexose absorption and gluconeogenesis have been studied in relation to refeeding after two different fasting phases: a long period of protein sparing during which energy expenditure is derived from lipid oxidation (phase II), and a later phase characterized by a rise in plasma corticosterone triggering protein catabolism (phase III). Such a switch in body fuel uses, leading to changes in body reserves and gluconeogenic precursors, could modulate intestinal gluconeogenesis and glucose transport. The gene and protein levels, and the cellular localization of the sodium–glucose cotransporter SGLT1, and of GLUT5 and GLUT2, as well as that of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (Glc6Pase) were measured. PEPCK and Glc6Pase activities were also determined. In phase III fasted rats, SGLT1 was up-regulated and intestinal glucose uptake rates were higher than in phase II fasted and fed rats. PEPCK and Glc6Pase mRNA, protein levels and activities also increased in phase III. GLUT5 and GLUT2 were down-regulated throughout the fast, but increased after refeeding, with GLUT2 recruited to the apical membrane. The increase in SGLT1 expression during phase III may allow glucose absorption at low concentrations as soon as food is available. Furthermore, an increased epithelial permeability due to fasting may induce a paracellular movement of glucose. In the absence of intestinal GLUT2 during fasting, Glc6Pase could be involved in glucose release to the bloodstream via membrane trafficking. Finally, refeeding triggered GLUT2 and GLUT5 synthesis and apical recruitment of GLUT2, to absorb larger amounts of hexoses. PMID:15878950

  3. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein

    SciTech Connect

    Fukumoto, Hirofumi; Seino, Susumu; Imura, Hiroo; Seino, Yutaka; Eddy, R.L.; Fukushima, Yoshimitsu; Byers, M.G.; Shows, T.B.; Bell, G.I. )

    1988-08-01

    Recombinant DNA clones encoding a glucose transporter-like protein have been isolated from adult human liver and kidney cDNA libraries by cross-hybridization with the human HepG2/erythrocyte glucose transporter cDNA. Analysis of the sequence of this 524-amino acid glucose transporter-like protein indicates that is has 55.5% identity with the HepG2/erythrocyte glucose transporter as well as a similar structural organization. Studies of the tissue distribution of the mRNA coding for this glucose transporter-like protein in adult human tissues indicate that the highest amounts are present in liver with lower amounts in kidney and small intestine. The amounts of glucose transporter-like mRNA in other tissues, including colon, stomach, cerebrum, skeletal muscle, and adipose tissue, were below the level of sensitivity of our assay. The single-copy gene encoding this glucose transporter-like protein has been localized to the q26.1{yields}q26.3 region of chromosome 3.

  4. Sodium glucose co-transporter inhibitors - A new class of old drugs.

    PubMed

    Malhotra, Aneeta; Kudyar, Surbhi; Gupta, Anil K; Kudyar, Rattan P; Malhotra, Pavan

    2015-01-01

    Sodium glucose co-transporter (SGLT) inhibitors are a new class of drugs which are used in the pharmacotherapy of Type-II diabetes, which happens to be a major risk factor for developing both micro as well as macro-vascular complications. These drugs inhibit the glucose reabsorption by inhibiting SGLT, which exhibits a novel and promising mechanism of action by promoting the urinary glucose excretion hence providing a basis of therapeutic intervention. Results of SGLT-II inhibitors are very encouraging as there is a significant elevation of GLP-1 level, which forms the basis of relevance in treatment of diabetes. It targets the HbA1C and keeps a check on its levels. It also exerts other positive benefits such as weight loss, reduction in blood glucose levels, reduction in blood pressure and improvement in insulin resistance and β-cell dysfunction: All contributing to effective glycemic control. SGLT inhibition will develop as effective modality as it has the capability of inhibiting reabsorption of greater percentage of filtered glucose load. PMID:26539362

  5. Sodium glucose co-transporter inhibitors – A new class of old drugs

    PubMed Central

    Malhotra, Aneeta; Kudyar, Surbhi; Gupta, Anil K.; Kudyar, Rattan P.; Malhotra, Pavan

    2015-01-01

    Sodium glucose co-transporter (SGLT) inhibitors are a new class of drugs which are used in the pharmacotherapy of Type-II diabetes, which happens to be a major risk factor for developing both micro as well as macro-vascular complications. These drugs inhibit the glucose reabsorption by inhibiting SGLT, which exhibits a novel and promising mechanism of action by promoting the urinary glucose excretion hence providing a basis of therapeutic intervention. Results of SGLT-II inhibitors are very encouraging as there is a significant elevation of GLP-1 level, which forms the basis of relevance in treatment of diabetes. It targets the HbA1C and keeps a check on its levels. It also exerts other positive benefits such as weight loss, reduction in blood glucose levels, reduction in blood pressure and improvement in insulin resistance and β-cell dysfunction: All contributing to effective glycemic control. SGLT inhibition will develop as effective modality as it has the capability of inhibiting reabsorption of greater percentage of filtered glucose load. PMID:26539362

  6. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    SciTech Connect

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.

    1987-06-15

    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.

  7. A receptor state space model of the insulin signalling system in glucose transport.

    PubMed

    Gray, Catheryn W; Coster, Adelle C F

    2015-12-01

    Insulin is a potent peptide hormone that regulates glucose levels in the blood. Insulin-sensitive cells respond to insulin stimulation with the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM), enabling the clearance of glucose from the blood. Defects in this process can give rise to insulin resistance and ultimately diabetes. One widely cited model of insulin signalling leading to glucose transport is that of Sedaghat et al. (2002) Am. J. Physiol. Endocrinol. Metab. 283, E1084-E1101. Consisting of 20 deterministic ordinary differential equations (ODEs), it is the most comprehensive model of insulin signalling to date. However, the model possesses some major limitations, including the non-conservation of key components. In the current work, we detail mathematical and sensitivity analyses of the Sedaghat model. Based on the results of these analyses, we propose a reduced state space model of the insulin receptor subsystem. This reduced model maintains the input-output relation of the original model but is computationally more efficient, analytically tractable and resolves some of the limitations of the Sedaghat model. PMID:25673317

  8. Modulation of Glucose Transporter Protein by Dietary Flavonoids in Type 2 Diabetes Mellitus

    PubMed Central

    Hajiaghaalipour, Fatemeh; Khalilpourfarshbafi, Manizheh; Arya, Aditya

    2015-01-01

    Diabetes mellitus (DM) is a metabolic diseases characterized by hyperglycemia due to insufficient or inefficient insulin secretory response. This chronic disease is a global problem and there is a need for greater emphasis on therapeutic strategies in the health system. Phytochemicals such as flavonoids have recently attracted attention as source materials for the development of new antidiabetic drugs or alternative therapy for the management of diabetes and its related complications. The antidiabetic potential of flavonoids are mainly through their modulatory effects on glucose transporter by enhancing GLUT-2 expression in pancreatic β cells and increasing expression and promoting translocation of GLUT-4 via PI3K/AKT, CAP/Cb1/TC10 and AMPK pathways. This review highlights the recent findings on beneficial effects of flavonoids in the management of diabetes with particular emphasis on the investigations that explore the role of these compounds in modulating glucose transporter proteins at cellular and molecular level. PMID:25892959

  9. Low Red Blood Cell Vitamin C Concentrations Induce Red Blood Cell Fragility: A Link to Diabetes Via Glucose, Glucose Transporters, and Dehydroascorbic Acid

    PubMed Central

    Tu, Hongbin; Li, Hongyan; Wang, Yu; Niyyati, Mahtab; Wang, Yaohui; Leshin, Jonathan; Levine, Mark

    2015-01-01

    Strategies to prevent diabetic microvascular angiopathy focus on the vascular endothelium. Because red blood cells (RBCs) are less deformable in diabetes, we explored an original concept linking decreased RBC deformability to RBC ascorbate and hyperglycemia. We characterized ascorbate concentrations from human and mouse RBCs and plasma, and showed an inverse relationship between RBC ascorbate concentrations and deformability, measured by osmotic fragility. RBCs from ascorbate deficient mice were osmotically sensitive, appeared as spherocytes, and had decreased β-spectrin. These aberrancies reversed with ascorbate repletion in vivo. Under physiologic conditions, only ascorbate's oxidation product dehydroascorbic acid (DHA), a substrate for facilitated glucose transporters, was transported into mouse and human RBCs, with immediate intracellular reduction to ascorbate. In vitro, glucose inhibited entry of physiologic concentrations of dehydroascorbic acid into mouse and human RBCs. In vivo, plasma glucose concentrations in normal and diabetic mice and humans were inversely related to respective RBC ascorbate concentrations, as was osmotic fragility. Human RBC β-spectrin declined as diabetes worsened. Taken together, hyperglycemia in diabetes produced lower RBC ascorbate with increased RBC rigidity, a candidate to drive microvascular angiopathy. Because glucose transporter expression, DHA transport, and its inhibition by glucose differed for mouse versus human RBCs, human experimentation is indicated. PMID:26870799

  10. Gene expression pattern of glucose transporters in the skeletal muscles of newly hatched chicks.

    PubMed

    Shimamoto, Saki; Ijiri, Daichi; Kawaguchi, Mana; Nakashima, Kazuki; Ohtsuka, Akira

    2016-07-01

    The gene expression pattern of the glucose transporters (GLUT1, GLUT3, GLUT8, and GLUT12) among pectoralis major and minor, biceps femoris, and sartorius muscles from newly hatched chicks was examined. GLUT1 mRNA level was higher in pectoralis major muscle than in the other muscles. Phosphorylated AKT level was also high in the same muscle, suggesting a relationship between AKT and GLUT1 expression. PMID:27008100

  11. Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.

    PubMed

    Gross, J J; van Dorland, H A; Wellnitz, O; Bruckmaier, R M

    2015-08-01

    In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long-term manipulated glucose and insulin concentrations in combination with a LPS-induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT-qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha-lactalbumin, insulin-induced gene 1, κ-casein and acetyl-CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS-induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG. PMID:25319117

  12. Experimental type II diabetes and related models of impaired glucose metabolism differentially regulate glucose transporters at the proximal tubule brush border membrane.

    PubMed

    Chichger, Havovi; Cleasby, Mark E; Srai, Surjit K; Unwin, Robert J; Debnam, Edward S; Marks, Joanne

    2016-06-01

    What is the central question of this study? Although SGLT2 inhibitors represent a promising treatment for patients suffering from diabetic nephropathy, the influence of metabolic disruption on the expression and function of glucose transporters is largely unknown. What is the main finding and its importance? In vivo models of metabolic disruption (Goto-Kakizaki type II diabetic rat and junk-food diet) demonstrate increased expression of SGLT1, SGLT2 and GLUT2 in the proximal tubule brush border. In the type II diabetic model, this is accompanied by increased SGLT- and GLUT-mediated glucose uptake. A fasted model of metabolic disruption (high-fat diet) demonstrated increased GLUT2 expression only. The differential alterations of glucose transporters in response to varying metabolic stress offer insight into the therapeutic value of inhibitors. SGLT2 inhibitors are now in clinical use to reduce hyperglycaemia in type II diabetes. However, renal glucose reabsorption across the brush border membrane (BBM) is not completely understood in diabetes. Increased consumption of a Western diet is strongly linked to type II diabetes. This study aimed to investigate the adaptations that occur in renal glucose transporters in response to experimental models of diet-induced insulin resistance. The study used Goto-Kakizaki type II diabetic rats and normal rats rendered insulin resistant using junk-food or high-fat diets. Levels of protein kinase C-βI (PKC-βI), GLUT2, SGLT1 and SGLT2 were determined by Western blotting of purified renal BBM. GLUT- and SGLT-mediated d-[(3) H]glucose uptake by BBM vesicles was measured in the presence and absence of the SGLT inhibitor phlorizin. GLUT- and SGLT-mediated glucose transport was elevated in type II diabetic rats, accompanied by increased expression of GLUT2, its upstream regulator PKC-βI and SGLT1 protein. Junk-food and high-fat diet feeding also caused higher membrane expression of GLUT2 and its upstream regulator PKC

  13. Dissociation of in vitro sensitivities of glucose transport and antilipolysis to insulin in NIDDM

    SciTech Connect

    Yki-Jaervinen, H.; Kubo, K.; Zawadzki, J.; Lillioja, S.; Young, A.; Abbott, W.; Foley, J.E.

    1987-09-01

    It is unclear from previous studies whether qualitative or only quantitative differences exist in insulin action in adipocytes obtained from obese subjects with non-insulin-dependent diabetes mellitus (NIDDM) when compared with equally obese nondiabetic subjects. In addition, the role of changes in insulin binding as a cause of insulin resistance in NIDDM is still controversial. The authors compared the sensitivities of (/sup 14/C)-glucose transport and antilipolysis to insulin and measured (/sup 125/I)-insulin binding in abdominal adipocytes obtained from 45 obese nondiabetic, obese diabetic, and 15 nonobese female southwestern American Indians. Compared with the nonobese group, the sensitivities of glucose transport antilipolysis were reduced in both the obese nondiabetic and obese diabetic groups. Compared with the obese nondiabetic subjects, the ED/sub 50/ for stimulation of glucose transport was higher in the obese patients with NIDDM. In contrast, the ED/sub 50/S for antilipolysis were similar in obese diabetic patients and obese nondiabetic subjects. No differences was found in insulin binding in patients with NIDDM when compared with the equally obese nondiabetic subjects. These data indicate 1) the mechanism of insulin resistance differs in NIDDM and obesity, and 2) the selective loss of insulin sensitivity in NIDDM precludes changes in insulin binding as a cause of insulin resistance in this disorder.

  14. Photoaffinity labeling of the human erythrocyte glucose transporter with /sup 4/H-labelled forskolin

    SciTech Connect

    Shanahan, M.F.; Edwards, B.M.; Morris, D.P.

    1986-05-01

    Forskolin, a potent activator of adenylate cyclase, is also known to inhibit glucose transport in a number of cells. The authors have investigated photoincorporation of (/sup 3/H)forskolin into erythrocyte membrane proteins using a technique they previously developed for photolabeling the erythrocyte glucose transporter with cytochalasin B (CB). A 30-40s irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into all of the major membrane protein bands. However, most of the incorporation occurred in only three regions of the gel. Peak 1 was a sharp peak near the top of the gel in the region corresponding to spectrin, peak 2 appeared to be associated with band 3 (approx. 90kDa), and the third region labeled was between 41-60 kDa which corresponds to the region of the glucose transporter. This region appeared to contain several overlapping peaks with the largest incorporation of label occurring around 45 kDa in the area of red cell actin. When photolabeling was performed in the presence of 400 ..mu..M cytochalasin B (8.0 ..mu..M forskolin) the labeling in the 41-60 kDa region was totally inhibited while labeling of the 90 kDa peak was partially blocked. CB had no effect on the photolabeling of peak 1 by forskolin.

  15. Coupling between insulin binding and activation of glucose transport in rat adipocytes

    SciTech Connect

    Ahn, N.G.; Lipkin, E.W.; Teller, D.C.; de Haeen, C.

    1986-05-01

    Previous studies have shown that the kinetics of binding of insulin (I) to its receptor (R) in isolated rat adipocytes at 15/sup 0/C, where insulin degradation was observed to be negligible, could best be described by the model: R+I in equilibrium RI in equilibrium R'I. According to this model, bound insulin is distributed between two kinetically distinct states of the occupied receptor, RI and R'I. The quantities of RI and R'I contributing to the observed total binding of insulin to cells can be obtained from the four rate constants describing the model. In order to examine the possible roles of RI and R'I in mediating hormone action, insulin stimulation of carrier-mediated 3-0-methyl-(U-/sup 14/C) glucose transport at 15/sup 0/C was studied. The results show that insulin activation of the rate of glucose transport was sigmoidal with time, and this was qualitatively similar to the formation of R'I with time. In contrast, formation of RI was described by an exponential approach to a plateau. This finding raises the possibility that R'I is the form of the insulin receptor directly mediating insulin activation of glucose transport.

  16. A sodium-indpendent low affinity transport system for neutral amino acids in rabbit ileal mucosa.

    PubMed Central

    Paterson, J Y; Sepúlveda, F V; Smith, M W

    1980-01-01

    1. The kinetic parameters for serine, alanine and methionine uptake by rabbit ileal mucosa have been determined in the absence of Na. 2. Uptake of all three amino acids took place through a single mediated system. The apparent Km values of serine, alanine and methionine for this system were equal to their respective apparent K1 values (approximately 89, 75 and 23 mM respectively). 3. Autoradiography was used to measure the cellular location of alanine uptake by rabbit ileum. Approximately 80% of the total uptake took place in the upper third of each villus. This uptake was reduced by 75% either by removal of Na or addition of serine. The proportional distribution of Na-dependent and Na-independent alanine uptakes along the villus was found to be equal. 4. The kinetic properties of the low affinity uptake mechanism for neutral amino acids, seen in the absence of Na, were virtually identical with those of one of the uptake mechanisms seen previously in the presence of Na. 5. The low affinity uptake mechanism appears to be Na-independent. It is suggested that the Na-coupled uptake of amino acid takes place through the high affinity system. PMID:7359411

  17. Diet effects on glucose absorption in the small intestine of neonatal calves: importance of intestinal mucosal growth, lactase activity, and glucose transporters.

    PubMed

    Steinhoff-Wagner, Julia; Zitnan, Rudolf; Schönhusen, Ulrike; Pfannkuche, Helga; Hudakova, Monika; Metges, Cornelia C; Hammon, Harald M

    2014-10-01

    Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters. PMID:25108868

  18. Role of villus microcirculation in intestinal absorption of glucose: coupling of epithelial with endothelial transport

    PubMed Central

    Pappenheimer, J R; Michel, C C

    2003-01-01

    Capillaries in jejunal villi can absorb nutrients at rates several hundred times greater (per gram tissue) than capillaries in other tissues, including contracting skeletal muscle and brain. We here present an integrative hypothesis to account for these exceptionally large trans-endothelial fluxes and their relation to epithelial transport. Equations are developed for estimating concentration gradients of glucose across villus capillary walls, along paracellular channels and across subjunctional lateral membranes of absorptive cells. High concentrations of glucose discharged across lateral membranes to subjunctional intercellular spaces are delivered to abluminal surfaces of villus capillaries by convection-diffusion in intercellular channels without significant loss of concentration. Post-junctional paracellular transport thus provides the series link between epithelial and endothelial transport and makes possible the large trans-endothelial concentration gradients required for absorption to blood. Our analysis demonstrates that increases of villus capillary blood flow and permeability-surface area product (PS) are essential components of absorptive mechanisms: epithelial transport of normal digestive loads could not be sustained without concomitant increases in capillary blood flow and PS. The low rates of intestinal absorption found in anaesthetised animals may be attributed to inhibition of normal villus microvascular responses to epithelial transport. PMID:12937296

  19. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  20. Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli.

    PubMed

    Tao, Junyi; Diaz, Rebecca K; Teixeira, César R V; Hackmann, Timothy J

    2016-05-10

    Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [(14)C]glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the Vmax of 2-NBDG transport was 2.9-fold lower than that for [(14)C]glucose, and the Km was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [(14)C]mannose and [(14)C]deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement. PMID:27096355

  1. N-Indolylglycosides bearing modifications at the glucose C6-position as sodium-dependent glucose co-transporter 2 inhibitors.

    PubMed

    Chu, Kuang-Feng; Yao, Chun-Hsu; Song, Jen-Shin; Chen, Chiung-Tong; Yeh, Teng-Kuang; Hsieh, Tsung-Chih; Huang, Chung-Yu; Wang, Min-Hsien; Wu, Szu-Huei; Chang, Wei-En; Chao, Yu-Sheng; Lee, Jinq-Chyi

    2016-05-15

    Suppression of glucose reabsorption through the inhibition of sodium-dependent glucose co-transporter 2 (SGLT2) is a promising therapeutic approach for the treatment of type 2 diabetes. To investigate the effect of C6-substitution on inhibition of SGLT2 by N-indolylglucosides, a small library of 6-triazole, 6-amide, 6-urea, and 6-thiourea N-indolylglycosides were synthesized and tested. A detailed structure-activity relationship (SAR) study culminated in the identification of 6-amide derivatives 6a and 6o as potent SGLT2 inhibitors, which were further tested for inhibitory activity against SGLT1. The data obtained indicated that 6a and 6o are mildly to moderately selective for SGLT2 over SGLT1. Both compounds were also evaluated in a urinary glucose excretion test and pharmacokinetic study; 6a was found capable of inducing urinary glucose excretion in normal SD rats. PMID:27075813

  2. Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4.

    PubMed Central

    Ishii, K; Hayashi, H; Todaka, M; Kamohara, S; Kanai, F; Jinnouchi, H; Wang, L; Ebina, Y

    1995-01-01

    Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7543750

  3. Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

    PubMed

    Ahn, Seong Kyu; Cho, Pyo Yun; Na, Byoung-Kuk; Hong, Sung-Jong; Nam, Ho-Woo; Sohn, Woon-Mok; Ardelli, Bernadette F; Park, Yun-Kyu; Kim, Tong-Soo; Cha, Seok Ho

    2016-01-01

    A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis. PMID:26450594

  4. Gender differences in the gene expression profiles of glucose transporter GLUT class I and SGLT in mouse tissues.

    PubMed

    Nagai, K; Yoshida, S; Konishi, H

    2014-11-01

    Glucose transporters play key roles in controlling blood sugar levels and are recognized as the pharmacological targets of antidiabetic agents. In the present study, we compared the gene expression profiles of glucose transporter GLUT class I and SGLT isoforms in the skeletal muscle, heart, liver, kidney, and brain of male and female mice. The expression profiles of GLUT1 -4 and SGLT1 -2 in male mouse tissues were similar to those previously reported. Significant gender differences were observed in mRNA expression in terms of individual these glucose transport systems and the tissues examined. Especially, all of the corresponding mRNAs of renal GLUT class I and SGLT isoforms were expressed at higher levels in female mice than in male mice. However, no significant differences were observed in serum glucose concentrations between male and female mice. These results strongly suggest that prominent gender differences exist in the gene expression profiles of these glucose transporters in mouse tissues, and that the quantitative and functional multiplicities of glucose transporters may contribute to the successful regulation of blood glucose concentrations irrespective of gender differences. PMID:25985584

  5. Energy balance and metabolic changes with sodium-glucose co-transporter 2 inhibition.

    PubMed

    Rajeev, S P; Cuthbertson, D J; Wilding, J P H

    2016-02-01

    Sodium-glucose co-transporter 2 (SGLT2) inhibitors are the latest addition to the class of oral glucose-lowering drugs. They have been rapidly adopted into clinical practice because of therapeutic advantages, including weight loss and reduction in blood pressure, in addition to glycaemic benefits and a low intrinsic risk of hypoglycaemia. Although there are extensive data on the clinical effects of SGLT2 inhibition, the metabolic effects of inhibiting renal glucose reabsorption have not been fully described. Recent studies have identified compensatory metabolic effects, such as an increase in endogenous glucose production, and have also shown an increase in glucagon secretion during SGLT2 inhibition. In addition, there is a discrepancy between the expected and observed weight loss found in clinical studies on SGLT2 inhibitors, probably as a result of changes in energy balance with this treatment approach. SGLT2 inhibition is likely to have intriguing effects on whole body metabolism which have not been fully elucidated, and which, if explained, might help optimize the use of this new class of medicines. PMID:26403227

  6. Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter: Intervention Approaches.

    PubMed

    Alam, Fahmida; Islam, Md Asiful; Khalil, Md Ibrahim; Gan, Siew Hua

    2016-01-01

    Type 2 diabetes mellitus (T2DM), the most common form of diabetes, is characterized by insulin resistance in the hepatic and peripheral tissues. Glucose transporter 4 (GLUT4) plays a major role in the pathophysiology of T2DM. Its defective expression or translocation to the peripheral cell plasma membrane in T2DM patients hinders the entrance of glucose into the cell for energy production. In addition to suitable drugs, an appropriate diet and/or exercise can be implemented to target the increase in GLUT4 expression, GLUT4 concentrations and GLUT4 translocation to the cell surface when managing the glucose metabolism of T2DM patients. In this review, we discussed successful intervention strategies that were individually administered or coupled with diet and/or exercise and affected the expression and translocation of GLUT4 in T2DM while reducing the excess glucose load from the blood. Additionally, some potentially good synthetic and natural compounds, which can activate the insulin-independent GLUT4 signaling pathways for the efficient management of T2DM, are highlighted as possible targets or emerging alternative sources for future anti-diabetic drug development. PMID:26951104

  7. Membrane topology of the high-affinity L-glutamate transporter (GLAST- 1) of the central nervous system

    PubMed Central

    1996-01-01

    The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L- aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane- spanning domains. The results of our experiments suggest a novel 10- transmembrane domain topology of GLAST-1, a representative of the L- glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets. PMID:8991097

  8. Allelic isoforms of the H+/nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides.

    PubMed

    Slugoski, Melissa D; Loewen, Shaun K; Ng, Amy M L; Baldwin, Stephen A; Cass, Carol E; Young, James D

    2004-11-01

    Contigs 19-10196 and 19-20196 of the Stanford Candida albicans genome sequence databank encode two putative allelic isoforms of C. albicans CaCNT, a recently characterized 608 amino acid residue H+-coupled fungal member of the CNT family of concentrative nucleoside transport proteins. The single Ser/Gly difference between CaCNT/19-20196 and CaCNT occurs at position 328 in putative TM 7, and corresponds to a Ser/Gly substitution previously shown to contribute to the contrasting pyrimidine and purine nucleoside selectivities of human (h) and rat (r) Na+-dependent CNT1 and CNT2. CaCNT/19-10196 differs from CaCNT by four amino acids, but has Gly at position 328. These new proteins were recreated by site-directed mutagenesis of CaCNT and characterized functionally by heterologous expression in Xenopus laevis oocytes. In marked contrast to h/rCNT1/2, both CaCNT/19-10196 and CaCNT/19-20196 exhibited permeant selectivities for purine nucleosides (adenosine, guanosine and inosine) and uridine similar to that of CaCNT. However, although H+-coupled, CaCNT/19-20196 exhibited a approximately 10-fold higher apparent Km for uridine than either CaCNT or CaCNT/19-10196. CaCNT/19-20196 also exhibited a low apparent affinity for inosine. We conclude that the three proteins correspond to high-affinity (CaCNT, CaCNT/19-10196) and low-affinity (CaCNT/19-20196) allelic isoforms of the C. albicans CNT nucleoside transporter. This is the first example of a single amino acid residue substitution altering a CNT protein's overall apparent affinity for nucleosides. PMID:15543539

  9. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    PubMed Central

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  10. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.

    PubMed

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  11. The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter.

    PubMed

    Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H; Ecker, Gerhard F; Freissmuth, Michael; Sucic, Sonja

    2010-12-01

    The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na(+)/Cl(-)-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERT(Y95F) mutation, which greatly reduced the affinity for [(3)H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [(3)H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP(+), paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

  12. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  13. MondoA senses non-glucose sugars: regulation of thioredoxin-interacting protein (TXNIP) and the hexose transport curb.

    PubMed

    Stoltzman, Carrie A; Kaadige, Mohan R; Peterson, Christopher W; Ayer, Donald E

    2011-11-01

    Glucose is required for cell growth and proliferation. The MondoA·Mlx transcription factor is glucose-responsive and accumulates in the nucleus by sensing glucose 6-phosphate. One direct and glucose-induced target of MondoA·Mlx complexes is thioredoxin-interacting protein (TXNIP). TXNIP is a potent negative regulator of glucose uptake, and hence its regulation by MondoA·Mlx triggers a feedback loop that restricts glucose uptake. This feedback loop is similar to the "hexose transport curb" first described almost 30 years ago. We show here that MondoA responds to the non-glucose hexoses, allose, 3-O-methylglucose, and glucosamine by accumulating in the nucleus and activating TXNIP transcription. The metabolic inhibitor 3-bromopyruvate blocks the transcriptional response to allose and 3-O-methylglucose, indicating that their metabolism, or a parallel pathway, is required to stimulate MondoA activity. Our dissection of the hexosamine biosynthetic pathway suggests that in addition to sensing glucose 6-phosphate, MondoA can also sense glucosamine 6-phosphate. Analysis of glucose uptake in wild-type, MondoA-null, or TXNIP-null murine embryonic fibroblasts indicates a role for the MondoA-TXNIP regulatory circuit in the hexose transport curb, although other redundant pathways also contribute. PMID:21908621

  14. Dopamine transporters, D2 receptors, and glucose metabolism in corticobasal degeneration.

    PubMed

    Klaffke, Stefanie; Kuhn, Andrea A; Plotkin, Michail; Amthauer, Holger; Harnack, Daniel; Felix, Roland; Kupsch, Andreas

    2006-10-01

    Alterations in presynaptic and postsynaptic dopaminergic system and cerebral glucose metabolism in corticobasal degeneration (CBD) were assessed to evaluate the potential usefulness of different imaging methods for CBD. (123)I-FP-CIT/(123)I-beta-CIT SPECT and (123)I-IBZM SPECT as well as (18)F-FDG PET were performed in eight CBD patients. Decreased presynaptic dopamine transporter binding was found in all CBD patients while D2 receptor binding was reduced in only one patient. (18)F-FDG PET displayed a contralateral hypometabolism in cortical and subcortical areas in seven out of eight patients. Our results demonstrate that glucose metabolism and DAT are reduced, while D2 receptors may be frequently preserved in CBD. PMID:16773621

  15. Interaction of Peptide Transporter 1 With D-Glucose and L-Glutamic Acid; Possible Involvement of Taste Receptors.

    PubMed

    Arakawa, Hiroshi; Ohmachi, Taichi; Ichiba, Kiko; Kamioka, Hiroki; Tomono, Takumi; Kanagawa, Masahiko; Idota, Yoko; Hatano, Yasuko; Yano, Kentaro; Morimoto, Kaori; Ogihara, Takuo

    2016-01-01

    We investigated the influence of sweet and umami (savory) tastants on the intestinal absorption of cephalexin (CEX), a substrate of peptide transporter 1 (PEPT1, SLC15A1) in rats. After oral administration of glucose or mannitol to rats, CEX was administered together with a second dose of glucose or mannitol. Western blot analysis indicated that expression of PEPT1 in rat jejunum membrane was decreased by glucose, compared to mannitol. Furthermore, the maximum plasma concentration (Cmax) of orally administered CEX was reduced by glucose compared to mannitol. The effect of glucose was diminished by nifedipine, a L-type Ca(2+) channel blocker. We also found that Cmax of orally administered CEX was reduced by treatment with L-glutamic acid, compared to D-glutamic acid. Thus, excessive intake of glucose and L-glutamic acid may impair oral absorption of PEPT1 substrates. PMID:26852864

  16. Sodium glucose co-transporter 2 (SGLT2) inhibitors: new among antidiabetic drugs.

    PubMed

    Opie, L H

    2014-08-01

    Type 2 diabetes is characterized by decreased insulin secretion and sensitivity. The available oral anti-diabetic drugs act on many different molecular sites. The most used of oral anti-diabetic agents is metformin that activates glucose transport vesicles to the cell surface. Others are: the sulphonylureas; agents acting on the incretin system; GLP-1 agonists; dipetidylpeptidase-4 inhibitors; meglinitide analogues; and the thiazolidinediones. Despite these many drugs acting by different mechanisms, glycaemic control often remains elusive. None of these drugs have a primary renal mechanism of action on the kidneys, where almost all glucose excreted is normally reabsorbed. That is where the inhibitors of glucose reuptake (sodium-glucose cotransporter 2, SGLT2) have a unique site of action. Promotion of urinary loss of glucose by SGLT2 inhibitors embodies a new principle of control in type 2 diabetes that has several advantages with some urogenital side-effects, both of which are evaluated in this review. Specific approvals include use as monotherapy, when diet and exercise alone do not provide adequate glycaemic control in patients for whom the use of metformin is considered inappropriate due to intolerance or contraindications, or as add-on therapy with other anti-hyperglycaemic medicinal products including insulin, when these together with diet and exercise, do not provide adequate glycemic control. The basic mechanisms are improved β-cell function and insulin sensitivity. When compared with sulphonylureas or other oral antidiabetic agents, SGLT2 inhibitors provide greater HbA1c reduction. Urogenital side-effects related to the enhanced glycosuria can be troublesome, yet seldom lead to discontinuation. On this background, studies are analysed that compare SGLT2 inhibitors with other oral antidiabetic agents. Their unique mode of action, unloading the excess glycaemic load, contrasts with other oral agents that all act to counter the effects of diabetic

  17. UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation

    PubMed Central

    Vozza, Angelo; Parisi, Giovanni; De Leonardis, Francesco; Lasorsa, Francesco M.; Castegna, Alessandra; Amorese, Daniela; Marmo, Raffaele; Calcagnile, Valeria M.; Palmieri, Luigi; Ricquier, Daniel; Paradies, Eleonora; Scarcia, Pasquale; Palmieri, Ferdinando; Bouillaud, Frédéric; Fiermonte, Giuseppe

    2014-01-01

    Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA–producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties. PMID:24395786

  18. Relationship between cerebral sodium-glucose transporter and hyperglycemia in cerebral ischemia.

    PubMed

    Yamazaki, Yui; Harada, Shinichi; Tokuyama, Shogo

    2015-09-14

    Post-ischemic hyperglycemia exacerbates the development of cerebral ischemia. To elucidate this exacerbation mechanism, we focused on sodium-glucose transporter (SGLT) as a mediator that lead hyperglycemia to cerebral ischemia. SGLT transport glucose into the cell, together with sodium ion, using the sodium concentration gradient. We have previously reported that suppression of cerebral SGLT ameliorates cerebral ischemic neuronal damage. However, detail relationship cerebral between SGLT and post-ischemic hyperglycemia remain incompletely defined. Therefore, we examined the involvement of cerebral SGLT on cerebral ischemic neuronal damage with or without hyperglycemic condition. Cell survival rate of primary cultured neurons was assessed by biochemical assay. A mouse model of focal ischemia was generated using a middle cerebral artery occlusion (MCAO). Neuronal damage was assessed with histological and behavioral analyses. Concomitant hydrogen peroxide/glucose treatment exacerbated hydrogen peroxide alone-induced cell death. Although a SGLT family-specific inhibitor, phlorizin had no effect on developed hydrogen peroxide alone-induced cell death, it suppressed cell death induced by concomitant hydrogen peroxide/glucose treatment. α-MG induced a concentration-dependent and significant decrease in neuronal survival. PHZ administered on immediately after reperfusion had no effect, but PHZ given at 6h after reperfusion had an effect. Our in vitro study indicates that SGLT is not involved in neuronal cell death in non-hyperglycemic condition. We have already reported that post-ischemic hyperglycemia begins to develop at 6h after MCAO. Therefore, current our in vivo study show post-ischemic hyperglycemic condition may be necessary for the SGLT-mediated exacerbation of cerebral ischemic neuronal damage. PMID:26254165

  19. Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

    PubMed

    Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

    2015-12-01

    The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses. PMID:26402434

  20. Experimental characterization of the transport phenomena, adsorption, and elution in a protein A affinity monolithic medium.

    PubMed

    Herigstad, M Omon; Dimartino, Simone; Boi, Cristiana; Sarti, Giulio C

    2015-08-14

    A commercially available convective interaction media (CIM) Protein A monolithic column was fully characterized in view of its application for the affinity capture of IgG in monoclonal antibody production processes. By means of moment analysis, the interstitial porosity and axial dispersion coefficient were determined. The frontal analysis method of characteristic points was employed, for the first time with monolithic media, to determine the dynamic binding capacity. The effects of the flow rate and pH on the total recovery of polyclonal IgG and elution profile were evaluated. A comparison with literature data for Protein A chromatography beads demonstrate the superior bed utilization of monolithic media, which gave better performance at lower residence times. PMID:26143608

  1. The effect of insulin on plasma glucose concentrations, expression of hepatic glucose transporters and key gluconeogenic enzymes during the perinatal period in broiler chickens.

    PubMed

    Franssens, Lies; Lesuisse, Jens; Wang, Yufeng; Willems, Els; Willemsen, Hilke; Koppenol, Astrid; Guo, Xiaoquan; Buyse, Johan; Decuypere, Eddy; Everaert, Nadia

    2016-06-01

    Chickens have blood glucose concentrations that are twofold higher than those observed in mammals. Moreover, the insulin sensitivity seems to decrease with postnatal age in both broiler and layer chickens. However, little is known about the response of insulin on plasma glucose concentrations and mRNA abundance of hepatic glucose transporters 1, 2, 3, 8, 9 and 12 (GLUT1, 2, 3, 8, 9 and 12) and three regulatory enzymes of the gluconeogenesis, phosphoenolpyruvate carboxykinase 1 and 2 (PCK1 and 2) or fructose-1,6-biphosphatase 1 (FBP1) in chicks during the perinatal period. In the present study, broiler embryos on embryonic day (ED)16, ED18 or newly-hatched broiler chicks were injected intravenously with bovine insulin (1μg/g body weight (BW)) to examine plasma glucose response and changes in hepatic mRNA abundance of the GLUTs, PCK1 and 2 and FBP1. Results were compared with a non-treated control group and a saline-injected sham group. Plasma glucose levels of insulin-treated ED18 embryos recovered faster from their minimum level than those of insulin-treated ED16 embryos or newly-hatched chicks. In addition, at the minimum plasma glucose level seven hours post-injection (PI), hepatic GLUT2, FBP1 and PCK2 mRNA abundance was decreased in insulin-injected embryos, compared to sham and control groups, being most pronounced when insulin injection occurred on ED16. PMID:26723190

  2. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

    PubMed Central

    He, Xiao; Garza, Denisse; Nigam, Sanjay K.; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M-1s-1 and an even slower dissociation rate (kd) in the range of 10−4 s-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not

  3. Phosphorylation of RS1 (RSC1A1) Steers Inhibition of Different Exocytotic Pathways for Glucose Transporter SGLT1 and Nucleoside Transporter CNT1, and an RS1-Derived Peptide Inhibits Glucose Absorption.

    PubMed

    Veyhl-Wichmann, Maike; Friedrich, Alexandra; Vernaleken, Alexandra; Singh, Smriti; Kipp, Helmut; Gorboulev, Valentin; Keller, Thorsten; Chintalapati, Chakravarthi; Pipkorn, Rüdiger; Pastor-Anglada, Marçal; Groll, Jürgen; Koepsell, Hermann

    2016-01-01

    Cellular uptake adapts rapidly to physiologic demands by changing transporter abundance in the plasma membrane. The human gene RSC1A1 codes for a 67-kDa protein named RS1 that has been shown to induce downregulation of the sodium-D-glucose cotransporter 1 (SGLT1) and of the concentrative nucleoside transporter 1 (CNT1) in the plasma membrane by blocking exocytosis at the Golgi. Injecting RS1 fragments into Xenopus laevis oocytes expressing SGLT1 or CNT1 and measuring the expressed uptake of α-methylglucoside or uridine 1 hour later, we identified a RS1 domain (RS1-Reg) containing multiple predicted phosphorylation sites that is responsible for this post-translational downregulation of SGLT1 and CNT1. Dependent on phosphorylation, RS1-Reg blocks the release of SGLT1-containing vesicles from the Golgi in a glucose-dependent manner or glucose-independent release of CNT1-containing vesicles. We showed that upregulation of SGLT1 in the small intestine after glucose ingestion is promoted by glucose-dependent disinhibition of the RS1-Reg-blocked exocytotic pathway of SGLT1 between meals. Mimicking phosphorylation of RS1-Reg, we obtained a RS1-Reg variant that downregulates SGLT1 in the brush-border membrane at high luminal glucose concentration. Because RS1 mediates short-term regulation of various transporters, we propose that the RS1-Reg-navigated transporter release from Golgi represents a basic regulatory mechanism of general importance, which implies the existence of receptor proteins that recognize different phosphorylated forms of RS1-Reg and of complex transporter-specific sorting in the trans-Golgi. RS1-Reg-derived peptides that downregulate SGLT1 at high intracellular glucose concentrations may be used for downregulation of glucose absorption in small intestine, which has been proposed as strategy for treatment of type 2 diabetes. PMID:26464324

  4. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  5. Berry anthocyanins and anthocyanidins exhibit distinct affinities for the efflux transporters BCRP and MDR1

    PubMed Central

    Dreiseitel, A; Oosterhuis, B; Vukman, KV; Schreier, P; Oehme, A; Locher, S; Hajak, G; Sand, PG

    2009-01-01

    Background and purpose: Dietary anthocyanins hold great promise in the prevention of chronic disease but factors affecting their bioavailability remain poorly defined. Specifically, the role played by transport mechanisms at the intestinal and blood–brain barriers (BBB) is currently unknown. Experimental approach: In the present study, 16 anthocyanins and anthocyanidins were exposed to the human efflux transporters multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), using dye efflux, ATPase and, for BCRP, vesicular transport assays. Key results: All test compounds interacted with the BCRP transporter in vitro. Of these, seven emerged as potential BCRP substrates (malvidin, petunidin, malvidin-3-galactoside, malvidin-3,5-diglucoside, cyanidin-3-galactoside, peonidin-3-glucoside, cyanidin-3-glucoside) and 12 as potential inhibitors of BCRP (cyanidin, peonidin, cyanidin-3,5-diglucoside, malvidin, pelargonidin, delphinidin, petunidin, delphinidin-3-glucoside, cyanidin-3-rutinoside, malvidin-3-glucoside, pelargonidin-3,5-diglucoside, malvidin-3-galactoside). Malvidin, malvidin-3-galactoside and petunidin exhibited bimodal activities serving as BCRP substrates at low concentrations and, at higher concentrations, as BCRP inhibitors. Effects on MDR1, in contrast, were weak. Only aglycones exerted mild inhibitory activity. Conclusions and implications: Although the anthocyanidins under study may alter pharmacokinetics of drugs that are BCRP substrates, they are less likely to interfere with activities of MDR1 substrates. The present data suggest that several anthocyanins and anthocyanidins may be actively transported out of intestinal tissues and endothelia, limiting their bioavailability in plasma and brain. PMID:19922539

  6. Chemical constituents from Eucalyptus citriodora Hook leaves and their glucose transporter 4 translocation activities.

    PubMed

    Wang, Chao; Yang, Jing; Zhao, Ping; Zhou, Qi; Mei, Zhinan; Yang, Guangzhong; Yang, Xinzhou; Feng, Yunjiang

    2014-07-15

    Bioassay-guided phytochemical investigation of the EtOAc fraction from the leaves of a Chinese medicinal herb, Eucalyptus citriodora Hook, resulted in the isolation of a new compound rhodomyrtosone E (1), along with 12 known compounds (2-13). The structure of the new compound was established by 1D and 2D NMR, MS data and X-ray crystallographic analysis. Betulinic acid (2) and corosolic acid (5) increased glucose transporter 4 (GLUT-4) translocation by 2.38 and 1.78-fold, respectively. PMID:24894556

  7. Use of a high electron-affinity molybdenum dithiolene complex to p-dope hole-transport layers.

    PubMed

    Qi, Yabing; Sajoto, Tissa; Barlow, Stephen; Kim, Eung-Gun; Brédas, Jean-Luc; Marder, Seth R; Kahn, Antoine

    2009-09-01

    Experimental and theoretical results are presented on the electronic structure of molybdenum tris[1,2-bis(trifluoromethyl) ethane-1,2-dithiolene] (Mo(tfd)(3)), a high electron-affinity organometallic complex that constitutes a promising candidate as a p-dopant for organic molecular semiconductors. The electron affinity of the compound, determined via inverse photoemission spectroscopy, is 5.6 eV, which is 0.4 eV larger than that of the commonly used p-dopant F(4)-TCNQ. The LUMO level of Mo(tfd)(3) is calculated to be delocalized over the whole molecule, which is expected to lead to low pinning potential. Efficient p-doping of a standard hole transport material (alpha-NPD) is demonstrated via measurements of Fermi level shifts and enhanced conductivity in alpha-NPD:1% Mo(tfd)(3). Rutherford backscattering measurements show good stability of the three-dimensional Mo(tfd)(3) molecule in the host matrix with respect to diffusion. PMID:19678703

  8. Effect of absorption of D-glucose and water on paracellular transport in rat duodenum-jejunum.

    PubMed

    O'Rourke, M; Shi, X; Gisolfi, C; Schedl, H

    1995-03-01

    Paracellular transport is thought to be a major absorptive pathway for small nutrient molecules. The authors used in vivo in situ perfusion of rat duodenum-proximal jejunum to examine paracellular transport using lactulose as a probe. They perfused solutions with a constant lactulose concentration but varied initial D-glucose concentration (range 12-176 mM) to open paracellular pathways and to increase water absorption, thereby optimizing potential for paracellular transport of lactulose and other solutes in its molecular weight range. All solutions contained sodium chloride to approach isotonicity. Water absorption was measured as the difference in weight of solution perfused and sample collected. Absorption of D-glucose increased with mean luminal D-glucose concentration, and water absorption more than doubled (from 0.12 +/- 0.03 to 0.26 +/- 0.05 mL/min per g dry wt of segment) as mean luminal glucose concentration was increased from 10 to 80 mM. Lactulose absorption was at the threshold of detection and did not correlate with D-glucose or water absorption. Expressed as percent per segment, D-glucose absorption ranged from 29-50%, and the lactulose absorption rate was 4-5%. The fraction of D-glucose absorption that could be attributed to lactulose absorptive pathways was 12% at the highest rate of water absorption. In conclusion, based on lactulose as a probe, under conditions of opening tight junctions by D-glucose, the paracellular component of D-glucose absorption was of the order of 1/10 of total D-glucose absorption (ie, not a major absorptive pathway. PMID:7879819

  9. Insulin and insulin-like growth factor I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes.

    PubMed Central

    Mora, S; Kaliman, P; Chillarón, J; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation. Images Figure 2 Figure 3 PMID:7575481

  10. Insulin and insulin-like growth factor I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes.

    PubMed

    Mora, S; Kaliman, P; Chillarón, J; Testar, X; Palacín, M; Zorzano, A

    1995-10-01

    1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation. PMID:7575481

  11. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    PubMed

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

  12. Sodium glucose transporter protein 2 inhibitors: focusing on the kidney to treat type 2 diabetes

    PubMed Central

    Peene, Bernard

    2014-01-01

    Type 2 diabetes mellitus (T2DM) is increasing worldwide. Treatment of T2DM continues to present challenges, with a significant proportion of patients failing to achieve and maintain glycemic targets. Despite the availability of many oral antidiabetic agents, therapeutic efficacy is also offset by side effects such as weight gain and hypoglycemia. Therefore, the search for novel therapeutic agents with an improved benefit–risk profile continues. In the following review we focus on a novel class of oral antidiabetic drugs, the sodium glucose transporter protein 2 (SGLT2) inhibitors, which have unique characteristics. SGLT2 inhibitors focus on the kidney as a therapeutic target, where they inhibit the reabsorption of glucose in the proximal tubule, causing an increase in urinary glucose excretion. Doing this, they reduce plasma glucose independently of the β-cell function of the pancreas. SGLT2 inhibitors are effective at lowering hemoglobin A1c, but also induce weight loss and reduce blood pressure, with a low risk of hypoglycemia. In general, the SGLT2 inhibitors are well tolerated, with the most frequent adverse events being mild urinal and genital infections. Since their primary site of effect is the kidney, these drugs are less effective in patients with impaired kidney function but evidence is emerging that these drugs may also have a protective effect against diabetic nephropathy. This review focuses on the most extensively studied SGLT2 inhibitors dapagliflozin, canagliflozin and empagliflozin. Dapagliflozin and canagliflozin have already been approved for marketing by the US Food and Drug Administration. The European Medicines Agency has accepted all three drugs for marketing. PMID:25419452

  13. Sodium glucose transporter protein 2 inhibitors: focusing on the kidney to treat type 2 diabetes.

    PubMed

    Peene, Bernard; Benhalima, Katrien

    2014-10-01

    Type 2 diabetes mellitus (T2DM) is increasing worldwide. Treatment of T2DM continues to present challenges, with a significant proportion of patients failing to achieve and maintain glycemic targets. Despite the availability of many oral antidiabetic agents, therapeutic efficacy is also offset by side effects such as weight gain and hypoglycemia. Therefore, the search for novel therapeutic agents with an improved benefit-risk profile continues. In the following review we focus on a novel class of oral antidiabetic drugs, the sodium glucose transporter protein 2 (SGLT2) inhibitors, which have unique characteristics. SGLT2 inhibitors focus on the kidney as a therapeutic target, where they inhibit the reabsorption of glucose in the proximal tubule, causing an increase in urinary glucose excretion. Doing this, they reduce plasma glucose independently of the β-cell function of the pancreas. SGLT2 inhibitors are effective at lowering hemoglobin A1c, but also induce weight loss and reduce blood pressure, with a low risk of hypoglycemia. In general, the SGLT2 inhibitors are well tolerated, with the most frequent adverse events being mild urinal and genital infections. Since their primary site of effect is the kidney, these drugs are less effective in patients with impaired kidney function but evidence is emerging that these drugs may also have a protective effect against diabetic nephropathy. This review focuses on the most extensively studied SGLT2 inhibitors dapagliflozin, canagliflozin and empagliflozin. Dapagliflozin and canagliflozin have already been approved for marketing by the US Food and Drug Administration. The European Medicines Agency has accepted all three drugs for marketing. PMID:25419452

  14. Chronic heart failure selectively induces regional heterogeneity of insulin-responsive glucose transporters

    PubMed Central

    Ware, Bruce; Bevier, Marie; Nishijima, Yoshinori; Rogers, Suzanne; Carnes, Cynthia A.

    2011-01-01

    Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT4 (the major cardiac isoform) and GLUT12 (a novel, second insulin-sensitive isoform). Potential regional patterns in glucose transport across the cardiac chambers have not been examined; thus, we hypothesized that insulin-responsive GLUT4 and -12 protein and gene expression would be chamber specific in healthy subjects and during chronic heart failure (HF). Using a canine model of tachypacing-induced, progressive, chronic HF, total GLUT protein and messenger RNA in both ventricles and atria (free wall and appendage) were investigated by immunoblotting and real-time PCR. In controls, GLUT4, but not GLUT12, protein content was significantly higher in the atria compared with the ventricles, with the highest content in the right atrium (RA; P < 0.001). GLUT4 and GLUT12 mRNA levels were similar across the cardiac chambers. During chronic HF, GLUT4 and GLUT12 protein content was highest in the left ventricle (LV; by 2.5- and 4.2-fold, respectively, P < 0.01), with a concomitant increase in GLUT4 and GLUT12 mRNA (P < 0.001). GLUT4, but not GLUT12, protein content was decreased in RA during chronic HF (P = 0.001). In conclusion, GLUT4 protein was differentially expressed across the chambers in the healthy heart, and this regional pattern was reversed during HF. Our data suggest that LV was the primary site dependent on both GLUT4 and GLUT12 during chronic HF. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF. PMID:21849635

  15. Galanin antagonist increases insulin resistance by reducing glucose transporter 4 effect in adipocytes of rats.

    PubMed

    Guo, Lili; Shi, Mingyi; Zhang, Ling; Li, Guangzhi; Zhang, Lingxiang; Shao, Hu; Fang, Penghua; Ma, Yingping; Li, Jian; Shi, Qiaojia; Sui, Yumei

    2011-08-01

    Seeing that galanin increases animal body weight on the conditions of inhibiting insulin secretion and animals with metabolic disorder of galanin easily suffer from diabetes, we postulate that endogenous galanin is necessary to reduce insulin resistance in adipocytes. To test this hypothesis, we compared four groups of rats to examine whether an increase in galanin secretion stimulated by swimming may reduce insulin resistance. The rats from sedentary and trained drug groups were injected by M35, a galanin antagonist. The rats from trained control and trained drug groups swam after each injection for four weeks. We found that exercise significantly elevated plasma galanin contents and glucose transporter 4 (GLUT4) mRNA levels in adipocytes. Meanwhile, M35 treatment reduced GLUT4 and GLUT4 mRNA levels, and glucose infusing rates in euglycemic-hyperinsulinemic clamp tests. The ratios of GLUT4 concentrations at plasma membranes to total cell membranes in both drug groups were lower compared with each control group, respectively. These observations suggest that endogenous galanin reduces insulin resistance by increasing GLUT4 contents and promoting GLUT4 transportation from intracellular membranes to plasma membranes in adipocytes. Galanin is an important hormone to reduce insulin resistance in rats. PMID:21664358

  16. Expression and Regulation of Facilitative Glucose Transporters in Equine Insulin-Sensitive Tissue: From Physiology to Pathology

    PubMed Central

    Lacombe, Véronique A.

    2014-01-01

    Glucose uptake is the rate-limiting step in glucose utilization in mammalians and is tightly regulated by a family of specialized proteins, called the facilitated glucose transporters (GLUTs/SLC2). GLUT4, the major isoform in insulin-responsive tissue, translocates from an intracellular pool to the cell surface and as such determines insulin-stimulated glucose uptake. However, despite intensive research over 50 years, the insulin-dependent and -independent pathways that mediate GLUT4 translocation are not fully elucidated in any species. Insulin resistance (IR) is one of the hallmarks of equine metabolic syndrome and is the most common metabolic predisposition for laminitis in horses. IR is characterized by the impaired ability of insulin to stimulate glucose disposal into insulin-sensitive tissues. Similar to other species, the functional capability of the insulin-responsive GLUTs is impaired in muscle and adipose tissue during IR in horses. However, the molecular mechanisms of altered glucose transport remain elusive in all species, and there is still much to learn about the physiological and pathophysiological functions of the GLUT family members, especially in regard to class III. Since GLUTs are key regulators of whole-body glucose homeostasis, they have received considerable attention as potential therapeutic targets to treat metabolic disorders in human and equine patients. PMID:24977043

  17. Expression and regulation of facilitative glucose transporters in equine insulin-sensitive tissue: from physiology to pathology.

    PubMed

    Lacombe, Véronique A

    2014-01-01

    Glucose uptake is the rate-limiting step in glucose utilization in mammalians and is tightly regulated by a family of specialized proteins, called the facilitated glucose transporters (GLUTs/SLC2). GLUT4, the major isoform in insulin-responsive tissue, translocates from an intracellular pool to the cell surface and as such determines insulin-stimulated glucose uptake. However, despite intensive research over 50 years, the insulin-dependent and -independent pathways that mediate GLUT4 translocation are not fully elucidated in any species. Insulin resistance (IR) is one of the hallmarks of equine metabolic syndrome and is the most common metabolic predisposition for laminitis in horses. IR is characterized by the impaired ability of insulin to stimulate glucose disposal into insulin-sensitive tissues. Similar to other species, the functional capability of the insulin-responsive GLUTs is impaired in muscle and adipose tissue during IR in horses. However, the molecular mechanisms of altered glucose transport remain elusive in all species, and there is still much to learn about the physiological and pathophysiological functions of the GLUT family members, especially in regard to class III. Since GLUTs are key regulators of whole-body glucose homeostasis, they have received considerable attention as potential therapeutic targets to treat metabolic disorders in human and equine patients. PMID:24977043

  18. Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine.

    PubMed

    Jung, Jin Hyuk; Iwabuchi, Kumiko; Yang, Zhihong; Loeken, Mary R

    2016-01-01

    The hexose transporter, GLUT2 (SLC2A2), which is expressed by mouse embryos, is important for survival before embryonic day 10.5, but its function in embryos is unknown. GLUT2 can transport the amino sugar glucosamine (GlcN), which could increase substrate for the hexosamine biosynthetic pathway (HBSP) that produces UDP-N-acetylglucosamine for O-linked N-acetylglucosamine modification (O-GlcNAcylation) of proteins. To understand this, we employed a novel murine embryonic stem cell (ESC) line that, like mouse embryos, expresses functional GLUT2 transporters. GlcN stimulated ESC proliferation in a GLUT2-dependent fashion but did not regulate pluripotency. Stimulation of proliferation was not due to increased O-GlcNAcylation. Instead, GlcN decreased dependence of the HBSP on fructose-6-PO4 and glutamine. Consequently, glycolytic- and glutamine-derived intermediates that are needed for anabolic metabolism were increased. Thus, maternally obtained GlcN may increase substrates for biomass accumulation by embryos, as exogenous GlcN does for GLUT2-expressing ESC, and may explain the need for GLUT2 expression by embryos. PMID:27311888

  19. Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine

    PubMed Central

    Jung, Jin Hyuk; Iwabuchi, Kumiko; Yang, Zhihong; Loeken, Mary R.

    2016-01-01

    The hexose transporter, GLUT2 (SLC2A2), which is expressed by mouse embryos, is important for survival before embryonic day 10.5, but its function in embryos is unknown. GLUT2 can transport the amino sugar glucosamine (GlcN), which could increase substrate for the hexosamine biosynthetic pathway (HBSP) that produces UDP-N-acetylglucosamine for O-linked N-acetylglucosamine modification (O-GlcNAcylation) of proteins. To understand this, we employed a novel murine embryonic stem cell (ESC) line that, like mouse embryos, expresses functional GLUT2 transporters. GlcN stimulated ESC proliferation in a GLUT2-dependent fashion but did not regulate pluripotency. Stimulation of proliferation was not due to increased O-GlcNAcylation. Instead, GlcN decreased dependence of the HBSP on fructose-6-PO4 and glutamine. Consequently, glycolytic- and glutamine-derived intermediates that are needed for anabolic metabolism were increased. Thus, maternally obtained GlcN may increase substrates for biomass accumulation by embryos, as exogenous GlcN does for GLUT2-expressing ESC, and may explain the need for GLUT2 expression by embryos. PMID:27311888

  20. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    PubMed

    Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S

    2013-04-16

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle. PMID:23517233

  1. The Structure of the Metal Transporter Tp34 and its Affinity for Divalent Metal Ions

    NASA Astrophysics Data System (ADS)

    Knutsen, Gregory; Deka, Ranjit; Brautigam, Chad; Tomchick, Diana; Machius, Mischa; Norgard, Michael

    2007-10-01

    Tp34 is periplasmic membrane protein of the nonculitvatable spirochete Treponema pallidum, the pathogen of syphillis. It was proposed that Tp34 is a divalent metal transporter, but the identity of the preferred metal ion(s) was unclear. In this study we investigated the ability of divalent metal ions to induce rTp34 dimerization using hydrodynamic techniques and determine the crystal structure of metal bound forms. Using analytical ultracentrifugation sedimentation velocity experiments, we determined that cobalt is superior to nickel at inducing the dimerization of rTp34. rTp34 was crystallized and selected crystals were incubated at a pH 7.5 with CuSO4 and NiSO4. Diffraction experiments were conducted and the processed electron density maps showed that copper was bound to the major metal binding site as well as to three additional minor binding sites. By contrast nickel was only bound to the major metal binding site in one monomer and to three additional minor sites. These results along with previous findings support evidence of Tp34 being involved with metal transport and/or iron utilization.

  2. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  3. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    PubMed Central

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  4. Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate.

    PubMed

    Li, Hsin-Fen; Knutson, Barbara L; Nokes, Sue E; Lynn, Bert C; Flythe, Michael D

    2012-02-01

    Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H(2)) are also produced. The effect of hydrogenase inhibitors (H(2), carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N(2) to H(2), acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H(2) or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD(+) from NADH by H(2), lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H(2) production for the conversion of synthetic gases to chemicals. PMID:22218768

  5. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

    PubMed Central

    Riskin, Arieh; Mond, Yehudit

    2015-01-01

    Background Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture. Methods Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. Results GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation. PMID:26886772

  6. Minireview: recent developments in the regulation of glucose transporter-4 traffic: new signals, locations, and partners.

    PubMed

    Ishiki, Manabu; Klip, Amira

    2005-12-01

    Glucose transporter (GLUT) 4 is the major glucose transporter of muscle and adipose cells, exquisitely regulated by insulin through posttranslational events. Twenty years after the seminal observations that GLUT4 levels rapidly rise at the plasma membrane (PM) and drop in endomembranes in response to an acute insulin challenge, we are still mapping the intracellular traffic of the transporter and the regulatory events that insulin unleashes. Newly synthesized GLUT4 enters an insulin-responsive compartment aided by GGA2 (an Arf-binding protein). In cultured adipocytes and myocytes, GLUT4 concentrates in a perinuclear pole through participation of microtubules and the EHD1 Eps15 homology domain-containing protein 1. In the absence of stimuli, GLUT4 distributes between recycling endosomes and the insulin-responsive compartment. A handful of proteins that bind to GLUT4 appear to regulate its half-life (e.g. Ubc9) and tethering within endomembranes (e.g. TUG). Insulin-derived signals promote not only GLUT4 mobilization toward the PM but also its traffic between endosomal compartments and internalization from the PM. Class IA phosphatidylinositol (PI) 3-kinase plays a pivotal role at several steps of GLUT4 mobilization. The PI 3-kinase --> atypical PKC and --> Akt/PKB --> AS160 signaling cascades are major regulators of GLUT4 exocytosis aided by small GTPases. At the cell periphery, GLUT4-containing vesicles tether, dock, and fuse with the PM assisted by the exocyst complex followed by engagement of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex [with vesicle-associated membrane protein (VAMP)2 as the vesicular (v)-SNARE and soluble NSF-attachment protein (SNAP)23 and syntaxin4 as target (t)-SNAREs] regulated by the accessory proteins Munc18c, Synip and Tomosyn. Vesicle tethering and fusion are regulated by insulin through input from class IA PI 3-kinase. PMID:16150904

  7. Glucose-6-phosphate transport activity in liver microsomes exposed to stilbene disulfonate derivatives

    SciTech Connect

    Countaway, J.L.; Arion, W.J.

    1986-05-01

    Glucose-6-P (G6P) hydrolysis by hepatic microsomes (MS) is mediated by a coupled system composed of the G6P transporter (T1), the enzyme (E) and a phosphate transporter (T2). Zoccoli et al. concluded that T1 is a 54 kDa protein based on a linear correlation of labeling by /sup 3/H-4,4'diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (/sup 3/H-H/sub 2/DIDS) and inhibition of system activity. The authors cannot support this conclusion: (1) in their hands the reaction of /sup 3/H-H/sub 2/DIDS with MS proteins is extremely nonspecific, and (2) the linear correlation must be between labeling and inhibition of T1 activity, because transport per se is not the absolute rate limiting step in hydrolysis by the system. Point 2 is readily demonstrated by examining the influence of the enzyme inhibitor, D-glucose, on the sensitivity of the system to inhibition by H/sub 2/DIDS. Studies of H/sub 2/DIDS inhibition of the system in MS from fasted and diabetic rats revealed that the observed inhibition constant for the system, K/sub i(S)/, is inversely proportional to the fraction of latent G6Pase activity (LF) seen before exposure to H/sub 2/DIDS, and K/sub i(S)/ x LF - K/sub i(T1)/, the inhibition constant for T1 activity. This relationship is derived from the equation 1/V/sub (S)/ - 1/V/sub (E)/ = 1/V/sub (T1)/, where V denotes the initial rates of S, E and T1, respectively. The latter equation can be used to calculate V/sub (T1)/ for any preparation of intact MS, and it predicts that labeling and inhibition of T1 will be linearly correlated with V/sub (T1)/ but not V/sub (S)/.

  8. Glucose-Induced Down Regulation of Thiamine Transporters in the Kidney Proximal Tubular Epithelium Produces Thiamine Insufficiency in Diabetes

    PubMed Central

    Larkin, James R.; Zhang, Fang; Godfrey, Lisa; Molostvov, Guerman; Zehnder, Daniel; Rabbani, Naila; Thornalley, Paul J.

    2012-01-01

    Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy. PMID:23285265

  9. Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis▿

    PubMed Central

    Bao, Wei-Guo; Guiard, Bernard; Fang, Zi-An; Donnini, Claudia; Gervais, Michel; Passos, Flavia M. Lopes; Ferrero, Iliana; Fukuhara, Hiroshi; Bolotin-Fukuhara, Monique

    2008-01-01

    The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. PMID:18806211

  10. Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

    PubMed

    Hopfer, U; Sigrist-Nelson, K; Ammann, E; Murer, H

    1976-12-01

    A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism. PMID:137908

  11. Functional role of glucose metabolism, osmotic stress, and sodium-glucose cotransporter isoform-mediated transport on Na+/H+ exchanger isoform 3 activity in the renal proximal tubule.

    PubMed

    Pessoa, Thaissa Dantas; Campos, Luciene Cristina Gastalho; Carraro-Lacroix, Luciene; Girardi, Adriana C C; Malnic, Gerhard

    2014-09-01

    Na(+)-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na(+)-H(+) exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule. PMID:24652792

  12. Phylogenetic analysis and tissue distribution of elasmobranch glucose transporters and their response to feeding.

    PubMed

    Deck, Courtney A; LeMoine, Christophe M R; Walsh, Patrick J

    2016-01-01

    Elasmobranch diets consist of high quantities of protein and lipids, but very low levels of carbohydrates including glucose. Reflecting this diet, most tissues use lipids and ketone bodies as their main metabolic fuel. However, the rectal gland has been shown to be dependent on glucose as a fuel, so we hypothesized that glucose transporters (GLUTs) would be present and upregulated in the gland during times of activation (e.g. following a meal). In this study, we searched for and identified putative class I GLUTs in three elasmobranchs and a holocephalan using transcriptomes, and used these to reconstruct a Bayesian phylogeny. We determined that each of the four species possessed three of the four class I GLUT sequences, but the identities of the isoforms present in each species differed between the elasmobranchs (GLUT1, 3 and 4) and the holocephalan (GLUT1, 2 and 3). We then used qPCR to measure mRNA levels of these GLUTs in the rectal gland, liver, intestine, and muscle of fed and starved spiny dogfish (Squalus suckleyi). The rectal gland data showed higher mRNA levels of GLUT4 in the starved relative to the fed fish. In the muscle, both GLUT1 and 4 were significantly elevated at 24 h post-feeding, as was the case for GLUT4 in the liver. In the intestine on the other hand, GLUT4 was significantly elevated by 6 h post-feeding, remaining elevated through 48 h. We suggest that GLUT4 has taken on the role of GLUT2 in elasmobranchs as the expression patterns observed in the liver and intestine are representative of GLUT2 in other vertebrates. PMID:26873951

  13. Diminished glucose transport and phosphorylation in Alzheimer`s disease determined by dynamic FDG-PET

    SciTech Connect

    Piert, M.; Koeppe, R.A.; Giordani, B.; Berent, S.; Kuhl, D.E.

    1996-02-01

    Using dynamic [{sup 18}F] fluorodeoxyglucose (FDG) and PET, kinetic rate constants that describe influx (K{sub 1}) and efflux (k{sub 2}) of FDG as well s phosphorylation (k{sub 3}) and dephosphorylation (k{sub 4}) were determined in patients with probable Alzheimer`s disease and similarly aged normal controls. The regional cerebral metabolic rate for glucose (CMR{sub glu}) was calculated from individually fitted rate constants in frontal, temporal, parietal and occipital cerebral cortex, caudate nucleus, putamen, thalamus and cerebellar cortex. Dynamic PET scans were obtained in normal controls (n = 10, mean age = 67) and Alzheimer`s disease patients (n = 8, mean age = 67) for 60 min following injection of 10 mCi of FDG. The Alzheimer`s disease group was characterized by decreases of the CMR{sub glu} ranging from 13.3% in the frontal to 40.9% in the parietal cortex, which achieved significance in all regions except the thalamus. K{sub 1} was significantly reduced in the parietal (p < 0.01) and temporal cortices (p < 0.005), temporal and occipital cortex, and in the putamen and cerebellum (p < 0.05). The rate constants k{sub 2} and k{sub 4} were unchanged in the Alzheimer`s disease group. These data suggest that hypometabolism in Alzheimer`s disease is related to reduced glucose phosphorylation activity as well as diminished glucose transport, particularly in the most metabolically affected areas of the brain, the parietal and temporal cortex. 60 refs., 2 figs., 2 tabs.

  14. Phylogenetic analysis and tissue distribution of elasmobranch glucose transporters and their response to feeding

    PubMed Central

    Deck, Courtney A.; LeMoine, Christophe M. R.; Walsh, Patrick J.

    2016-01-01

    ABSTRACT Elasmobranch diets consist of high quantities of protein and lipids, but very low levels of carbohydrates including glucose. Reflecting this diet, most tissues use lipids and ketone bodies as their main metabolic fuel. However, the rectal gland has been shown to be dependent on glucose as a fuel, so we hypothesized that glucose transporters (GLUTs) would be present and upregulated in the gland during times of activation (e.g. following a meal). In this study, we searched for and identified putative class I GLUTs in three elasmobranchs and a holocephalan using transcriptomes, and used these to reconstruct a Bayesian phylogeny. We determined that each of the four species possessed three of the four class I GLUT sequences, but the identities of the isoforms present in each species differed between the elasmobranchs (GLUT1, 3 and 4) and the holocephalan (GLUT1, 2 and 3). We then used qPCR to measure mRNA levels of these GLUTs in the rectal gland, liver, intestine, and muscle of fed and starved spiny dogfish (Squalus suckleyi). The rectal gland data showed higher mRNA levels of GLUT4 in the starved relative to the fed fish. In the muscle, both GLUT1 and 4 were significantly elevated at 24 h post-feeding, as was the case for GLUT4 in the liver. In the intestine on the other hand, GLUT4 was significantly elevated by 6 h post-feeding, remaining elevated through 48 h. We suggest that GLUT4 has taken on the role of GLUT2 in elasmobranchs as the expression patterns observed in the liver and intestine are representative of GLUT2 in other vertebrates. PMID:26873951

  15. Colloids in groundwater: Their mobilization, subsurface transport, and sorption affinity for toxic chemicals

    SciTech Connect

    Not Available

    1991-01-01

    During the initial project period, we have pursued several activities with the overall goal of characterizing the roles of colloid in groundwater. First, we have collected soil cores from a site where we have previously found large quantities of kaolinite colloids in the groundwater. We have intensely investigated these cores to test our hypothesis that the colloids have been mobilized as a result of iron oxide dissolution. Next, we have constructed a soil core system in our laboratory with which we are attempting to mimic the factors that we think are governing colloid transport in the subsurface. Finally, we have pursued the issue of how well organic chemicals bind to the kinds of colloids that we are seeing at field sites. Together, with our knowledge of colloid mobility, we anticipate that this sorption data will enable us to predict the influence of groundwater colloids on contaminant fates in the subsurface. Our progress in each of these activities is described in this report. 7 refs., 12 figs.

  16. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight.

    PubMed

    Singh, Awadhesh Kumar

    2015-01-01

    Sodium-glucose co-transporter-2 inhibitors (SGLT-2i) are newly approved class of oral anti-diabetic drugs, in the treatment of type 2 diabetes, which reduces blood glucose through glucouresis via the kidney, independent, and irrespective of available pancreatic beta-cells. Studies conducted across their clinical development program found, a modest reduction in glycated hemoglobin ranging from -0.5 to -0.8%, without any significant hypoglycemia. Moreover, head-to-head studies versus active comparators yielded comparable efficacy. Interestingly, weight and blood pressure reduction were additionally observed, which was not only consistent but significantly superior to active comparators, including metformin, sulfonylureas, and dipeptydylpeptide-4 inhibitors. Indeed, these additional properties makes this class a promising oral anti-diabetic drug. Surprisingly, a potentially fatal unwanted side effect of diabetic ketoacidosis has been noted with its widespread use, albeit rarely. Nevertheless, this has created a passé among the clinicians. This review is an attempt to pool those ketosis data emerging with SGLT-2i, and put a perspective on its implicated mechanism. PMID:26693421

  17. Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

    PubMed Central

    Lawrence, M S; Ho, D Y; Dash, R; Sapolsky, R M

    1995-01-01

    We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid. Images Fig. 2 Fig. 5 PMID:7638175

  18. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight

    PubMed Central

    Singh, Awadhesh Kumar

    2015-01-01

    Sodium-glucose co-transporter-2 inhibitors (SGLT-2i) are newly approved class of oral anti-diabetic drugs, in the treatment of type 2 diabetes, which reduces blood glucose through glucouresis via the kidney, independent, and irrespective of available pancreatic beta-cells. Studies conducted across their clinical development program found, a modest reduction in glycated hemoglobin ranging from −0.5 to −0.8%, without any significant hypoglycemia. Moreover, head-to-head studies versus active comparators yielded comparable efficacy. Interestingly, weight and blood pressure reduction were additionally observed, which was not only consistent but significantly superior to active comparators, including metformin, sulfonylureas, and dipeptydylpeptide-4 inhibitors. Indeed, these additional properties makes this class a promising oral anti-diabetic drug. Surprisingly, a potentially fatal unwanted side effect of diabetic ketoacidosis has been noted with its widespread use, albeit rarely. Nevertheless, this has created a passé among the clinicians. This review is an attempt to pool those ketosis data emerging with SGLT-2i, and put a perspective on its implicated mechanism. PMID:26693421

  19. Expression and clinical significance of glucose transporter-1 in pancreatic cancer

    PubMed Central

    LU, KAI; YANG, JIAN; LI, DE-CHUN; HE, SONG-BING; ZHU, DONG-MING; ZHANG, LI-FENG; ZHANG, XU; CHEN, XIAO-CHEN; ZHANG, BING; ZHOU, JIAN

    2016-01-01

    Increasing evidence has demonstrated that malignant cells exhibit increased glucose uptake, which facilitates survival and growth in a hypoxic environment. The glucose transporter-1 (GLUT-1) is overexpressed in a variety of malignant tumors. However, the association between GLUT-1 expression and clinicopathological factors, 18F-fluorodeoxyglucose uptake and tumor proliferation in pancreatic cancer has not been investigated to date. In the present study, the expression of GLUT-1 in 53 pancreatic cancer tissues was analyzed, which revealed that GLUT-1 was overexpressed in pancreatic tissue and correlated with poor prognosis and clinicopathological characteristics, including increased tumor size, clinical stage and lymph node metastasis, maximum standardized uptake value (SUVmax) and Ki-67 expression. The receiver operating characteristic curve analysis indicated that a cut-off SUVmax value of 4.830 was associated with optimal sensitivity (88%) and specificity (71.4%) for the detection of strong positive GLUT-1 expression. In addition, as the expression of GLUT-1 was found to correlate with Ki-67 expression, GLUT-1 may exhibit a significant effect on cell proliferation in pancreatic cancer. Overall, these findings indicate that GLUT-1 may represent a prognostic indicator, and a potential therapeutic target for pancreatic cancer. PMID:27347132

  20. Total Zinc Intake May Modify the Glucose-Raising Effect of a Zinc Transporter (SLC30A8) Variant

    PubMed Central

    Kanoni, Stavroula; Nettleton, Jennifer A.; Hivert, Marie-France; Ye, Zheng; van Rooij, Frank J.A.; Shungin, Dmitry; Sonestedt, Emily; Ngwa, Julius S.; Wojczynski, Mary K.; Lemaitre, Rozenn N.; Gustafsson, Stefan; Anderson, Jennifer S.; Tanaka, Toshiko; Hindy, George; Saylor, Georgia; Renstrom, Frida; Bennett, Amanda J.; van Duijn, Cornelia M.; Florez, Jose C.; Fox, Caroline S.; Hofman, Albert; Hoogeveen, Ron C.; Houston, Denise K.; Hu, Frank B.; Jacques, Paul F.; Johansson, Ingegerd; Lind, Lars; Liu, Yongmei; McKeown, Nicola; Ordovas, Jose; Pankow, James S.; Sijbrands, Eric J.G.; Syvänen, Ann-Christine; Uitterlinden, André G.; Yannakoulia, Mary; Zillikens, M. Carola; Wareham, Nick J.; Prokopenko, Inga; Bandinelli, Stefania; Forouhi, Nita G.; Cupples, L. Adrienne; Loos, Ruth J.; Hallmans, Goran; Dupuis, Josée; Langenberg, Claudia; Ferrucci, Luigi; Kritchevsky, Stephen B.; McCarthy, Mark I.; Ingelsson, Erik; Borecki, Ingrid B.; Witteman, Jacqueline C.M.; Orho-Melander, Marju; Siscovick, David S.; Meigs, James B.; Franks, Paul W.; Dedoussis, George V.

    2011-01-01

    OBJECTIVE Many genetic variants have been associated with glucose homeostasis and type 2 diabetes in genome-wide association studies. Zinc is an essential micronutrient that is important for β-cell function and glucose homeostasis. We tested the hypothesis that zinc intake could influence the glucose-raising effect of specific variants. RESEARCH DESIGN AND METHODS We conducted a 14-cohort meta-analysis to assess the interaction of 20 genetic variants known to be related to glycemic traits and zinc metabolism with dietary zinc intake (food sources) and a 5-cohort meta-analysis to assess the interaction with total zinc intake (food sources and supplements) on fasting glucose levels among individuals of European ancestry without diabetes. RESULTS We observed a significant association of total zinc intake with lower fasting glucose levels (β-coefficient ± SE per 1 mg/day of zinc intake: −0.0012 ± 0.0003 mmol/L, summary P value = 0.0003), while the association of dietary zinc intake was not significant. We identified a nominally significant interaction between total zinc intake and the SLC30A8 rs11558471 variant on fasting glucose levels (β-coefficient ± SE per A allele for 1 mg/day of greater total zinc intake: −0.0017 ± 0.0006 mmol/L, summary interaction P value = 0.005); this result suggests a stronger inverse association between total zinc intake and fasting glucose in individuals carrying the glucose-raising A allele compared with individuals who do not carry it. None of the other interaction tests were statistically significant. CONCLUSIONS Our results suggest that higher total zinc intake may attenuate the glucose-raising effect of the rs11558471 SLC30A8 (zinc transporter) variant. Our findings also support evidence for the association of higher total zinc intake with lower fasting glucose levels. PMID:21810599

  1. Zinc Finger Protein 407 (ZFP407) Regulates Insulin-stimulated Glucose Uptake and Glucose Transporter 4 (Glut4) mRNA*

    PubMed Central

    Buchner, David A.; Charrier, Alyssa; Srinivasan, Ethan; Wang, Li; Paulsen, Michelle T.; Ljungman, Mats; Bridges, Dave; Saltiel, Alan R.

    2015-01-01

    The glucose transporter GLUT4 facilitates insulin-stimulated glucose uptake in peripheral tissues including adipose, muscle, and heart. GLUT4 function is impaired in obesity and type 2 diabetes leading to hyperglycemia and an increased risk of cardiovascular disease and neuropathy. To better understand the regulation of GLUT4 function, a targeted siRNA screen was performed and led to the discovery that ZFP407 regulates insulin-stimulated glucose uptake in adipocytes. The decrease in insulin-stimulated glucose uptake due to ZFP407 deficiency was attributed to a reduction in GLUT4 mRNA and protein levels. The decrease in GLUT4 was due to both decreased transcription of Glut4 mRNA and decreased efficiency of Glut4 pre-mRNA splicing. Interestingly, ZFP407 coordinately regulated this decrease in transcription with an increase in the stability of Glut4 mRNA, resulting in opposing effects on steady-state Glut4 mRNA levels. More broadly, transcriptome analysis revealed that ZFP407 regulates many peroxisome proliferator-activated receptor (PPAR) γ target genes beyond Glut4. ZFP407 was required for the PPARγ agonist rosiglitazone to increase Glut4 expression, but was not sufficient to increase expression of a PPARγ target gene reporter construct. However, ZFP407 and PPARγ co-overexpression synergistically activated a PPARγ reporter construct beyond the level of PPARγ alone. Thus, ZFP407 may represent a new modulator of the PPARγ signaling pathway. PMID:25596527

  2. Zinc finger protein 407 (ZFP407) regulates insulin-stimulated glucose uptake and glucose transporter 4 (Glut4) mRNA.

    PubMed

    Buchner, David A; Charrier, Alyssa; Srinivasan, Ethan; Wang, Li; Paulsen, Michelle T; Ljungman, Mats; Bridges, Dave; Saltiel, Alan R

    2015-03-01

    The glucose transporter GLUT4 facilitates insulin-stimulated glucose uptake in peripheral tissues including adipose, muscle, and heart. GLUT4 function is impaired in obesity and type 2 diabetes leading to hyperglycemia and an increased risk of cardiovascular disease and neuropathy. To better understand the regulation of GLUT4 function, a targeted siRNA screen was performed and led to the discovery that ZFP407 regulates insulin-stimulated glucose uptake in adipocytes. The decrease in insulin-stimulated glucose uptake due to ZFP407 deficiency was attributed to a reduction in GLUT4 mRNA and protein levels. The decrease in GLUT4 was due to both decreased transcription of Glut4 mRNA and decreased efficiency of Glut4 pre-mRNA splicing. Interestingly, ZFP407 coordinately regulated this decrease in transcription with an increase in the stability of Glut4 mRNA, resulting in opposing effects on steady-state Glut4 mRNA levels. More broadly, transcriptome analysis revealed that ZFP407 regulates many peroxisome proliferator-activated receptor (PPAR) γ target genes beyond Glut4. ZFP407 was required for the PPARγ agonist rosiglitazone to increase Glut4 expression, but was not sufficient to increase expression of a PPARγ target gene reporter construct. However, ZFP407 and PPARγ co-overexpression synergistically activated a PPARγ reporter construct beyond the level of PPARγ alone. Thus, ZFP407 may represent a new modulator of the PPARγ signaling pathway. PMID:25596527

  3. Increased Rat Placental Fatty Acid, but Decreased Amino Acid and Glucose Transporters Potentially Modify Intrauterine Programming.

    PubMed

    Nüsken, Eva; Gellhaus, Alexandra; Kühnel, Elisabeth; Swoboda, Isabelle; Wohlfarth, Maria; Vohlen, Christina; Schneider, Holm; Dötsch, Jörg; Nüsken, Kai-Dietrich

    2016-07-01

    Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n=20 placentas; SOP, n=24; C, n=12), 24 h (LIG, n=28; SOP, n=20; C, n=12) and 72 h (LIG, n=20; SOP, n=20; C, n=24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. J. Cell. Biochem. 117: 1594-1603, 2016. © 2015 Wiley Periodicals, Inc. PMID:26590355

  4. Unusual phenotype of glucose transport protein type 1 deficiency syndrome: A case report and literature review

    PubMed Central

    Posar, Annio; Santucci, Margherita

    2014-01-01

    The glucose transport protein type 1 (GLUT1) deficit causes a chronic brain energy failure. The classic phenotype of GLUT1 deficiency syndrome is characterized by: Mild to severe motor delay and mental retardation; infantile-onset epilepsy; head growth deceleration; movement disorders (ataxia, dystonia, spasticity); and non-epileptic paroxysmal events (intermittent ataxia, periodic confusion, recurrent headaches). During last years the classic phenotype of this syndrome, as originally reported, has expanded. We report the atypical phenotype of a boy with GLUT1 deficiency syndrome, characterized by mild mental retardation and drug-resistant absence seizures with onset at the age of 6 years, without movement disorders nor decrease of head circumference. A prompt diagnosis of this disorder is mandatory since the ketogenic diet might represent an effective treatment. PMID:24891901

  5. Unusual phenotype of glucose transport protein type 1 deficiency syndrome: A case report and literature review.

    PubMed

    Posar, Annio; Santucci, Margherita

    2014-01-01

    The glucose transport protein type 1 (GLUT1) deficit causes a chronic brain energy failure. The classic phenotype of GLUT1 deficiency syndrome is characterized by: Mild to severe motor delay and mental retardation; infantile-onset epilepsy; head growth deceleration; movement disorders (ataxia, dystonia, spasticity); and non-epileptic paroxysmal events (intermittent ataxia, periodic confusion, recurrent headaches). During last years the classic phenotype of this syndrome, as originally reported, has expanded. We report the atypical phenotype of a boy with GLUT1 deficiency syndrome, characterized by mild mental retardation and drug-resistant absence seizures with onset at the age of 6 years, without movement disorders nor decrease of head circumference. A prompt diagnosis of this disorder is mandatory since the ketogenic diet might represent an effective treatment. PMID:24891901

  6. Interleukin-2 and concanavalin A upregulate a cat2 isoform encoding a high affinity L-arginine transporter in feline lymphocytes.

    PubMed Central

    Stevens, B R; Tellier, M; Harvey, W; Feldman, D H; Bosworth, J

    2000-01-01

    The immunological responses of activated lymphocytes are associated with increased nitric oxide (NO) biosynthesis. Studies in the literature have primarily approached control of NO by focusing on the regulation of the nitric oxide synthase (NOS) isoforms. However, the present study approaches the control of NO synthesis by addressing the regulation of L-arginine availability to lymphocytes via regulation of membrane transport. The guanidino nitrogen of L-arginine is the sole biosynthetic precursor of NO. We investigated cytokine and mitogen regulation of membrane L-arginine transporters for the first time in feline cells. Feline peripheral blood mononuclear cells were treated with interleukin-2 and concanavalin A, then alternatively spliced isoforms of L-arginine transporters known in other species were probed by RT-PCR, using various oligonucleotide primers that hybridized to several regions in common with the isoforms. Both high affinity and low affinity isoforms are encoded by mRNAs arising from mutually exclusive alternative splicing of the primary transcript. A region of 123 bp was obtained that encoded an extracellular polypeptide loop of 41 amino acids. The sequence of this region represented the high affinity L-arginine substrate binding site of a CAT2 transporter polypeptide isoform, but not the CAT2a isoform low affinity binding site. Neither of the inducible isoforms were constitutively expressed in unstimulated feline cells. This is the first report demonstrating that domestic cats possess the cat2 gene encoding an inducible L-arginine transporter, and, furthermore, that the high affinity isoform transcript is activated by interleukin-2 and concanavalin A in feline lymphocytes. Images Figure 1. Figure 3. PMID:10935886

  7. Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells

    PubMed Central

    Koch, Andreas; Lang, Sven Arke; Wild, Peter Johannes; Gantner, Susanne; Mahli, Abdo; Spanier, Gerrit; Berneburg, Mark; Müller, Martina; Bosserhoff, Anja Katrin; Hellerbrand, Claus

    2015-01-01

    The glucose transporter isoform 1 (GLUT1; SLC2A1) is a key rate-limiting factor in the transport of glucose into cancer cells. Enhanced GLUT1 expression and accelerated glycolysis have been found to promote aggressive growth in a range of tumor entities. However, it was unknown whether GLUT1 directly impacts metastasis. Here, we aimed at analyzing the expression and function of GLUT1 in malignant melanoma. Immunohistochemical analysis of 78 primary human melanomas on a tissue micro array showed that GLUT1 expression significantly correlated with the mitotic activity and a poor survival. To determine the functional role of GLUT1 in melanoma, we stably suppressed GLUT1 in the murine melanoma cell line B16 with shRNA. GLUT1 suppressed melanoma cells revealed significantly reduced proliferation, apoptosis resistance, migratory activity and matrix metalloproteinase 2 (MMP2) expression. In a syngeneic murine model of hepatic metastasis, GLUT1-suppressed cells formed significantly less metastases and showed increased apoptosis compared to metastases formed by control cells. Treatment of four different human melanoma cell lines with a pharmacological GLUT1 inhibitor caused a dose-dependent reduction of proliferation, apoptosis resistance, migratory activity and MMP2 expression. Analysis of MAPK signal pathways showed that GLUT1 inhibition significantly decreased JNK activation, which regulates a wide range of targets in the metastatic cascade. In summary, our study provides functional evidence that enhanced GLUT1 expression in melanoma cells favors their metastatic behavior. These findings specify GLUT1 as an attractive therapeutic target and prognostic marker for this highly aggressive tumor. PMID:26293674

  8. Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose-limited chemostat culture.

    PubMed

    Shah, Mihir V; van Mastrigt, Oscar; Heijnen, Joseph J; van Gulik, Walter M

    2016-04-01

    Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26683700

  9. A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport.

    PubMed

    Gual, Philippe; Shigematsu, Satoshi; Kanzaki, Makoto; Grémeaux, Thierry; Gonzalez, Teresa; Pessin, Jeffrey E; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2002-11-15

    Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein). We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgamma1, the p85 subunit of the phosphoinositide 3-kinase and Crk-II. It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins. We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake. Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process. Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport. In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains. These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake. PMID:12215429

  10. The yeast Aft2 transcription factor determines selenite toxicity by controlling the low affinity phosphate transport system.

    PubMed

    Pérez-Sampietro, María; Serra-Cardona, Albert; Canadell, David; Casas, Celia; Ariño, Joaquín; Herrero, Enrique

    2016-01-01

    The yeast Saccharomyces cerevisiae is employed as a model to study the cellular mechanisms of toxicity and defense against selenite, the most frequent environmental selenium form. We show that yeast cells lacking Aft2, a transcription factor that together with Aft1 regulates iron homeostasis, are highly sensitive to selenite but, in contrast to aft1 mutants, this is not rescued by iron supplementation. The absence of Aft2 strongly potentiates the transcriptional responses to selenite, particularly for DNA damage- and oxidative stress-responsive genes, and results in intracellular hyperaccumulation of selenium. Overexpression of PHO4, the transcriptional activator of the PHO regulon under low phosphate conditions, partially reverses sensitivity and hyperaccumulation of selenite in a way that requires the presence of Spl2, a Pho4-controlled protein responsible for post-transcriptional downregulation of the low-affinity phosphate transporters Pho87 and Pho90. SPL2 expression is strongly downregulated in aft2 cells, especially upon selenite treatment. Selenite hypersensitivity of aft2 cells is fully rescued by deletion of PHO90, suggesting a major role for Pho90 in selenite uptake. We propose that the absence of Aft2 leads to enhanced Pho90 function, involving both Spl2-dependent and independent events and resulting in selenite hyperaccumulation and toxicity. PMID:27618952

  11. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

    PubMed

    Miethke, Marcus; Monteferrante, Carmine G; Marahiel, Mohamed A; van Dijl, Jan Maarten

    2013-10-01

    Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states. PMID:23764491

  12. A high affinity kidney targeting by chitobionic acid-conjugated polysorbitol gene transporter alleviates unilateral ureteral obstruction in rats.

    PubMed

    Islam, Mohammad Ariful; Kim, Sanghwa; Firdous, Jannatul; Lee, Ah-Young; Hong, Seong-Ho; Seo, Min Kyeong; Park, Tae-Eun; Yun, Cheol-Heui; Choi, Yun-Jaie; Chae, Chanhee; Cho, Chong-Su; Cho, Myung-Haing

    2016-09-01

    Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P < 0.001) after systemic tail-vein injection and significantly reduced the UUO symptoms, returning the UUO rats to a normal health status. The kidney-targeted CBA-PSGT-delivered HGF also strikingly reduced various pathologic and molecular markers in vivo such as the level of collagens (type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting. PMID:27318934

  13. GintAMT3 – a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis

    PubMed Central

    Calabrese, Silvia; Pérez-Tienda, Jacob; Ellerbeck, Matthias; Arnould, Christine; Chatagnier, Odile; Boller, Thomas; Schüßler, Arthur; Brachmann, Andreas; Wipf, Daniel; Ferrol, Nuria; Courty, Pierre-Emmanuel

    2016-01-01

    Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a Vmax of 240 nmol-1 min-1 108 cells-1, which is regulated by substrate concentration and carbon supply. PMID:27252708

  14. Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli.

    PubMed Central

    Wengender, P A; Miller, K J

    1995-01-01

    The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins. PMID:7887605

  15. Caffeamide 36-13 Regulates the Antidiabetic and Hypolipidemic Signs of High-Fat-Fed Mice on Glucose Transporter 4, AMPK Phosphorylation, and Regulated Hepatic Glucose Production

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2014-01-01

    This study was to investigate the antidiabetic and antihyperlipidemic effects of (E)-3-[3, 4-dihydroxyphenyl-1-(piperidin-1-yl)prop-2-en-1-one] (36-13) (TS), one of caffeic acid amide derivatives, on high-fat (HF-) fed mice. The C57BL/6J mice were randomly divided into the control (CON) group and the experimental group, which was firstly fed a HF diet for 8 weeks. Then, the HF group was subdivided into four groups and was given TS orally (including two doses) or rosiglitazone (Rosi) or vehicle for 4 weeks. Blood, skeletal muscle, and tissues were examined by measuring glycaemia and dyslipidemia-associated events. TS effectively prevented HF diet-induced increases in the levels of blood glucose, triglyceride, insulin, leptin, and free fatty acid (FFA) and weights of visceral fa; moreover, adipocytes in the visceral depots showed a reduction in size. TS treatment significantly increased the protein contents of glucose transporter 4 (GLUT4) in skeletal muscle; TS also significantly enhanced Akt phosphorylation in liver, whereas it reduced the expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Moreover, TS enhanced phosphorylation of AMP-activated protein kinase (phospho-AMPK) both in skeletal muscle and liver tissue. Therefore, it is possible that the activation of AMPK by TS resulted in enhanced glucose uptake in skeletal muscle, contrasting with diminished gluconeogenesis in liver. TS exhibits hypolipidemic effect by decreasing the expressions of fatty acid synthase (FAS). Thus, antidiabetic properties of TS occurred as a result of decreased hepatic glucose production by PEPCK and G6Pase downregulation and improved insulin sensitization. Thus, amelioration of diabetic and dyslipidemic state by TS in HF-fed mice occurred by regulation of GLUT4, G6Pase, and FAS and phosphorylation of AMPK. PMID:25140189

  16. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1.

    PubMed

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  17. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1

    PubMed Central

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  18. Microscale enzymatic optical biosensors using mass transport limiting nanofilms. 1. Fabrication and characterization using glucose as a model analyte.

    PubMed

    Stein, Erich W; Grant, Patrick S; Zhu, Huiguang; McShane, Michael J

    2007-02-15

    "Smart tattoo" sensors-fluorescent microspheres that can be implanted intradermally and interrogated noninvasively using light-are being developed as potential tools for in vivo biochemical monitoring. In this work, a platform for enzymatic tattoo-type sensors is described and prototype devices evaluated using glucose as a model analyte. Sensor particles were prepared by immobilizing Pt(II) octaethylporphine (PtOEP), a phosphorescent dye readily quenched by molecular oxygen, into hybrid silicate microspheres, followed by loading and subsequent covalent immobilization of glucose oxidase. Rhodamine B-doped multilayer nanofilms were subsequently assembled on the surfaces of the particles to provide a reference signal and provide critical control of glucose transport into the particle. The enzymatic oxidation of glucose within the sensor results in the glucose concentration-dependent depletion of local oxygen levels, enabling indirect monitoring of glucose by measuring relative changes in PtOEP emission. A custom testing apparatus was used to monitor the dynamic sensor response to varying bulk oxygen and glucose levels, respectively. For the prototypes tested, dynamic test results indicate that the sensors respond rapidly (t(95) = 84 s) and reversibly to changes in bulk glucose levels, while demonstrating high baseline stability. The sensitivity (change in intensity ratio) of these devices was determined to be 4.16 +/- 0.57%/mg dL(-1). The analytical range for the prototypes was determined to be 2-120 mg/dl, though this can be extended to cover the physiologically relevant range by tailoring the nanofilm coatings. These findings confirm the potential for enzymatic microscale optical and pave the way for extension of this initial demonstration with glucose to target other biochemical species relevant to metabolic monitoring. PMID:17297932

  19. Microscale Enzymatic Optical Biosensors using Mass-Transport Limiting Nanofilms. 1. Fabrication and Characterization Using Glucose as a Model Analyte

    PubMed Central

    Stein, Erich W.; Grant, Patrick S.; Zhu, Huiguang; McShane, Michael J.

    2008-01-01

    “Smart tattoo” sensors – fluorescent microspheres which can be implanted intradermally and interrogated noninvasively using light – are being developed as potential tools for in vivo biochemical monitoring. In this work, a platform for enzymatic tattoo-type sensors is described, and prototype devices evaluated using glucose as a model analyte. Sensor particles were prepared by immobilizing Pt(II) octaethylporphine (PtOEP), a phosphorescent dye readily quenched by molecular oxygen, into hybrid silicate microspheres, followed by loading and subsequent covalent immobilization of glucose oxidase (GOx). Rhodamine B (RITC)-doped multilayer nanofilms were subsequently assembled on the surfaces of the particles to provide a reference signal and provide critical control of glucose transport into the particle. The enzymatic oxidation of glucose within the sensor results in the glucose concentration-dependent depletion of local oxygen levels, enabling indirect monitoring of glucose by measuring relative changes in PtOEP emission. A custom testing apparatus was used to monitor the dynamic sensor response to varying bulk oxygen and glucose levels, respectively. For the prototypes tested, dynamic test results indicate that the sensors respond rapidly (t95 = 84 sec) and reversibly to changes in bulk glucose levels, while demonstrating high baseline stability. The sensitivity (change in intensity ratio) of these devices was determined to be 4.16 ± 0.57 %/mg dL−1. The analytical range for the prototypes was determined to be 2 to 120 mg/dl, though this can be extended to cover the physiologically relevant range by tailoring the nanofilm coatings. These findings confirm the potential for enzymatic microscale optical, and pave the way for extension of this initial demonstration with glucose to target other biochemical species relevant to metabolic monitoring. PMID:17297932

  20. In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

    PubMed

    Mishra, Rama K; Wei, Changyong; Hresko, Richard C; Bajpai, Richa; Heitmeier, Monique; Matulis, Shannon M; Nooka, Ajay K; Rosen, Steven T; Hruz, Paul W; Schiltz, Gary E; Shanmugam, Mala

    2015-06-01

    Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics. PMID:25847249

  1. In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy*

    PubMed Central

    Mishra, Rama K.; Wei, Changyong; Hresko, Richard C.; Bajpai, Richa; Heitmeier, Monique; Matulis, Shannon M.; Nooka, Ajay K.; Rosen, Steven T.; Hruz, Paul W.; Schiltz, Gary E.; Shanmugam, Mala

    2015-01-01

    Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics. PMID:25847249

  2. SKELETAL MUSCLE SODIUM GLUCOSE CO-TRANSPORTERS IN OLDER ADULTS WITH TYPE 2 DIABETES UNDERGOING RESISTANCE TRAINING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined the expression of the sodium-dependent glucose co-transporter system (SGLT3) in skeletal muscle of Hispanic older adults with type 2 diabetes. Subjects (65+/-8 yr) were randomized to resistance training (3x/wk, n=13) or standard of care (controls, n=5) for 16 weeks. Skeletal muscle SGL...

  3. Aqueous extract of tamarind seeds selectively increases glucose transporter-2, glucose transporter-4, and islets' intracellular calcium levels and stimulates β-cell proliferation resulting in improved glucose homeostasis in rats with streptozotocin-induced diabetes mellitus.

    PubMed

    Sole, Sushant Shivdas; Srinivasan, B P

    2012-08-01

    Tamarindus indica Linn. has been in use for a long time in Asian food and traditional medicine for different diseases including diabetes and obesity. However, the molecular mechanisms of these effects have not been fully understood. In view of the multidimensional activity of tamarind seeds due to their having high levels of polyphenols and flavonoids, we hypothesized that the insulin mimetic effect of aqueous tamarind seed extract (TSE) might increase glucose uptake through improvement in the expression of genes of the glucose transporter (GLUT) family and sterol regulatory element-binding proteins (SREBP) 1c messenger RNA (mRNA) in the liver. Daily oral administration of TSE to streptozotocin (STZ)-induced (90 mg/kg intraperitoneally) type 2 diabetic male Wistar rats at different doses (120 and 240 mg/kg body weight) for 4 weeks showed positive correlation with intracellular calcium and insulin release in isolated islets of Langerhans. Tamarind seed extract supplementation significantly improved the GLUT-2 protein and SREBP-1c mRNA expression in the liver and GLUT-4 protein and mRNA expression in the skeletal muscles of diabetic rats. The elevated levels of serum nitric oxide (NO), glycosylated hemoglobin level (hemoglobin (A1c)) and tumor necrosis factor α (TNF-α) decreased after TSE administration. Immunohistochemical findings revealed that TSE abrogated STZ-induced apoptosis and increased β-cell neogenesis, indicating its effect on islets and β-cell mass. In conclusion, it was found that the antidiabetic effect of TSE on STZ-induced diabetes resulted from complex mechanisms of β-cell neogenesis, calcium handling, GLUT-2, GLUT-4, and SREBP-1c. These findings show the scope for formulating a new herbal drug for diabetes therapy. PMID:22935346

  4. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

    PubMed Central

    Agbanyo, F R; Vijayalakshmi, D; Craik, J D; Gati, W P; McAdam, D P; Asakura, J; Robins, M J; Paterson, A R; Cass, C E

    1990-01-01

    Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups. Images Fig. 5. Fig. 6. Fig. 7. PMID:2241896

  5. Effects of insulin and epinephrine on Na/sup +/-K/sup +/ and glucose transport in soleus muscle

    SciTech Connect

    Clausen, T.; Flatman, J.A.

    1987-04-01

    To identify possible cause-effect relationships between changes in active Na/sup +/-K/sup +/ transport, resting membrane potential, and glucose transport, the effects of insulin and epinephrine were compared in rat soleus muscle. Epinephrine, which produced twice as large a hyperpolarization as insulin, induced only a modest increase in /sup 14/C-labeled sugar transport. Ouabain, at a concentration (10/sup -3/ M) sufficient to block active Na/sup +/-K/sup +/ transport and the hyperpolarization induced by the two hormones, did not interfere with sugar transport stimulation. After Na/sup +/ loading in K/sup +/-free buffer, the return to K/sup +/-containing standard buffer caused marked stimulation of active /sup 22/Na/sup +/-/sup 42/K/sup +/ transport, twice the hyperpolarization produced by insulin but no change in sugar transport. The insulin-induced activation of the /sup 22/Na/sup +/-/sup 42/K/sup +/ pump leads to decreased intracellular /sup 22/Na/sup +/ concentration and hyperpolarization, but none of these events can account for the concomitant activation of the glucose transport system. The stimulating effect of insulin on active Na/sup +/-K/sup +/ transport was not suppressed by amiloride, indicating that in intact skeletal muscle it is not elicited by a primary increase in Na/sup +/ influx via the Na/sup +//H/sup +/-exchange system.

  6. Synthesis and Development of Poly(N-Hydroxyethyl Acrylamide)-Ran-3-Acrylamidophenylboronic Acid Polymer Fluid for Potential Application in Affinity Sensing of Glucose

    PubMed Central

    Li, Siqi; Davis, Erin N; Huang, Xian; Song, Bing; Peltzman, Rebecca; Sims, David M; Lin, Qiao; Wang, Qian

    2011-01-01

    Background In previous work, we described viscosity and permittivity microelectromechanical systems (MEMS) sensors for continuous glucose monitoring (CGM) using poly[acrylamide-ran-3-acrylamidophenylboronic acid (PAA-ran-PAAPBA). In order to enhance our MEMS device antifouling properties, a novel, more hydrophilic polymer-sensing fluid was developed. Method To optimize sensing performance, we synthesized biocompatible copolymers poly(N-hydroxyethyl acrylamide)-ran-3-acrylamidophenylboronic acid (PHEAA-ran-PAAPBA) and developed its sensing fluid for viscosity-based glucose sensing. Key factors such as polymer composition and molecular weight were investigated in order to optimize viscometric responses. Results Compared with PAA-ran-PAAPBA fluid of a similar binding moiety percentage, PHEAA-ran-PAAPBA showed comparable high binding specificity to glucose in a reversible manner and even better performance in glucose sensing in terms of glucose sensing range (27–468 mg/ml) and sensitivity (within 3% standard error of estimate). Preliminary experiment on a MEMS viscometer demonstrated that the polymer fluid was able to sense the glucose concentration. Conclusions Our MEMS systems using PHEAA-ran-PAAPBA will possess enhanced implantable traits necessary to enable CGM in subcutaneous tissues. PMID:22027298

  7. Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate

    PubMed Central

    Hackmann, Timothy J.; Firkins, Jeffrey L.

    2015-01-01

    From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio sp.), we have re-evaluated the contribution of electron transport phosphorylation (ETP) to ATP formation in this group. This group is unique in that most (76%) genomes were predicted to possess genes for both Ech and Rnf transmembrane ion pumps. These pumps act in concert with the NifJ and Bcd-Etf to form a electrochemical potential (ΔμH+ and ΔμNa+), which drives ATP synthesis by ETP. Of the 62 total butyrivibrio genomes currently available from the Hungate 1000 project, all 62 were predicted to possess NifJ, which reduces oxidized ferredoxin (Fdox) during pyruvate conversion to acetyl-CoA. All 62 possessed all subunits of Bcd-Etf, which reduces Fdox and oxidizes reduced NAD during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH+ or ΔμNa+ from oxidation of reduced Fd (Fdred) and reduction of oxidized NAD. Further, 47 genomes possessed all six subunits of the Ech, which generates ΔμH+ from oxidation of Fdred. For glucose fermentation to butyrate and H2, the electrochemical potential established should drive synthesis of ∼1.5 ATP by the F0F1-ATP synthase (possessed by all 62 genomes). The total yield is ∼4.5 ATP/glucose after accounting for three ATP formed by classic substrate-level phosphorylation, and it is one the highest yields for any glucose fermentation. The yield was the same when unsaturated fatty acid bonds, not H+, served as the electron acceptor (as during biohydrogenation). Possession of both Ech and Rnf had been previously documented in only a few sulfate-reducers, was rare in other rumen prokaryotic genomes in our analysis, and may confer an energetic advantage to rumen butyrivibrios. This unique energy conservation system might enhance the butyrivibrios’ ability to overcome growth inhibition by unsaturated fatty acids, as postulated herein. PMID:26157432

  8. The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

    PubMed Central

    Qiao, Dan; Nikitina, Lyudmila A; Buznikov, Gennady A; Lauder, Jean M; Seidler, Frederic J; Slotkin, Theodore A

    2003-01-01

    Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants. PMID:14594623

  9. Psy2 Targets the PP4 Family Phosphatase Pph3 To Dephosphorylate Mth1 and Repress Glucose Transporter Gene Expression

    PubMed Central

    Ma, Hui; Han, Bong-Kwan; Guaderrama, Marisela; Aslanian, Aaron; Yates, John R.; Hunter, Tony

    2014-01-01

    The reversible nature of protein phosphorylation dictates that any protein kinase activity must be counteracted by protein phosphatase activity. How phosphatases target specific phosphoprotein substrates and reverse the action of kinases, however, is poorly understood in a biological context. We address this question by elucidating a novel function of the conserved PP4 family phosphatase Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity is required for the timely dephosphorylation of the downstream transcriptional repressor Rgt1 upon glucose withdrawal, a critical event in the repression of HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1, an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein kinase activated in response to glucose, in vitro and regulates Mth1 function via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2 phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene expression by glucose. PMID:24277933

  10. Psy2 targets the PP4 family phosphatase Pph3 to dephosphorylate Mth1 and repress glucose transporter gene expression.

    PubMed

    Ma, Hui; Han, Bong-Kwan; Guaderrama, Marisela; Aslanian, Aaron; Yates, John R; Hunter, Tony; Wittenberg, Curt

    2014-02-01

    The reversible nature of protein phosphorylation dictates that any protein kinase activity must be counteracted by protein phosphatase activity. How phosphatases target specific phosphoprotein substrates and reverse the action of kinases, however, is poorly understood in a biological context. We address this question by elucidating a novel function of the conserved PP4 family phosphatase Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity is required for the timely dephosphorylation of the downstream transcriptional repressor Rgt1 upon glucose withdrawal, a critical event in the repression of HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1, an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein kinase activated in response to glucose, in vitro and regulates Mth1 function via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2 phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene expression by glucose. PMID:24277933

  11. [(18) F]-Fluorodeoxy-d-glucose uptake-positive seborrhoeic keratosis on positron emission tomography may result from high expression of glucose transporter.

    PubMed

    Kariya, T; Kato, Y; Kanzaki, A; Kanda, Y; Ohara, T; Tsuboi, R

    2016-07-01

    [(18) F]-Fluorodeoxy-d-glucose (FDG) positron emission tomography-computed tomography (PET-CT) is known to be highly accurate in differentiating benign lesions from malignant lesions. In rare cases, benign tumours, viral infections and sarcoidosis of the skin have been reported to show FDG uptake, but the mechanism remains unclear. Here we report the first documented case of seborrhoeic keratosis (SK) showing increased FDG uptake. FDG PET-CT can be used to detect enhanced glycolysis of tumour cells by measuring increased levels of glucose transporters (GLUTs) indicative of higher glucose uptake. GLUT1 and GLUT3 expression in this case was compared with that in PET-negative SK and two normal skin samples using quantitative polymerase chain reaction with paraffin-embedded tissue. The expression of GLUT1 and GLUT3 was higher in PET-positive SK than in PET-negative SK or normal skin. More specifically, the expression of GLUT3 was observed only in the PET-positive case. This study revealed that high GLUT1 and GLUT3 expression in SK might be associated with the uptake of FDG. PMID:26801868

  12. Preventive effects of procyanidin A2 on glucose homeostasis, pancreatic and duodenal homebox 1, and glucose transporter 2 gene expression disturbance induced by bisphenol A in male mice.

    PubMed

    Ahangarpour, A; Afshari, G; Mard, S A; Khodadadi, A; Hashemitabar, M

    2016-04-01

    Procyanidins (PCs) as oligomeric compounds with antidiabetic properties formed from catechin and epicatechin molecules. Bisphenol A(BPA) is a common chemical material use in food and beverage packaging. The aim of this study was to explore the protective effects of procyanidin A2 (PCA2) against glucose homeostasis disturbance and gene expression of pancreatic and duodenal homebox 1 (Pdx1) as well as glucose transporter 2 (Glut2) induced by BPA in male mice. First tested these five concentrations of PCA2 (3 - 300 μM) alone and in combination with BPA(100 μg/L), on insulin secretion from isolated islets at in vitro condition. Next, examined the influence of BPA and PCA2 on islet apoptosis using flowcytometry. At in vivo condition, the BPA (100 μg/kg) and PCA2 (10 μmol/kg) administered for 20 days then, blood glucose and insulin, Pdx1 and, Glut2 genes expression, and oxidative stress markers examined. The results indicated that PCA2 strongly prevents islet cells apoptosis induced by BPA and, co-administration of PCA2 and BPA modified hyperglycemia. BPA reduced Pdx1 and Glut2 mRNA expression and antioxidant level in pancreas tissue, whereas PCA2 prevented from these effects. The findings from these studies suggest that use of PCA2 rich plants have preventive effects on hyperglycemia, and type 2 diabetes. PMID:27226184

  13. Catalytic amplification based on hole-transporting materials as efficient metal-free electrocatalysts for non-enzymatic glucose sensing.

    PubMed

    Gu, Yue; Yuan, Rongrong; Yan, Xiaoyi; Li, Cong; Liu, Weilu; Chen, Ruixue; Tang, Liu; Zheng, Bo; Li, Yaru; Zhang, Zhiquan; Yang, Ming

    2015-08-19

    Hole-transporting materials with tunable structures and properties are mainly applied in organic light-emitting diodes as transport layer. But their catalytic properties as signal amplifiers in biological assays are seldom reported. In this paper, a starburst molecule, 4,4,4″-tri(N-carbazolyl)-triphenylamine (TCT), containing a triphenylamine as the central core and three carbazoles as the peripheral functional groups was designed and synthesized. Subsequently, the hole-transporting material based on the TCT polymer, poly(TCT) (PTCT), was achieved via a low-cost electrochemical method and exploited as an efficient metal-free electrocatalyst for non-enzymatic glucose detection. Here, this hole-transporting material served three purposes: electrochemical recognition (owing to hydrogen bonding interaction and the biomimetic microenvironment created by the polymer), electrocatalysis (owing to the hole-transporting capability of triphenylamine and the catalytic property of carbazole), and signal amplification (owing to energy migration along the conductive polymer backbone). The electrocatalytic and sensing performances of the sensor based on PTCT were evaluated in detail. Results revealed that the PTCT film could efficiently catalyze the oxidation of glucose at a less-positive potential (+0.20 V) in the absence of any enzymes. The response to glucose was linear in the concentration range of 1.0-6000 μM, and the detection limit was 0.20 μM. With good stability and selectivity, the proposed sensor could be feasibly applied to detect glucose in practical samples. The encouraging sensing performances suggest that the hole-transporting material is one of the promising biomimetic catalysts for electrocatalysis and relevant fields. PMID:26343433

  14. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    PubMed

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP. PMID:26958868

  15. Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

    PubMed Central

    Coudert, Edouard; Pascal, Géraldine; Dupont, Joëlle; Simon, Jean; Cailleau-Audouin, Estelle; Crochet, Sabine; Duclos, Michel Jacques; Tesseraud, Sophie; Métayer-Coustard, Sonia

    2015-01-01

    In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals. PMID:26431526

  16. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

    PubMed Central

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min−1•mg−1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  17. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae.

    PubMed

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  18. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

    DOE PAGESBeta

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-01-19

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min-1•mg-1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

  19. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

    PubMed Central

    Munday, Jane C.; Eze, Anthonius A.; Baker, Nicola; Glover, Lucy; Clucas, Caroline; Aguinaga Andrés, David; Natto, Manal J.; Teka, Ibrahim A.; McDonald, Jennifer; Lee, Rebecca S.; Graf, Fabrice E.; Ludin, Philipp; Burchmore, Richard J. S.; Turner, C. Michael R.; Tait, Andy; MacLeod, Annette; Mäser, Pascal; Barrett, Michael P.; Horn, David; De Koning, Harry P.

    2014-01-01

    Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter. PMID:24235095

  20. Do Glut1 (glucose transporter type 1) defects exist in epilepsy patients responding to a ketogenic diet?

    PubMed

    Becker, Felicitas; Schubert, Julian; Weckhuysen, Sarah; Suls, Arvid; Grüninger, Steffen; Korn-Merker, Elisabeth; Hofmann-Peters, Anne; Sperner, Jürgen; Cross, Helen; Hallmann, Kerstin; Elger, Christian E; Kunz, Wolfram S; Madeleyen, René; Lerche, Holger; Weber, Yvonne G

    2015-08-01

    In the recent years, several neurological syndromes related to defects of the glucose transporter type 1 (Glut1) have been descried. They include the glucose transporter deficiency syndrome (Glut1-DS) as the most severe form, the paroxysmal exertion-induced dyskinesia (PED), a form of spastic paraparesis (CSE) as well as the childhood (CAE) and the early-onset absence epilepsy (EOAE). Glut1, encoded by the gene SLC2A1, is the most relevant glucose transporter in the brain. All Glut1 syndromes respond well to a ketogenic diet (KD) and most of the patients show a rapid seizure control. Ketogenic Diet developed to an established treatment for other forms of pharmaco-resistant epilepsies. Since we were interested in the question if those patients might have an underlying Glut1 defect, we sequenced SLC2A1 in a cohort of 28 patients with different forms of pharmaco-resistant epilepsies responding well to a KD. Unfortunately, we could not detect any mutations in SLC2A1. The exact action mechanisms of KD in pharmaco-resistant epilepsy are not well understood, but bypassing the Glut1 transporter seems not to play an important role. PMID:26088884

  1. High-affinity K(+) transport in Arabidopsis: AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.

    PubMed

    Pyo, Young Jae; Gierth, Markus; Schroeder, Julian I; Cho, Myeon Haeng

    2010-06-01

    Potassium (K(+)) is a major plant nutrient required for growth and development. It is generally accepted that plant roots absorb K(+) through uptake systems operating at low concentrations (high-affinity transport) and/or high external concentrations (low-affinity transport). To understand the molecular basis of high-affinity K(+) uptake in Arabidopsis (Arabidopsis thaliana), we analyzed loss-of-function mutants in AtHAK5 and AKT1, two transmembrane proteins active in roots. Compared with the wild type under NH(4)(+)-free growth conditions, athak5 mutant plants exhibited growth defects at 10 mum K(+), but at K(+) concentrations of 20 mum and above, athak5 mutants were visibly indistinguishable from the wild type. While germination, scored as radicle emergence, was only slightly decreased in athak5 akt1 double mutants on low-K(+) medium, double mutants failed to grow on medium containing up to 100 mum K(+) and growth was impaired at concentrations up to 450 mum K(+). Moreover, transfer of 3-d-old plants from high to low K(+) concentrations led to growth defects and leaf chlorosis at 10 mum K(+) in athak5 akt1 double mutant plants. Determination of Rb(+)(K(+)) uptake kinetics in wild-type and mutant roots using rubidium ((86)Rb(+)) as a tracer for K(+) revealed that high-affinity Rb(+)(K(+)) uptake into roots is almost completely abolished in double mutants and impaired in single mutants. These results strongly indicate that AtHAK5 and AKT1 are the two major, physiologically relevant molecular entities mediating high-affinity K(+) uptake into roots during seedling establishment and postgermination growth and that residual Rb(+)(K(+)) uptake measured in athak5 akt1 double mutant roots is insufficient to enable plant growth. PMID:20413648

  2. Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

    PubMed Central

    Maria, Zahra; Campolo, Allison R.; Lacombe, Veronique A.

    2015-01-01

    Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes. PMID:26720696

  3. Genipin stimulates glucose transport in C2C12 myotubes via an IRS-1 and calcium-dependent mechanism.

    PubMed

    Ma, Chan-Juan; Nie, Ai-Fang; Zhang, Zhi-Jian; Zhang, Zhi-Guo; Du, Li; Li, Xiao-Ying; Ning, Guang

    2013-03-01

    Genipin, a compound derived from Gardenia jasminoides Ellis fruits, has been used over the years in traditional Chinese medicine to treat symptoms of type 2 diabetes. However, the molecular basis for its antidiabetic effect has not been fully revealed. In this study, we investigated the effects of genipin on glucose uptake and signaling pathways in C(2)C(12) myotubes. Our study demonstrates that genipin stimulated glucose uptake in a time- and dose-dependent manner. The maximal effect was achieved at 2 h with a concentration of 10 μM. In myotubes, genipin promoted glucose transporter 4 (GLUT4) translocation to the cell surface, which was observed by analyzing their distribution in subcellular membrane fraction, and increased the phosphorylation of insulin receptor substrate-1 (IRS-1), AKT, and GSK3β. Meanwhile, genipin increased ATP levels, closed K(ATP) channels, and then increased the concentration of calcium in the cytoplasm in C(2)C(12) myotubes. Genipin-stimulated glucose uptake could be blocked by both the PI3-K inhibitor wortmannin and calcium chelator EGTA. Moreover, genipin increases the level of reactive oxygen species and ATP in C(2)C(12) myotubes. These results suggest that genipin activates IRS-1, PI3-K, and downstream signaling pathway and increases concentrations of calcium, resulting in GLUT4 translocation and glucose uptake increase in C(2)C(12) myotubes. PMID:23257267

  4. Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes

    SciTech Connect

    Harrison, S.A.; Buxton, J.M.; Czech, M.P. )

    1991-09-01

    Previous studies indicated that the erythroid-type (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labeling (with 3-({sup 125}I)iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. The authors conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.

  5. Further evidence for a two-step model of glucose-transport regulation. Inositol phosphate-oligosaccharides regulate glucose-carrier activity.

    PubMed Central

    Obermaier-Kusser, B; Mühlbacher, C; Mushack, J; Seffer, E; Ermel, B; Machicao, F; Schmidt, F; Häring, H U

    1989-01-01

    The insulin effect on glucose uptake is not sufficiently explained by a simple glucose-carrier translocation model. Recent studies rather suggest a two-step model of carrier translocation and carrier activation. We used several pharmacological tools to characterize the proposed model further. We found that inositol phosphate (IP)-oligosaccharides isolated from the drug Actovegin, as well as the alkaloid vinblastine, show a partial insulin-like effect on glucose-transport activity of fat-cells (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 5.8%, insulin 59%, IP-oligosaccharides 30%, vinblastine 29%) without inducing carrier translocation. On the other hand, two newly developed anti-diabetic compounds (alpha-activated carbonic acids, BM 130795 and BM 13907) induced carrier translocation to the same extent as insulin and phorbol esters [cytochalasin-B-binding sites in plasma membranes: basal 5 pmol/mg of protein, insulin 13 pmol/mg of protein, TPA (12-O-tetradecanoylphorbol 13-acetate) 11.8 pmol/mg of protein, BM 130795 10.8 pmol/mg of protein], but produce also only 40-50% of the insulin effect on glucose-transport activity (basal 5.8%, insulin 59%, TPA 23%, BM 130795 35%). Almost the full insulin effect was mimicked by a combination of phorbol esters and IP-oligosaccharides (basal 7%, insulin 50%, IP-oligosaccharides 30%, TPA 23%, IP-oligosaccharides + TPA 45%). None of these substances stimulated insulin-receptor kinase in vitro or in vivo, suggesting a post-kinase site of action. The data confirm the following aspects of the proposed model: (1) carrier translocation and carrier activation are two independently regulated processes; (2) the full insulin effect is mimicked only by a simultaneous stimulation of carrier translocation and intrinsic carrier activity, suggesting that insulin acts through a synergism of both mechanisms; (3) IP-oligosaccharides might be involved in the transmission of a stimulatory signal on carrier activity

  6. Effect of physiological stress on expression of glucose transporter 2 in liver of the wood frog, Rana sylvatica.

    PubMed

    Rosendale, Andrew J; Lee, Richard E; Costanzo, Jon P

    2014-12-01

    Glucose transporters (GLUTs) have been implicated in the survival of various physiological stresses in mammals; however, little is known about the role of these proteins in stress tolerance in lower vertebrates. The wood frog (Rana sylvatica), which survives multiple winter-related stresses by copiously mobilizing hepatic glycogen stores, is an interesting subject for the study of glucose transport in amphibians. We examined the effects of several physiological stresses on GLUT2 protein and mRNA levels in the liver of R. sylvatica. Using immunoblotting techniques to measure relative GLUT2 abundance, we found that GLUT2 numbers increased in response to organismal freezing, hypoxia exposure, and glucose loading; whereas, experimental dehydration and urea loading did not affect GLUT2 abundance. GLUT2 mRNA levels, assessed using quantitative real-time polymerase chain reaction, changed in accordance with protein abundance for most stresses, indicating that transcriptional regulation of GLUT2 occurs in response to stress. Overall, hepatic GLUT2 seems to be important in stress survival in R. sylvatica and is regulated to meet the physiological need to accumulate glucose. PMID:25384572

  7. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    PubMed Central

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  8. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    NASA Astrophysics Data System (ADS)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  9. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  10. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders

    SciTech Connect

    Smith, C.P.; Kanai, Y.; Stelzner, M.; Hediger, M.A.; Weremowicz, S.; Morton, C.C. )

    1994-03-15

    Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid. Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.

  11. Glucose transporter 3 is a rab11-dependent trafficking cargo and its transport to the cell surface is reduced in neurons of CAG140 Huntington's disease mice.

    PubMed

    McClory, Hollis; Williams, Dana; Sapp, Ellen; Gatune, Leah W; Wang, Ping; DiFiglia, Marian; Li, Xueyi

    2014-01-01

    Huntington's disease (HD) disturbs glucose metabolism in the brain by poorly understood mechanisms. HD neurons have defective glucose uptake, which is attenuated upon enhancing rab11 activity. Rab11 regulates numerous receptors and transporters trafficking onto cell surfaces; its diminished activity in HD cells affects the recycling of transferrin receptor and neuronal glutamate/cysteine transporter EAAC1. Glucose transporter 3 (Glut3) handles most glucose uptake in neurons. Here we investigated rab11 involvement in Glut3 trafficking. Glut3 was localized to rab11 positive puncta in primary neurons and immortalized striatal cells by immunofluorescence labeling and detected in rab11-enriched endosomes immuno-isolated from mouse brain by Western blot. Expression of dominant active and negative rab11 mutants in clonal striatal cells altered the levels of cell surface Glut3 suggesting a regulation by rab11. About 4% of total Glut3 occurred at the cell surface of primary WT neurons. HD(140Q/140Q) neurons had significantly less cell surface Glut3 than did WT neurons. Western blot analysis revealed comparable levels of Glut3 in the striatum and cortex of WT and HD(140Q/140Q) mice. However, brain slices immunolabeled with an antibody recognizing an extracellular epitope to Glut3 showed reduced surface expression of Glut3 in the striatum and cortex of HD(140Q/140Q) mice compared to that of WT mice. Surface labeling of GABAα1 receptor, which is not dependent on rab11, was not different between WT and HD(140Q/140Q) mouse brain slices. These data define Glut3 to be a rab11-dependent trafficking cargo and suggest that impaired Glut3 trafficking arising from rab11 dysfunction underlies the glucose hypometabolism observed in HD. PMID:25526803

  12. Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose

    PubMed Central

    Gopal, Shubha; Berg, Daniela; Hagen, Nicole; Schriefer, Eva-Maria; Stoll, Regina; Goebel, Werner; Kreft, Jürgen

    2010-01-01

    Background In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems. Methodology/Principal Findings We could show that Listeria monocytogenes and other Listeria species contain genes/gene products with high homology to the maltodextrin ABC transporter and utilization system of B. subtilis. Mutant construction and growth tests revealed that the L. monocytogenes gene cluster was required for the efficient utilization of maltodextrins as well as maltose. The gene for the ATP binding protein of the transporter was located distant from the cluster. Transcription analyses demonstrated that the system was induced by maltose/maltodextrins and repressed by glucose. Its induction was dependent on a LacI type transcriptional regulator. Repression by glucose was independent of the catabolite control protein CcpA, but was relieved in a mutant defective for Hpr kinase/phosphorylase. Conclusions/Significance The data obtained show that in L. monocytogenes the uptake of maltodextrin and, in contrast to B. subtilis, also maltose is exclusively mediated by an ABC transporter. Furthermore, the results suggest that glucose repression of the uptake system possibly is by inducer exclusion, a mechanism not described so far in this organism. PMID:20436965

  13. Salicylate acutely stimulates 5'-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles.

    PubMed

    Serizawa, Yasuhiro; Oshima, Rieko; Yoshida, Mitsuki; Sakon, Ichika; Kitani, Kazuto; Goto, Ayumi; Tsuda, Satoshi; Hayashi, Tatsuya

    2014-10-10

    Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5'-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr(172) phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-D-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL. PMID:25256746

  14. Identification of the glucose transporter in mammalian cell membranes using an /sup 125/(I)-forskolin photoaffinity label

    SciTech Connect

    Ruoho, A.; Wadzinski, B.; Shanahan, M.

    1987-05-01

    The glucose transporter has been identified in a variety of mammlian cell membranes using a carrier-free photoactivatable radioiodinated derivative of forskolin, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin, (I-125)IAPS-Fsk, at 1-10 nM. The membranes which have been photolabeled with (I-125)IAPS-Fsk are: rat cardiac sarcolemmal membranes, rat cortex and cerebellum synaptic membranes, human placental membranes, and wild type S49 lymphoma cell membranes. The glucose transporter in rat cardiac sarcolemmal membranes and rat cortex and cerebellum synaptic membranes was determined to be 45 kDa by SDS-PAGE. Photolysis of human placental membranes and S49 lymphoma membranes with (I-125)IAPS-Fsk followed by SDS-PAGE indicated specific derivatization of a broad band (45-55 kDa) in placental membranes and a narrower band (45 kDa) in the S49 lymphoma membranes. Digestion of the (I-125)IPAS-Fsk labelled placental and S49 lymphoma membranes with endo-B-galactosidase showed a reduction in the apparent molecular weight of the radiolabelled band to 40 kDa. Trypsinization of labelled placental and lymphoma membranes produced an 18 kDa radiolabelled proteolytic fragment. (I-125)IAPS-Fsk is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.

  15. Does apical membrane GLUT2 have a role in intestinal glucose uptake?

    PubMed Central

    Naftalin, Richard J

    2014-01-01

    It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border. The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route. As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface. These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations. PMID:25671087

  16. Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

    PubMed Central

    Rhoads, D B; Takano, M; Gattoni-Celli, S; Chen, C C; Isselbacher, K J

    1988-01-01

    The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment. Images PMID:3194405

  17. The Glucose Transporter (GLUT4) Enhancer Factor Is Required for Normal Wing Positioning in Drosophila

    PubMed Central

    Yazdani, Umar; Huang, Zhiyu; Terman, Jonathan R.

    2008-01-01

    Many of the transcription factors and target genes that pattern the developing adult remain unknown. In the present study, we find that an ortholog of the poorly understood transcription factor, glucose transporter (GLUT4) enhancer factor (Glut4EF, GEF) [also known as the Huntington's disease gene regulatory region-binding protein (HDBP) 1], plays a critical role in specifying normal wing positioning in adult Drosophila. Glut4EF proteins are zinc-finger transcription factors named for their ability to regulate expression of GLUT4 but nothing is known of Glut4EF's in vivo physiological functions. Here, we identify a family of Glut4EF proteins that are well conserved from Drosophila to humans and find that mutations in Drosophila Glut4EF underlie the wing-positioning defects seen in stretch mutants. In addition, our results indicate that previously uncharacterized mutations in Glut4EF are present in at least 11 publicly available fly lines and on the widely used TM3 balancer chromosome. These results indicate that previous observations utilizing these common stocks may be complicated by the presence of Glut4EF mutations. For example, our results indicate that Glut4EF mutations are also present on the same chromosome as two gain-of-function mutations of the homeobox transcription factor Antennapedia (Antp) and underlie defects previously attributed to Antp. In fact, our results support a role for Glut4EF in the modulation of morphogenetic processes mediated by Antp, further highlighting the importance of Glut4EF transcription factors in patterning and morphogenesis. PMID:18245850

  18. Immunoreactivity of glucose transporter 8 is localized in the epithelial cells of the choroid plexus and in ependymal cells.

    PubMed

    Murakami, Ryuta; Chiba, Yoichi; Tsuboi, Kazuhito; Matsumoto, Koichi; Kawauchi, Machi; Fujihara, Ryuji; Mashima, Masato; Kanenishi, Kenji; Yamamoto, Tetsuji; Ueno, Masaki

    2016-08-01

    High fructose intake is known to be associated with increased plasma triglyceride concentration, impaired glucose tolerance, insulin resistance, and high blood pressure. In addition, excess fructose intake is also thought to be a risk factor for dementia. Previous immunohistochemical studies have shown the presence of glucose transporter 5 (GLUT5), a major transporter of fructose, in the epithelial cells of the choroid plexus and ependymal cells in the brains of humans, rats, and mice, while GLUT2, a minor transporter of fructose, was localized in the ependymal cells of rat brain. In this study, immunoreactivity for the fructose transporter GLUT8 was observed in the cytoplasm of the epithelial cells in the choroid plexus and in the ependymal cells of the brains of humans and mice. These structures were not immunoreactive for GLUT7, GLUT11, and GLUT12. Our findings support the hypothesis of the transport of intravascular fructose through the epithelial cells of the choroid plexus and the ependymal cells. PMID:27160096

  19. Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

    NASA Astrophysics Data System (ADS)

    Li, Zhiming; Zhu, Guohui; Liu, Jiaming; Lu, Qiaomei; Yang, Minlan; Wu, Hong; Shi, Xiumei; Chen, Xinhua

    2007-08-01

    A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the Δ Ip of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot -1 for direct method and 0.18 fg spot -1 for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.

  20. GLUT2 (SLC2A2) is not the principal glucose transporter in human pancreatic beta cells: implications for understanding genetic association signals at this locus.

    PubMed

    McCulloch, Laura J; van de Bunt, Martijn; Braun, Matthias; Frayn, Keith N; Clark, Anne; Gloyn, Anna L

    2011-12-01

    SLC2A2 encoding glucose transporter -2 (GLUT2) acts as the primary glucose transporter and sensor in rodent pancreatic islets and is widely assumed to play a similar role in humans. In healthy adults SLC2A2 variants are associated with elevated fasting plasma glucose (fpg) concentrations but physiological characterisation does not support a defect in pancreatic beta-cell function. Interspecies differences can create barriers for the follow up of disease association signals. We hypothesised that GLUT2 is not the principal glucose transporter in human beta-cells and that SLC2A2 variants exert their effect on fpg levels through defects in other tissues. SLC2A1-4 (GLUT 1-4) mRNA expression levels were determined in human and mouse islets, beta-cells, liver, muscle and adipose tissue by qRT-PCR whilst GLUT1-3 protein levels were examined by immunohistochemistry. The presence of all three glucose transporters was demonstrated in human and mouse islets and purified beta-cells. Quantitative expression profiling demonstrated that Slc2a2 is the predominant glucose transporter (expression >10 fold higher that Slc2a1) in mouse islets whilst SLC2A1 and SLC2A3 predominate in both human islets and beta-cells (expression 2.8 and 2.7 fold higher than SLC2A2 respectively). Our data therefore suggest that GLUT2 is unlikely to be the principal glucose transporter in human beta-cells and that SLC2A2 defects in other metabolic tissues drive the observed differences in glucose levels between carriers of SLC2A2 variants. Direct extrapolation from rodent to human islet glucose transporter activity is unlikely to be appropriate. PMID:21920790

  1. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake.

    PubMed Central

    Huang, N C; Liu, K H; Lo, H J; Tsay, Y F

    1999-01-01

    The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis. PMID:10449574

  2. Regulation of glucose transport and transporter 4 (GLUT-4) in muscle and adipocytes of sucrose-fed rats: effects of N-3 poly- and monounsaturated fatty acids.

    PubMed

    Peyron-Caso, E; Fluteau-Nadler, S; Kabir, M; Guerre-Millo, M; Quignard-Boulangé, A; Slama, G; Rizkalla, S W

    2002-07-01

    The goal of this study was to compare the short-term effects of dietary n-3 polyunsaturated (fish oil) and monounsaturated (olive oil) fatty acids on glucose transport, plasma glucose and lipid controls in a dietary insulin resistance model using sucrose-fed rats. The underlying cellular and molecular mechanisms were also determined in the muscle and adipose tissue. Male Sprague-Dawley rats (5 weeks old) were randomized for diets containing 57.5 % (w/w) sucrose and 14 % lipids as either fish oil (SF), olive oil (SO) or a mixture of standard oils (SC) for 3 weeks. A fourth control group (C) was fed a diet containing 57.5 % starch and 14 % standard oils. After three weeks on the diet, body weight was comparable in the four groups. The sucrose-fed rats were hyperglycemic and hyperinsulinemic in response to glucose load. The presence of fish oil in the sucrose diet prevented sucrose-induced hyperinsulinemia and hypertriglyceridemia, but had no effect on plasma glucose levels. Insulin-stimulated glucose transport in adipocytes increased after feeding with fish oil (p < 0.005). These modifications were associated with increased Glut-4 protein (p < 0.05) and mRNA levels in adipocytes. In the muscle, no effect was found on Glut-4 protein levels. Olive oil, however, could not bring about any improvement in plasma insulin, plasma lipids or Glut-4 protein levels. We therefore conclude that the presence of fish oil, in contrast to olive oil, prevents insulin resistance and hypertriglyceridemia in rats on a sucrose diet, and restores Glut-4 protein quantity in adipocytes but not in muscle at basal levels. Dietary regulation of Glut-4 proteins appears to be tissue specific and might depend on insulin stimulation and/or duration of dietary interventions. PMID:12189582

  3. Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action.

    PubMed Central

    Tsakiridis, T; Vranic, M; Klip, A

    1995-01-01

    In L6 myotubes insulin stimulates glucose transport through the translocation of glucose transporters GLUT1, GLUT3 and GLUT4 from intracellular stores to the plasma membrane. An intact actin network and phosphatidylinositol 3-kinase activity are required for this process. Glucose transport is also stimulated by the mitochondrial ATP-production uncoupler dinitrophenol. We show here that, in serum-depleted myotubes, dinitrophenol induced translocation of GLUT1 and GLUT4, but not GLUT3. This response was not affected by inhibiting phosphatidylinositol 3-kinase or disassembling the actin network. Insulin, but not dinitrophenol, caused tyrosine phosphorylation of several polypeptides, including the insulin-receptor substrate-1 and mitogen-activated protein kinase. Similarly, insulin, but not dinitrophenol, caused actin reorganization, which was inhibited by wortmannin. We conclude that insulin and dinitrophenol stimulate glucose transport by different mechanisms. Images Figure 2 Figure 3 Figure 4 PMID:7619042

  4. Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    Wang, Q; Bilan, P J; Tsakiridis, T; Hinek, A; Klip, A

    1998-01-01

    Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85alpha and p110beta (but not p110alpha) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85alpha and p110beta with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85-p110 PI 3-kinase with glucose-transporter-containing compartments. PMID:9560323

  5. Sodium-glucose co-transporter-2 inhibitors as add-on therapy to insulin: rationale and evidences.

    PubMed

    Singh, Awadhesh Kumar; Singh, Ritu

    2016-01-01

    Sodium-glucose co-transporter-2 inhibitors (SGLT-2I) are recently approved class of anti-hyperglycaemic agents for the treatment of type 2 diabetes mellitus (T2DM). SGLT-2I inhibits renal glucose reabsorption, thereby ensuing urinary glucose excretion in a dose-dependent manner. This caloric loss and osmotic diuresis, secondary to increased urinary glucose excretion, has a unique potential to counter insulin induced weight gain and fluid retention, with little potential of hypoglycemic exacerbation. Also, as these agents act independently of insulin secretion or action, they are effective even in long-standing diabetes with depleted β-cell reserve. Improvement in insulin sensitivity, as observed with SGLT-2I can also facilitate insulin action. Furthermore, significant reduction in total daily insulin dosage and reduction of body weight as observed during combination therapy renders SGLT-2I, a near-ideal partner to insulin. This review aims to evaluate the safety and efficacy of currently used SGLT-2I as an add-on to insulin therapy in the treatment of T2DM. PMID:26732230

  6. TGF-beta transcriptionally activates the gene encoding the high-affinity adenosine transporter CNT2 in rat liver parenchymal cells.

    PubMed

    Valdés, R; Fernández-Veledo, S; Aymerich, I; Casado, F J; Pastor-Anglada, M

    2006-11-01

    The nucleoside transporter CNT2 is the highest-affinity adenosine transporter identified so far. Recent evidence suggests that CNT2 has functions other than salvage (i.e. modulation of purinergic responses). Here we identified TGF-beta1 as a potent inducer of CNT2 protein expression in liver parenchymal cells. By contrast, CNT1, which is a target of multifunctional cytokines involved in liver cell proliferation, does not respond to TGF-beta1 treatment. Cloning of a murine CNT2 gene sequence with promoter-like activity enabled us to demonstrate that this cytokine exerts this effect by transcriptionally activating the CNT2-encoding gene in a JNK-dependent manner. The evidence that CNT2 is not a target of multifunctional cytokines involved in hepatocyte proliferation, but instead, of a cytokine that plays major roles in differentiation and apoptosis, further supports the view that the main physiological role of this transporter protein is not nucleoside salvage. PMID:17013559

  7. Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT).

    PubMed

    Hosford, Patrick S; Millar, Julian; Ramage, Andrew G

    2015-04-01

    The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12-50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg(-1) ). However, decynium-22 (600 μg kg(-1) ), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not noradrenaline, whereas desipramine had the reverse action. The OCT3 inhibitor corticosterone (10 mg kg(-1) ) had no effect. Blockade of glutamate receptors with topical kynurenate (10-50 nm) reduced the vagally evoked 5-HT signal by 50%, indicating that this release was from at least two sources. It is concluded that vagally evoked 5-HT release is under the regulation of the high-capacity, low-affinity transporter PMAT, not the low-capacity, high-affinity transporter SERT. This is the first demonstration that PMAT may be playing a physiological role in the regulation of 5-HT transmission and this could indicate that 5-HT is acting, in part, as a volume transmitter within the NTS. PMID:25694117

  8. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    PubMed

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  9. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K⁺ uptake and Na⁺ transport in yeast.

    PubMed

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K(+), Na(+), Ca(2+), Mg(2+), Fe(2+), Cu(2+), Co(2+), Cd(2+), Mn(2+), Ba(2+), Ni(2+), Zn(2+), and Li(+)) were analyzed. AtCCX5 expression was found to affect the response to K(+) and Na(+) in yeast. The AtCCX5 transformant also showed a little better growth to Zn(2+). The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K(+) (0.5mM), and also suppressed its Na(+) sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K(+) uptake and was also involved in Na(+) transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K(+) uptake and Na(+) transport in yeast. PMID:21945443

  10. Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy*

    PubMed Central

    Neundlinger, Isabel; Puntheeranurak, Theeraporn; Wildling, Linda; Rankl, Christian; Wang, Lai-Xi; Gruber, Hermann J.; Kinne, Rolf K. H.; Hinterdorfer, Peter

    2014-01-01

    Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. PMID:24962566

  11. Changes in renal glucose transporters in an animal model of metabolic syndrome.

    PubMed

    Lehnen, A M; Leguisamo, N M; Dias, L D; Pinto, G H; Okamoto, N M; Machado, U F; Schaan, B D

    2013-10-01

    Considering the similarity between structural, hemodynamic, and functional changes of obesity-related renal disease and diabetic nephropathy, we hypothesized that renal glucose transporter changes occur in obesity as in diabetes. The aim of the work was to evaluate GLUT1 and GLUT2 in kidneys of an animal model of metabolic syndrome. Neonate spontaneously hypertensive rats (SHR), n=15/group, were treated with monosodium glutamate (5 mg/g) (MetS) for 9 days and compared with saline-treated Wistar-Kyoto (C) and SHR (H) rats. Lee index, systolic arterial pressure (SAP), glycemia, insulin resistance, triglycerides, and HDL cholesterol were evaluated at 3 and 6 months. Medullar GLUT1 and cortical GLUT2 were analyzed by Western blot. MetS vs. C and H rats had the highest Lee index (p<0.001) and insulin resistance (3-months C: 4.3±0.7, H: 3.9±0.9, MetS: 2.7±0.6; 6-months C: 4.2±0.6, H: 3.8±0.5, MetS: 2.4±0.6% · min⁻¹, p<0.001), similar glycemia, and the lowest HDL-cholesterol at 6-months (p<0.001). In the MetS and H rats, SAP was higher vs. C at 3-months (p<0.001) and 6-months (C: 151±15, H: 190±11, MetS: 185±13 mm Hg, p<0.001) of age. GLUT1 was ̴ 13× lower (p<0.001) at 3-months, reestablishing its content at 6-months in MetS group, while GLUT2 was 2× higher (p<0.001) in this group at 6-months of age. Renal GLUT1 and GLUT2 are modulated in kidney of rats with metabolic syndrome, where obesity, insulin resistance and hypertension coexist, despite normoglycemia. Like in diabetes, cortical GLUT2 overexpression may contribute to the development of kidney disease. PMID:24062089

  12. Active transport of C-11-Methyl-D-Glucose and 3-F-18-Deoxyglucose in acute ischemic brain disease and Huntington's chorea, studied by positron-emission-tomography (PET)

    SciTech Connect

    Vyska, K.; Magloire, R.; Schuier, F.; Machulla, H.J.; Knust, E.J.; Lange, W.; Becker, V.; Spohr, G.; Notohamiprodjo, G.; Feinendegen, L.E.

    1984-01-01

    C-11-Methyl-D-Glucose (CMG) and 3-F-18-Deoxyglucose (3FDG) were demonstrated to be non-metabolizable glucose analogues which are transported across the blood-brain-barrier into and out of tissue via the glucose carrier system (GCS). These two substances were used as indicators for determining the perfusion-independent rate constant of GCS in the brain. Five normals with informed consent, 12 patients with acute ischemic brain disease and 9 patients with initial and advanced Huntington's chorea were examined by PET after i.v. application of 5 mCi of GMG or 3FDG. In each patient 30 transaxial images were registered in 1 selected plane, image collection time being 1 min. Time-activity curves were created from different regions of interest. The slope to tracer steady state between tissue and blood yields the perfusion-independent rate constant of GCS from tissue to blood (k/sub 2/). In normals k/sub 2/ for CMG was 0.235 +- 0.03/min, as expected, and for 3FDG 0.47 +- 0.07/min indicating a higher affinity to GCS for 3FDG than CMG. In acute ischemic brain disease k/sub 2/ was normal or reduced at the site of insult for both CMG and 3FDG. In Huntington's chorea, k/sub 2/ was reduced in the basal ganglia but normal or occasionally significantly increased in frontal or occipital cortical areas, for CMG and 3FDG. The authors conclude that CMG permits noninvasive analysis of the perfusion-independent rate constant of CCS. 3FDG shows a higher affinity for CCS than CMC but gives comparable information.

  13. Chronic growth hormone treatment in normal rats reduces post-prandial skeletal muscle plasma membrane GLUT1 content, but not glucose transport or GLUT4 expression and localization.

    PubMed Central

    Napoli, R; Cittadini, A; Chow, J C; Hirshman, M F; Smith, R J; Douglas, P S; Horton, E S

    1996-01-01

    Whether skeletal muscle glucose transport system is impaired in the basal, post-prandial state during chronic growth hormone treatment is unknown. The current study was designed to determine whether 4 weeks of human growth hormone (hGH) treatment (3.5 mg/kg per day) would impair glucose transport and/or the number of glucose transporters in plasma membrane vesicles isolated from hindlimb skeletal muscle of Sprague-Dawley rats under basal, post-prandial conditions. hGH treatment was shown to have no effect on glucose influx (Vmax or K(m)) determined under equilibrium exchange conditions in isolated plasma membrane vesicles. Plasma membrane glucose transporter number (Ro) measured by cytochalasin B binding was also unchanged by hGH treatment. Consequently, glucose transporter turnover number (Vmax/Ro), a measure of average glucose transporter intrinsic activity, was similar in hGH-treated and control rats. hGH did not change GLUT4 protein content in whole muscle or in the plasma membrane, and muscle content of GLUT4 mRNA also was unchanged. In contrast, GLUT1 protein content in the plasma membrane fraction was significantly reduced by hGH treatment. This was associated with a modest, although not significant, decrease in muscle content of GLUT1 mRNA. In conclusion, high-dose hGH treatment for 4 weeks did not alter post-prandial skeletal muscle glucose transport activity. Neither the muscle level nor the intracellular localization of GLUT4 was changed by the hormone treatment. On the contrary, the basal post-prandial level of GLUT1 in the plasma membrane was reduced by hGH. The mRNA data suggest that this reduction might result from a decrease in the synthesis of GLUT1. PMID:8645183

  14. Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment*

    PubMed Central

    Belman, Jonathan P.; Bian, Rachel R.; Habtemichael, Estifanos N.; Li, Don T.; Jurczak, Michael J.; Alcázar-Román, Abel; McNally, Leah J.; Shulman, Gerald I.; Bogan, Jonathan S.

    2015-01-01

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake. PMID:25561724

  15. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    PubMed

    Belman, Jonathan P; Bian, Rachel R; Habtemichael, Estifanos N; Li, Don T; Jurczak, Michael J; Alcázar-Román, Abel; McNally, Leah J; Shulman, Gerald I; Bogan, Jonathan S

    2015-02-13

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake. PMID:25561724

  16. Pseudomonas syringae BetT Is a Low-Affinity Choline Transporter That Is Responsible for Superior Osmoprotection by Choline over Glycine Betaine▿

    PubMed Central

    Chen, Chiliang; Beattie, Gwyn A.

    2008-01-01

    The plant pathogen Pseudomonas syringae derives better osmoprotection from choline than from glycine betaine, unlike most bacteria that have been characterized. In this report, we identified a betaine/carnitine/choline family transporter (BCCT) in P. syringae pv. tomato strain DC3000 that mediates the transport of choline and acetylcholine. This transporter has a particularly low affinity (Km of 876 μM) and high capacity (Vmax of 80 nmol/min/mg of protein) for choline transport relative to other known BCCTs. Although BetT activity increased in response to hyperosmolarity, BetT mediated significant uptake under low-osmolarity conditions, suggesting a role in transport for both osmoprotection and catabolism. Growth studies with mutants deficient in BetT and other choline transporters demonstrated that BetT was responsible for the superior osmoprotection conferred to P. syringae by choline over glycine betaine when these compounds were provided at high concentrations (>100 μM). These results suggest that P. syringae has evolved to survive in relatively choline-rich habitats, a prediction that is supported by the common association of P. syringae with plants and the widespread production of choline, but genus- and species-specific production of glycine betaine, by plants. Among the three putative BCCT family transporters in Pseudomonas aeruginosa and six in Pseudomonas putida, different transporters were predicted to function based on similarity to Escherichia coli BetT than to P. syringae BetT. Functional P. putida and P. aeruginosa transporters were identified, and their possession of a long C-terminal tail suggested an osmoregulatory function for this tail; this function was confirmed for P. syringae BetT using deletion derivatives. PMID:18156257

  17. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    PubMed

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway. PMID:17588539

  18. Apparent lack of beta 3-adrenoceptors and of insulin regulation of glucose transport in brown adipose tissue of guinea pigs.

    PubMed

    Himms-Hagen, J; Triandafillou, J; Begin-Heick, N; Ghorbani, M; Kates, A L

    1995-01-01

    Norepinephrine-induced thermogenesis was substantial in adipocytes from brown adipose tissue (BAT) of cold-acclimated guinea pigs but absent in adipocytes from BAT of warm-acclimated guinea pigs. There was no thermogenic response to any beta 3-adrenergic agonist (CL-316,243, ZD-7114, BRL-28410, CGP-12177). The receptor was characterized as a beta 1-adrenoceptor. Adrenergic agonists stimulated adenylate cyclase in membranes from BAT of both warm- and cold-acclimated guinea pigs also via a beta 1-adrenoceptor; beta 3-adrenergic agonists had no effect. Glucose transport by brown adipocytes from warm-acclimated guinea pigs was not stimulated by either norepinephrine or insulin. Cold acclimation induced the appearance of stimulation of glucose transport by norepinephrine in association with the appearance of a large capacity for thermogenesis, but there was little improvement in response to insulin. GLUT4 was present in membranes from BAT of both warm- and cold-acclimated guinea pigs. Insulin is known to have an antilipolytic effect on both BAT and white adipose tissue of guinea pigs. Thus there is a selective lack of insulin-regulated glucose transport that is not improved by cold acclimation. Guinea pigs may have a mutated component of the translocation mechanism for GLUT4. beta 3-Adrenoceptors appear to be absent in brown adipocytes of adult guinea pigs, as in white adipocytes of guinea pigs, yet are known to be present in the gut. Tissue-specific expression of beta 3-adrenergic receptors in guinea pigs may differ from that in rats, in which receptors are expressed in the adipose tissues and gut. PMID:7840345

  19. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

    SciTech Connect

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.

  20. Stereoselective Binding of Chiral Ligands to Single Nucleotide Polymorphs (SNPs) of the Human Organic Cation Transporter-1 Determined Using Cellular Membrane Affinity Chromatography

    PubMed Central

    Moaddel, R.; Bighi, F.; Yamaguchi, R.; Patel, S.; Ravichandran, S.; Wainer, I.W.

    2010-01-01

    Membranes from stably transfected cell lines that expresses two point mutations of the human organic cation 1 transporter (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the Cellular Membrane Affinity Chromatography (CMAC) (hOCT1G465R) and CMAC(hOCT1R488M). Columns were created using both stationary phases and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium, [3H]-MPP+, as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol and isoproterenol. The chromatographic data obtained was used to refine a previously developed pharmacophore for the hOCT1 transporter. PMID:20206116

  1. Synthesis of L-rhamnose derived chiral bicyclic triazoles as novel sodium-glucose transporter (SGLT) inhibitors.

    PubMed

    Putapatri, Siddamal Reddy; Kanwal, Abhinav; Sridhar, Balasubramanian; Banerjee, Sanjay K; Kantevari, Srinivas

    2014-11-14

    Herein we describe the synthesis of a series of novel fused bicyclic 1,2,3-triazoles from commercially available, natural deoxy sugar, L-rhamnose. The key reactions involved are (i) Zn(OTf)2 catalyzed enantioselective alkynylation of L-rhamnose derived azidoaldehyde and (ii) deprotection of the acid sensitive 1,2-isopropylidene group followed by in situ intramolecular click-cycloaddition of azidoalkynols. Some compounds exhibit excellent sodium-glucose transporter (SGLT1 and SGLT2) inhibition activity. PMID:25175761

  2. Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae.

    PubMed

    Bianchi, Frans; Klooster, Joury S van 't; Ruiz, Stephanie J; Luck, Katja; Pols, Tjeerd; Urbatsch, Ina L; Poolman, Bert

    2016-01-01

    The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels. PMID:27550794

  3. Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae

    PubMed Central

    Bianchi, Frans; Klooster, Joury S. van ‘t; Ruiz, Stephanie J.; Luck, Katja; Pols, Tjeerd; Urbatsch, Ina L.; Poolman, Bert

    2016-01-01

    The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels. PMID:27550794

  4. Glucose Transporter Type 4 Redistribution on the Membrane Induced by Insulin through Akt in Hydrocortisone Treatment in Rat Skeletal Muscles.

    PubMed

    Chen, Chien-Min; Chiu, Lian; Chen, Hung-Chi; Cheng, Chun-Yuan; Shyu, Woei-Cherng; Chou, Chii-Wen; Lu, Cheng-You; Lin, Chung-Tien

    2015-10-31

    Hydrocortisone is a growth hormone frequently used in the treatment of low back pain. Hydrocortisone treatment has an anti-inflammation effect, which also inactivates glucose transporter type 4 (GLUT4) by p38 mitogen-activated protein kinase (MAPK) inhibition. Translocation of GLUT4 regulates body glucose homeostasis and muscle repair and is induced by insulin. In this study, 56 SD rats were divided into seven groups, and were treated with insulin or hydrocortisone in sedentary or exercise training groups. The muscle proteins and biochemical blood parameters were analyzed after 7 days of treatments. The results showed that the serum glucose increased in hydrocortisone treatment accompanied by GLUT4 inactivation in both the sedentary and exercise training rats. In the exercise training groups, GLUT4 was redistributed on the plasma membrane on co-treatment with insulin and hydrocortisone through Akt phosphorylation. Insulin treatment exerted a compensatory feedback effect on the GLUT4 translocation on hydrocortisone co-treatment, which was the cause of GLUT4 inactivation. PMID:26387653

  5. Autoantibodies to the GLUT-2 glucose transporter of beta cells in insulin-dependent diabetes mellitus of recent onset.

    PubMed Central

    Inman, L R; McAllister, C T; Chen, L; Hughes, S; Newgard, C B; Kettman, J R; Unger, R H; Johnson, J H

    1993-01-01

    Purified immunoglobulin G (IgG) from the serum of patients with insulin-dependent diabetes mellitus (IDDM) of recent onset inhibits high-Km uptake of 3-O-methyl-beta-D-glucose by rat pancreatic islets. To determine if the inhibition is the result of antibodies against GLUT-2, the high-Km glucose transporter of beta cells, we incubated IDDM sera with rat islet cells and with AtT-20ins cells transfected to express GLUT-2. IDDM sera inhibited glucose uptake in islet cells and in GLUT-2-expressing AtT-20ins cells but not in AtT-20ins cells transfected to express the low-Km isoform, GLUT-1. In 24 of 30 (77%) patients with newly diagnosed IDDM, IgG binding as measured by immunofluorescence and flow cytometry of the cells transfected to express GLUT-2 was > 2 standard deviations from the mean of the nondiabetic population; 29 of 31 (96%) of nondiabetic children were negative (P < 0.0001). Increased IgG binding could be removed by absorption with GLUT-2-expressing cells but not with GLUT-1-expressing cells. We conclude that most patients with IDDM of recent onset have autoantibodies to GLUT-2. PMID:8433987

  6. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  7. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  8. FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Duggan, Kelli D; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A

    2014-08-29

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  9. FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter*

    PubMed Central

    Boswell-Casteel, Rebba C.; Johnson, Jennifer M.; Duggan, Kelli D.; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A.

    2014-01-01

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  10. Accumulation of ascorbate by endocrine-regulated and glucose-sensitive transport of dehydroascorbic acid in luteinized rat ovarian cells.

    PubMed

    Kodaman, P H; Aten, R F; Behrman, H R

    1998-02-01

    The corpus luteum is notable for very high levels of ascorbic acid. In luteal cells, ascorbic acid depletion occurs as a result of consumption during radical scavenging, inhibition of ascorbic acid uptake, and stimulation of its secretion. Oxidation of ascorbic acid generates dehydroascorbic acid (DHAA). Although levels of DHAA in blood are much lower than those of ascorbic acid, DHAA serves as the major transportable form of ascorbate for certain cell types. The aim of the present studies was to investigate whether DHAA transport is a potential mechanism for conserving ascorbic acid in the corpus luteum. DHAA uptake by rat luteal cells precultured for 24 h was linear for up to 30 min. Kinetics studies showed that uptake of DHAA was a concentration-dependent and saturable process with an estimated Michaelis constant (Km) of 830 microM and a maximum velocity (Vmax) of 700 pmol/min per 10(6) cells, a rate 50 times that of ascorbate transport. More than 90% of DHAA was reduced to ascorbic acid within 2 h of cellular uptake. DHAA uptake was energy- and microfilament-dependent, as transport was inhibited by 2,4-dinitrophenol (1 mM) and cytochalasin B (10 microM). Menadione (50 microM), an intracellular generator of reactive oxygen species, also markedly reduced DHAA uptake. In contrast to ascorbic acid transport, DHAA uptake was potently inhibited by glucose and phloretin, an inhibitor of glucose transporters, with IC50s of approximately 5 mM and 10 microM, respectively. DHAA uptake appears to occur via an insulin-insensitive transporter, as insulin (10 nM) had no effect on uptake. However, 24-h preincubation with insulin-like growth factor (IGF)-I dose-dependently (10-100 ng/ml) stimulated DHAA uptake; similar concentrations of IGF-II had no effect. The secretion of radioactivity by cells preloaded with radiolabeled DHAA was significantly increased by prostaglandin F2alpha (1 microM). The ability of luteal cells to transport DHAA in a regulated manner may serve to

  11. Roles of the NMDA Receptor and EAAC1 Transporter in the Modulation of Extracellular Glutamate by Low and High Affinity AMPA Receptors in the Cerebellum in Vivo: Differential Alteration in Chronic Hyperammonemia.

    PubMed

    Cabrera-Pastor, Andrea; Taoro, Lucas; Llansola, Marta; Felipo, Vicente

    2015-12-16

    The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors. PMID:26428532

  12. Insulin signalling and glucose transport in the ovary and ovarian function during the ovarian cycle

    PubMed Central

    Dupont, Joëlle; Scaramuzzi, Rex J.

    2016-01-01

    Data derived principally from peripheral tissues (fat, muscle and liver) show that insulin signals via diverse interconnecting intracellular pathways and that some of the major intersecting points (known as critical nodes) are the IRSs (insulin receptor substrates), PI3K (phosphoinositide kinase)/Akt and MAPK (mitogen-activated protein kinase). Most of these insulin pathways are probably also active in the ovary and their ability to interact with each other and also with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) signalling pathways enables insulin to exert direct modulating influences on ovarian function. The present paper reviews the intracellular actions of insulin and the uptake of glucose by ovarian tissues (granulosa, theca and oocyte) during the oestrous/menstrual cycle of some rodent, primate and ruminant species. Insulin signals through diverse pathways and these are discussed with specific reference to follicular cell types (granulosa, theca and oocyte). The signalling pathways for FSH in granulosa cells and LH in granulosa and theca cells are summarized. The roles of glucose and of insulin-mediated uptake of glucose in folliculogenesis are discussed. It is suggested that glucose in addition to its well-established role of providing energy for cellular function may also have insulin-mediated signalling functions in ovarian cells, involving AMPK (AMP-dependent protein kinase) and/or hexosamine. Potential interactions of insulin signalling with FSH or LH signalling at critical nodes are identified and the available evidence for such interactions in ovarian cells is discussed. Finally the action of the insulin-sensitizing drugs metformin and the thiazolidinedione rosiglitazone on follicular cells is reviewed. PMID:27234585

  13. Insulin signalling and glucose transport in the ovary and ovarian function during the ovarian cycle.

    PubMed

    Dupont, Joëlle; Scaramuzzi, Rex J

    2016-06-01

    Data derived principally from peripheral tissues (fat, muscle and liver) show that insulin signals via diverse interconnecting intracellular pathways and that some of the major intersecting points (known as critical nodes) are the IRSs (insulin receptor substrates), PI3K (phosphoinositide kinase)/Akt and MAPK (mitogen-activated protein kinase). Most of these insulin pathways are probably also active in the ovary and their ability to interact with each other and also with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) signalling pathways enables insulin to exert direct modulating influences on ovarian function. The present paper reviews the intracellular actions of insulin and the uptake of glucose by ovarian tissues (granulosa, theca and oocyte) during the oestrous/menstrual cycle of some rodent, primate and ruminant species. Insulin signals through diverse pathways and these are discussed with specific reference to follicular cell types (granulosa, theca and oocyte). The signalling pathways for FSH in granulosa cells and LH in granulosa and theca cells are summarized. The roles of glucose and of insulin-mediated uptake of glucose in folliculogenesis are discussed. It is suggested that glucose in addition to its well-established role of providing energy for cellular function may also have insulin-mediated signalling functions in ovarian cells, involving AMPK (AMP-dependent protein kinase) and/or hexosamine. Potential interactions of insulin signalling with FSH or LH signalling at critical nodes are identified and the available evidence for such interactions in ovarian cells is discussed. Finally the action of the insulin-sensitizing drugs metformin and the thiazolidinedione rosiglitazone on follicular cells is reviewed. PMID:27234585

  14. Sodium-glucose linked transporter-2 inhibitors in chronic kidney disease.

    PubMed

    Zanoli, L; Granata, A; Lentini, P; Rastelli, S; Fatuzzo, P; Rapisarda, F; Castellino, P

    2015-01-01

    SGLT2 inhibitors are new antihyperglycaemic agents whose ability to lower glucose is directly proportional to GFR. Therefore, in chronic kidney disease (CKD) the blood glucose lowering effect is reduced. Unlike many current therapies, the mechanism of action of SGLT2 inhibitors is independent of insulin action or beta-cell function. In addition, the mechanism of action of SGLT2 inhibitors is complementary and not alternative to other antidiabetic agents. SGLT2 inhibitors could be potentially effective in attenuating renal hyperfiltration and, consequently, the progression of CKD. Moreover, the reductions in intraglomerular pressure, systemic blood pressure, and uric acid levels induced by SGLT inhibition may potentially be of benefit in CKD subjects without diabetes. However, at present, only few clinical studies were designed to evaluate the effects of SGLT2 inhibitors in CKD. Consequently, safety and potential efficacy beyond blood glucose lowering should be better clarified in CKD. In this paper we provide an updated review of the use of SGLT2 inhibitors in clinical practice, with particular attention on subjects with CKD. PMID:25785281

  15. Duodenal brush-border mucosal glucose transport and enzyme activities in aging man and effect of bacterial contamination of the small intestine.

    PubMed

    Wallis, J L; Lipski, P S; Mathers, J C; James, O F; Hirst, B H

    1993-03-01

    Duodenal biopsies were collected from 38 subjects (24 female and 14 male) ranging in age from 55 to 91 years. Evidence of bacterial contamination of the small bowel (BCSB) was sought at the same time by bacterial culture of duodenal aspirates and by hydrogen and [14C]glycocholic acid breath tests; subjects were considered to be positive for BCSB if any one of the three tests was abnormal. Biopsies were analyzed for six brush-border membrane enzyme activities: maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and alpha-glucosidase. Analysis of covariance with age as the covariate indicated no significant effect of age on the specific activities of these enzymes. Mucosal Na(+)-dependent glucose transport was quantified in brush-border membrane vesicles prepared from the biopsies. In all groups, glucose transport at 20-30 sec was greater (ranging from mean values of 2.45 to 3.66 times) than at 45 min, consistent with Na(+)-coupled glucose transport, and no significant effect of age was observed. BCSB had no significant effect on specific activities of any of the duodenal mucosal hydrolases but was associated with reduced (P = 0.05) brush-border glucose transport. None of the variables studied was significantly affected by the gender of subjects. In conclusion, these biochemical data do not support the contention that reduced capacity for carbohydrate absorption in the elderly is explained by reductions in duodenal brush-border mucosal disaccharidase activities or glucose transport. PMID:8444069

  16. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    PubMed

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium. PMID:19818021

  17. Inhibitory effects of alcohol on glucose transport across the blood–brain barrier leads to neurodegeneration: preventive role of acetyl-L-carnitine

    PubMed Central

    Muneer, P. M. Abdul; Alikunju, Saleena; Szlachetka, Adam M.; Haorah, James

    2011-01-01

    Purpose Evidence shows that alcohol intake causes oxidative neuronal injury and neurocognitive deficits that are distinct from the classical Wernicke-Korsakoff neuropathy. Our previous findings indicated that alcohol-elicited blood-brain barrier (BBB) damage leads to neuroinflammation and neuronal loss. The dynamic function of the BBB requires a constant supply and utilization of glucose. Here we examined whether interference of glucose uptake and transport at the endothelium by alcohol leads to BBB dysfunction and neuronal degeneration. Material and methods We tested the hypothesis in cell culture of human brain endothelial cells, neurons and alcohol intake in animal by immunofluorescence, Western blotting and glucose uptake assay methods. Results We found that decrease in glucose uptake correlates the reduction of glucose transporter protein 1 (GLUT1) in cell culture after 50 mM ethanol exposure. Decrease in GLUT1 protein levels was regulated at the translation process. In animal, chronic alcohol intake suppresses the transport of glucose into the frontal and occipital regions of the brain. This finding is validated by a marked decrease in GLUT1 protein expression in brain microvessel (the BBB). In parallel, alcohol intake impairs the BBB tight junction proteins occludin, zonula occludens-1, and claudin-5 in the brain microvessel. Permeability of sodium fluorescein and Evans Blue confirms the leakiness of the BBB. Further, depletion of trans-endothelial electrical resistance of the cell monolayer supports the disruption of BBB integrity. Administration of acetyl-L-carnitine (a neuroprotective agent) significantly prevents the adverse effects of alcohol on glucose uptake, BBB damage and neuronal degeneration. Conclusion These findings suggest that alcohol-elicited inhibition of glucose transport at the blood-brain interface leads to BBB malfunction and neurological complications. PMID:21079922

  18. OusB, a Broad-Specificity ABC-Type Transporter from Erwinia chrysanthemi, Mediates Uptake of Glycine Betaine and Choline with a High Affinity

    PubMed Central

    Choquet, Gwénaëlle; Jehan, Nathalie; Pissavin, Christine; Blanco, Carlos; Jebbar, Mohamed

    2005-01-01

    The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi. PMID:16000740

  19. Clinical Pharmacokinetic, Pharmacodynamic, and Drug-Drug Interaction Profile of Canagliflozin, a Sodium-Glucose Co-transporter 2 Inhibitor.

    PubMed

    Devineni, Damayanthi; Polidori, David

    2015-10-01

    The sodium-glucose co-transporter 2 (SGLT2) inhibitors represent novel therapeutic approaches in the management of type 2 diabetes mellitus; they act on kidneys to decrease the renal threshold for glucose (RTG) and increase urinary glucose excretion (UGE). Canagliflozin is an orally active, reversible, selective SGLT2 inhibitor. Orally administered canagliflozin is rapidly absorbed achieving peak plasma concentrations in 1-2 h. Dose-proportional systemic exposure to canagliflozin has been observed over a wide dose range (50-1600 mg) with an oral bioavailability of 65 %. Canagliflozin is glucuronidated into two inactive metabolites, M7 and M5 by uridine diphosphate-glucuronosyltransferase (UGT) 1A9 and UGT2B4, respectively. Canagliflozin reaches steady state in 4 days, and there is minimal accumulation observed after multiple dosing. Approximately 60 % and 33 % of the administered dose is excreted in the feces and urine, respectively. The half-life of orally administered canagliflozin 100 or 300 mg in healthy participants is 10.6 and 13.1 h, respectively. No clinically relevant differences are observed in canagliflozin exposure with respect to age, race, sex, and body weight. The pharmacokinetics of canagliflozin remains unaffected by mild or moderate hepatic impairment. Systemic exposure to canagliflozin is increased in patients with renal impairment relative to those with normal renal function; however, the efficacy is reduced in patients with renal impairment owing to the reduced filtered glucose load. Canagliflozin did not show clinically relevant drug interactions with metformin, glyburide, simvastatin, warfarin, hydrochlorothiazide, oral contraceptives, probenecid, and cyclosporine, while co-administration with rifampin modestly reduced canagliflozin plasma concentrations and thus may necessitate an appropriate monitoring of glycemic control. Canagliflozin increases UGE and suppresses RTG in a dose-dependent manner, thereby lowering the plasma glucose

  20. Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

    PubMed Central

    Lee, Na-Young; Park, Hyun-Joo; Kang, Young-Sook

    2016-01-01

    The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [3H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [3H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [3H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [3H]3-OMG uptake was increased at 48 h. However, [3H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [3H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [3H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition. PMID:26759707

  1. Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.

    PubMed

    Hresko, Richard C; Kraft, Thomas E; Tzekov, Anatoly; Wildman, Scott A; Hruz, Paul W

    2014-06-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  2. Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro

    PubMed Central

    Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V.; Manjunath, Kirangadur; Viswanatha, Gollapalle L.; Ashok, Godavarthi

    2016-01-01

    Aims: The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Materials and Methods: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. Results: The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. Conclusion: These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro. PMID:27104035

  3. Time-Course of Alterations in Myocardial Glucose Utilization in the Zucker Diabetic Fatty (ZDF) Rat with Correlation to Gene Expression of Glucose Transporters: A Small Animal PET Investigation

    PubMed Central

    Shoghi, Kooresh I.; Gropler, Robert J.; Sharp, Terry; Herrero, Pilar; Fettig, Nicole; Su, Yi; Mitra, Mayurranjan S.; Kovacs, Attila; Finck, Brian N.; Welch, Michael J.

    2010-01-01

    Objective Diabetic cardiomyopathy is associated with abnormalities in glucose metabolism. We evaluated myocardial glucose metabolism in a rodent model of Type 2 Diabetes (T2D), namely the Zucker Diabetic Rat (ZDF), and validated PET measures of glucose uptake against gene and protein expression of glucose transporters. Methods Six lean and ZDF rat underwent small animal PET imaging at the age of 14 weeks and at the age of 19 weeks. The imaging protocol consisted of a 60-minute dynamic acquisition with 18FDG (0.5–0.8mCi). Dynamic images were reconstructed using filtered back projection (FBP) with a 2.5 zoom on the heart and 40 frames per imaging session. PET measures of myocardial glucose uptake rate (MGUp) and utilization were determined with an input function derived by the Hybrid Image- Blood Sampling (HIBS) algorithm on recovery-corrected anterolateral myocardial regions of interest. Following the PET imaging session at week 19, hearts were extracted for gene and protein expression analysis of GLUT1 and GLUT4. The dependence of MGUp on gene expression of GLUT1 and GLUT4 was characterized by multiple-regression analysis. Results Compared to lean littermate control rats, MGUp was significantly depressed in ZDF rats at both week 14 and week 19 (P<0.006). Moreover, lean rats at week 19 displayed significantly higher MGUp than week 14 (P=0.007). Consistent with diminished MGUp, gene expression of GLUT4 was significantly (P=0.004) lower in ZDF rats. Finally, MGUp significantly (P=0.0003) correlated with gene expression of GLUT4. Conclusions Using small animal PET, we confirmed alterations in myocardial glucose utilization and validated PET measure of MGUp against gene and protein expression of glucose transporters in the diabetic heart of an animal model of T2D. PMID:18632819

  4. Effects of isoleucine on glucose uptake through the enhancement of muscular membrane concentrations of GLUT1 and GLUT4 and intestinal membrane concentrations of Na+/glucose co-transporter 1 (SGLT-1) and GLUT2.

    PubMed

    Zhang, Shihai; Yang, Qing; Ren, Man; Qiao, Shiyan; He, Pingli; Li, Defa; Zeng, Xiangfang

    2016-08-01

    Knowledge of regulation of glucose transport contributes to our understanding of whole-body glucose homoeostasis and human metabolic diseases. Isoleucine has been reported to participate in regulation of glucose levels in many studies; therefore, this study was designed to examine the effect of isoleucine on intestinal and muscular GLUT expressions. In an animal experiment, muscular GLUT and intestinal GLUT were determined in weaning pigs fed control or isoleucine-supplemented diets. Supplementation of isoleucine in the diet significantly increased piglet average daily gain, enhanced GLUT1 expression in red muscle and GLUT4 expression in red muscle, white muscle and intermediate muscle (P<0·05). In additional, expressions of Na+/glucose co-transporter 1 and GLUT2 were up-regulated in the small intestine when pigs were fed isoleucine-supplemented diets (P<0·05). C2C12 cells were used to examine the expressions of muscular GLUT and glucose uptake in vitro. In C2C12 cells supplemented with isoleucine in the medium, cellular 2-deoxyglucose uptake was increased (P<0·05) through enhancement of the expressions of GLUT4 and GLUT1 (P<0·05). The effect of isoleucine was greater than that of leucine on glucose uptake (P<0·05). Compared with newborn piglets, 35-d-old piglets have comparatively higher GLUT4, GLUT2 and GLUT5 expressions. The results of this study demonstrated that isoleucine supplementation enhanced the intestinal and muscular GLUT expressions, which have important implications that suggest that isoleucine could potentially increase muscle growth and intestinal development by enhancing local glucose uptake in animals and human beings. PMID:27464458

  5. Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites.

    PubMed Central

    Hennig, B; Koehler, H; Neupert, W

    1983-01-01

    Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria. PMID:6308663

  6. Site-specific antibodies as probes of the topology and function of the human erythrocyte glucose transporter.

    PubMed Central

    Davies, A; Ciardelli, T L; Lienhard, G E; Boyle, J M; Whetton, A D; Baldwin, S A

    1990-01-01

    Antibodies were raised against synthetic peptides corresponding to most of the regions of the human erythrocyte glucose transporter predicted to be extramembranous in the model of Mueckler, Caruso, Baldwin, Panico, Blench, Morris, Lienhard, Allard & Lodish [(1985) Science 229, 941-945]. Most of the antibodies (17 out of a total of 19) recognized the intact denatured protein on Western blots. However, only seven of the antibodies recognized the native membrane-bound protein, even after its deglycosylation. These antibodies, against peptides encompassing residues 217-272 and 450-492 in the hydrophilic central and C-terminal regions of the transporter, bound to the cytoplasmic surface of the erythrocyte membrane. This finding is in agreement with the prediction of the model that these regions of the sequence are cytoplasmic. Antibodies against peptides from the central cytoplasmic loop of the transporter were found to inhibit the binding of cytochalasin B to the membrane-bound protein, whereas antibodies against the C-terminal region had no effect. The anti-peptide antibodies were then used to map the sequence locations of fragments of the transporter arising from tryptic digestion of the membrane-bound protein. This in turn enabled the epitopes for a number of anti-transporter monoclonal antibodies to be located within either the central cytoplasmic loop or the C-terminal region of the protein. Of those monoclonal antibodies which inhibited cytochalasin B binding to the protein, all but one were found to have epitopes within the central region of the sequence. In conjunction with the results of the anti-peptide antibody studies, these findings indicate the importance of this part of the protein for transporter function. Images Fig. 7. PMID:1691633

  7. Dietary Lipid and Carbohydrate Interactions: Implications on Lipid and Glucose Absorption, Transport in Gilthead Sea Bream (Sparus aurata) Juveniles.

    PubMed

    Castro, Carolina; Corraze, Geneviève; Basto, Ana; Larroquet, Laurence; Panserat, Stéphane; Oliva-Teles, Aires

    2016-06-01

    A digestibility trial was performed with gilthead sea bream juveniles (IBW = 72 g) fed four diets differing in lipid source (fish oil, FO; or a blend of vegetable oil, VO) and starch content (0 %, CH-; or 20 %, CH+) to evaluate the potential interactive effects between carbohydrates and VO on the processes involved in digestion, absorption and transport of lipids and glucose. In fish fed VO diets a decrease in lipid digestibility and in cholesterol (C), High Density Lipoprotein(HDL)-C and Low Density Lipoprotein (LDL)-C (only in CH+ group) were recorded. Contrarily, dietary starch induced postprandial hyperglycemia and time related alterations on serum triacylglycerol (TAG), phospholipid (PL) and C concentrations. Fish fed a CH+ diet presented lower serum TAG than CH- group at 6 h post-feeding, and the reverse was observed at 12 h post-feeding for TAG and PL. Lower serum C and PL at 6 h post-feeding were recorded only in VOCH+ group. No differences between groups were observed in hepatic and intestinal transcript levels of proteins involved in lipid transport and hydrolysis (FABP, DGAT, GPAT, MTP, LPL, LCAT). Lower transcript levels of proteins related to lipid transport (ApoB, ApoA1, FABP2) were observed in the intestine of fish fed the CH+ diet, but remained unchanged in the liver. Overall, transcriptional mechanisms involved in lipid transport and absorption were not linked to changes in lipid serum and digestibility. Dietary starch affected lipid absorption and transport, probably due to a delay in lipid absorption. This study suggests that a combination of dietary VO and starch may negatively affect cholesterol absorption and transport. PMID:27023202

  8. Glucose and fructose uptake by Limulus polyphemus hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na+-dependent sugar transport activity.

    PubMed

    Sterling, Kenneth M; Ahearn, Gregory A

    2011-05-01

    [(3)H]-fructose and [(3)H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species. PMID:21184084

  9. A novel functional glucose transporter in the white shrimp Litopenaeus vannamei -LvGLUT2- is up-regulated during hypoxia in hepatopancreas.

    PubMed

    Martínez-Quintana, José A; Kikuta, Shingo; Felix-Portillo, Monserrath; Peregrino-Uriarte, Alma B; Yepiz-Plascencia, Gloria

    2015-12-01

    In hypoxia conditions, the white shrimp Litopenaeus vannamei shifts its energetic metabolism from aerobic to anaerobic, requiring more glucose uptake into the cells by GLUT proteins. We here report a novel glucose transporter in shrimp. The Lvglut2 cDNA is 2473 bp-long containing an ORF of 1458 bp encoding 486 amino acid residues. The deduced protein has the features of a facilitative sugar transporter. The Lvglut2 gene product tagged with GFP was expressed in the cell membrane of Xenopus oocytes. In the same expression system, untagged LvGLUT2 resulted to be a bidirectional glucose transporter that functions moving glucose down its concentration gradient in and out of the cell. Lvglut2 mRNA is expressed in hepatopancreas while in muscle and gills it was not detected. Hypoxia up-regulates the expression of Lvglut2 transcripts in hepatopancreas. These results provide a better understanding of facilitative glucose transporters and gene regulation during hypoxia in crustaceans. PMID:26415036

  10. Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system.

    PubMed Central

    Hoshino, T; Kose, K

    1990-01-01

    A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue. Images PMID:2120183

  11. Nucleotide sequences and characterization of liv genes encoding components of the high-affinity branched-chain amino acid transport system in Salmonella typhimurium.

    PubMed

    Matsubara, K; Ohnishi, K; Kiritani, K

    1992-07-01

    A 7.6-kb fragment of Salmonella typhimurium LT2 containing the liv gene cluster, which specifies the high-affinity branched-chain amino acid transport system (LIV-I), has been isolated. The upstream region contains the livB and livC genes encoding the leucine-isoleucine-valine-threonine and leucine-specific binding proteins, respectively. In this study, the nucleotide sequence of the 4-kb downstream segment was determined and found to contain four reading frames, designated as livA, livE, livF, and livG, that encode putative membrane-associated proteins. The livA and livE genes encode hydrophobic proteins composed of 308 and 425 amino acid residues, respectively. The livF and livG genes encode hydrophilic proteins of 255 and 237 amino acids, respectively; both the proteins contain consensus amino acid sequences found in proteins with ATP-binding sites. These four genes linked together have a potential rho-independent transcriptional terminator adjacent to the 3'-end of livG. No promoter sequence was found in the immediate upstream region of the livAEFG cluster. The livA, livE, livF, and livG gene products were identified as proteins with apparent M(r)s of 25,500, 34,500, 28,000, and 26,000, respectively, by SDS-polyacryl-amide gel electrophoresis. The deduced amino acid sequences of these four proteins showed strong homology to those of the corresponding membrane-associated proteins required for the high-affinity branched-chain amino acid transport systems from both Escherichia coli and Pseudomonas aeruginosa. PMID:1429514

  12. MicroRNAs overexpressed in growth-restricted rat skeletal muscles regulate the glucose transport in cell culture targeting central TGF-β factor SMAD4.

    PubMed

    Raychaudhuri, Santanu

    2012-01-01

    The micro-array profiling of micro-RNA has been performed in rat skeletal muscle tissues, isolated from male adult offspring of intrauterine plus postnatal growth restricted model (IPGR). Apparently, the GLUT4 mRNA expression in male sk. muscle was found to be unaltered in contrast to females. The over-expression of miR-29a and miR-23a in the experimental group of SMSP (Starved Mother Starved Pups) have been found to regulate the glucose transport activity with respect to their control counterparts CMCP (Control Mother Control Pups) as confirmed in rat L6 myoblast-myocyte cell culture system. The ex-vivo experimentation demonstrates an aberration in insulin signaling pathway in male sk. muscle that leads to the localization of the membrane-bound Glut4 protein. We have identified through a series of experiments one important protein factor SMAD4, a co-SMAD critical to the TGF-beta signaling pathway. This factor is targeted by miR-29a, as identified in an in vitro reporter-assay system in cell-culture experiment. The other micro-RNA, miR-23a, targets SMAD4 indirectly that seems to be critical in regulating insulin-dependent glucose transport activity. MicroRNA mimics, inhibitors and siRNA studies indicate the role of SMAD4 as inhibitory for glucose transport activities in normal physiological condition. The data demonstrate for the first time a critical function of microRNAs in fine-tuning the regulation of glucose transport in skeletal muscle. Chronic starved conditions (IPGR) in sk. muscle up-regulates microRNA changing the target protein expression patterns, such as SMAD4, to alter the glucose transport pathways for the survival. The innovative outcome of this paper identifies a critical pathway (TGF-beta) that may act negatively for the mammalian glucose transport machinery. PMID:22506032

  13. Effects of high-intensity swimming training on GLUT-4 and glucose transport activity in rat skeletal muscle.

    PubMed

    Terada, S; Yokozeki, T; Kawanaka, K; Ogawa, K; Higuchi, M; Ezaki, O; Tabata, I

    2001-06-01

    This study was performed to assess the effects of short-term, extremely high-intensity intermittent exercise training on the GLUT-4 content of rat skeletal muscle. Three- to four-week-old male Sprague-Dawley rats with an initial body weight ranging from 45 to 55 g were used for this study. These rats were randomly assigned to an 8-day period of high-intensity intermittent exercise training (HIT), relatively high-intensity intermittent prolonged exercise training (RHT), or low-intensity prolonged exercise training (LIT). Age-matched sedentary rats were used as a control. In the HIT group, the rats repeated fourteen 20-s swimming bouts with a weight equivalent to 14, 15, and 16% of body weight for the first 2, the next 4, and the last 2 days, respectively. Between exercise bouts, a 10-s pause was allowed. RHT consisted of five 17-min swimming bouts with a 3-min rest between bouts. During the first bout, the rat swam without weight, whereas during the following four bouts, the rat was attached to a weight equivalent to 4 and 5% of its body weight for the first 5 days and the following 3 days, respectively. Rats in the LIT group swam 6 h/day for 8 days in two 3-h bouts separated by 45 min of rest. In the first experiment, the HIT, LIT, and control rats were compared. GLUT-4 content in the epitrochlearis muscle in the HIT and LIT groups after training was significantly higher than that in the control rats by 83 and 91%, respectively. Furthermore, glucose transport activity, stimulated maximally by both insulin (2 mU/ml) (HIT: 48%, LIT: 75%) and contractions (25 10-s tetani) (HIT: 55%, LIT: 69%), was higher in the training groups than in the control rats. However, no significant differences in GLUT-4 content or in maximal glucose transport activity in response to both insulin and contractions were observed between the two training groups. The second experiment demonstrated that GLUT-4 content after HIT did not differ from that after RHT (66% higher in trained rats than

  14. Photoperiod and stress regulation of corticosteroid receptor, brain-derived neurotrophic factor, and glucose transporter GLUT3 mRNA in the hippocampus of male Siberian hamsters (Phodopus sungorus).

    PubMed

    Walton, J C; Grier, A J; Weil, Z M; Nelson, R J

    2012-06-28

    In response to changing day lengths, small photoperiodic rodents have evolved a suite of adaptations to survive the energetic bottlenecks of winter. Among these adaptations are changes in metabolism, adiposity, and energy balance. Whereas hypothalamic and neuroendocrine regulation of these adaptations has been extensively studied, the impact of day length, and interaction of day length and stress, on the energy balance of neurons within the central nervous system remains unspecified. Thus, we exposed male Siberian hamsters (Phodopus sungorus) to either short or long day lengths for 14 weeks to induce the full suite of adaptive responses, exposed them to 4h of restraint, and then measured relative mRNA expression in the hippocampus for low- and high-affinity glucocorticoid receptors (glucocorticoid receptor (GR), mineralocorticoid receptor (MR)), brain-derived neurotrophic factor (BDNF), and the neuron-specific glucose transporter GLUT3. Independent of photoperiod, restraint elevated plasma cortisol (CORT) concentrations and reduced expression of GR, MR, and BDNF. Neither restraint nor photoperiod significantly altered GLUT3 expression. Among all groups, plasma cortisol concentrations were negatively correlated with GR and MR expression. MR, BDNF, and GLUT3 levels were positively correlated with one another, even when controlling for photoperiod and CORT. Taken together, these results suggest that, as peripheral energy balance changes across day length in this photoperiodic species, the neurons of the hippocampus do not alter relative gene expression levels of three proteins involved in monitoring neuronal glucose regulation and morphology. PMID:22521589

  15. Procyanidin Promotes Translocation of Glucose Transporter 4 in Muscle of Mice through Activation of Insulin and AMPK Signaling Pathways.

    PubMed

    Yamashita, Yoko; Wang, Liuqing; Nanba, Fumio; Ito, Chiaki; Toda, Toshiya; Ashida, Hitoshi

    2016-01-01

    Procyanidins are the oligomeric or polymeric forms of epicatechin and catechin. In this study, we isolated and purified dimer to tetramer procyanidins from black soybean seed coat and investigated the anti-hyperglycemic effects by focusing on glucose transporter 4 (GLUT4) translocation and the underlying molecular mechanism in skeletal muscle of mice. The anti-hyperglycemic effects of pr