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Sample records for high-affinity glucose transport

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

    Science.gov (United States)

    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

  2. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available 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.

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

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    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. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available 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.

  5. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    DEFF Research Database (Denmark)

    Jørgensen, Thomas R; vanKuyk, Patricia A; Poulsen, Bjarne R

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mst......-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays...

  6. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    Science.gov (United States)

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence. PMID:23691177

  7. Glucose Transport in the Extremely Thermoacidophilic Sulfolobus solfataricus Involves a High-Affinity Membrane-Integrated Binding Protein

    NARCIS (Netherlands)

    Albers, Sonja-V.; Elferink, Marieke G.L.; Charlebois, Robert L.; Sensen, Christoph W.; Driessen, Arnold J.M.; Konings, Wil N.

    1999-01-01

    The archaeon Sulfolobus solfataricus grows optimally at 80°C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with

  8. A high-affinity molybdate transporter in eukaryotes.

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    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  9. Isolation and cloning of the gene encoding high affinity phosphate transporter in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High affinity phosphate transporter plays an important role in plant adapting to low phosphorus. Isolation of genes coding this kind of protein has attracted worldwide scholars to accomplish. We aimed to isolate the gene and transfer it to target plants for breeding.

  10. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; YASUHARA, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

  11. Characterization of a genetically reconstituted high-affinity system for serotonin transport

    Energy Technology Data Exchange (ETDEWEB)

    Chang, A.S.S.; Lam, D.M.K. (Baylor College of Medicine, Woodlands, TX (USA) Baylor College of Medicine, Houston, TX (USA)); Frnka, J.V.; Chen, D. (Baylor College of Medicine, Woodlands, TX (USA))

    1989-12-01

    By transfecting mouse fibroblast L-M cells with human genomic DNA, the authors have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-({sup 3}H)HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na{sup +} and temperature dependence; (iv) kinetic saturability; and (v) high affinity for 5-HT. This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, they postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neutrotransmitter-uptake systems as well as substrate for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.

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

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    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.

  13. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

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    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

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

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

    2007-11-20

    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 K(m) value of MOT1 for molybdate is approximately 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.

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

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    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.

  16. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

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    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

  17. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

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

    Science.gov (United States)

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

    2016-07-15

    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.

  19. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)prop...

  20. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  1. In silico analysis of high affinity potassium transporter (HKT) isoforms in different plants.

    Science.gov (United States)

    Zamani Babgohari, Mahbobeh; Ebrahimie, Esmaeil; Niazi, Ali

    2014-01-01

    High affinity potassium transporters (HKTs) are located in the plasma membrane of the vessels and have significant influence on salt tolerance in some plants. They exclude Na(+) from the parenchyma cells to reduce Na(+) concentration. Despite many studies, the underlying regulatory mechanisms and the exact functions of HKTs within different genomic backgrounds are relatively unknown. In this study, various bioinformatics techniques, including promoter analysis, identification of HKT-surrounding genes, and construction of gene networks, were applied to investigate the HKT regulatory mechanism. Promoter analysis showed that rice HKTs carry ABA response elements. Additionally, jasmonic acid response elements were detected on promoter region of TmHKT1;5. In silico synteny highlighted several unknown and new loci near rice, Arabidopsis thaliana and Physcomitrella patent HKTs, which may play a significant role in salt stress tolerance in concert with HKTs. Gene network prediction unravelled that crosstalk between jasmonate and ethylene reduces AtHKT1;1 expression. Furthermore, antiporter and transferase proteins were found in AtHKT1;1 gene network. Interestingly, regulatory elements on the promoter region of HKT in wild genotype (TmHKT1;5) were more frequent and variable than the ones in cultivated wheat (TaHKT1;5) which provides the possibility of rapid response and better understanding of environmental conditions for wild genotype. Detecting ABA and jasmonic acid response elements on promoter regions of HKTs provide valuable clues on underlying regulatory mechanisms of HKTs. In silico synteny and pathway discovery indicated several candidates which act in concert with HKTs in stress condition. We highlighted different arrangement of regulatory elements on promoter region of wild wheat (TmHKT1;5) compared to bread wheat (TaHKT1;5) in this study.

  2. Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

    Science.gov (United States)

    Luo, Dan; Chen, Lei; Yu, Baoping

    2017-06-17

    The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. We report for the first time that CHT1 may be involved

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

    Science.gov (United States)

    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.

  4. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.

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    Norholm, Morten H H; Nour-Eldin, Hussam H; Brodersen, Peter; Mundy, John; Halkier, Barbara A

    2006-04-17

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

  5. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    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...... 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...

  6. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) impacts lipid, bone, and glucose homeostasis. The GIP receptor belongs to G protein-coupled receptor family B1 and signals through GαS. High affinity ligands for in vivo use are needed to elucidate GIP's physiological...... functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

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

    Science.gov (United States)

    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.

  8. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

    Science.gov (United States)

    Stagg, Amy R.; Fleming, Judith C.; Baker, Meghan A.; Sakamoto, Massayuki; Cohen, Nadine; Neufeld, Ellis J.

    1999-01-01

    We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400–550 nM; Vmax 11 pmol/min/106 cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death. J. Clin. Invest. 103:723–729 (1999). PMID:10074490

  9. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    Science.gov (United States)

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

  10. Osmoprotectants in Halomonas elongata: High-affinity betaine transport system and choline-betaine pathway

    OpenAIRE

    Nieto Gutiérrez, Joaquín José; Cánovas, David; Vargas, C; Ventosa Ucero, Antonio; Csonka, Laszlo N.

    1996-01-01

    The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M...

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

    OpenAIRE

    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. Kine...

  12. Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

    Science.gov (United States)

    Erickson, Jeffrey D

    2017-07-01

    Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K(+) -stimulated MeAIB transport displays an affinity (Km ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca(2+) , and is blocked by inhibition of voltage-gated Ca(2+) channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca(2+) and voltage-gated calcium channels, but is also blocked by the Na(+) channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca(2+) , but on Na(+) ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K(+) -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017

  13. Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram

    DEFF Research Database (Denmark)

    Kumar, Vivek; Rahbek-Clemmensen, Troels; Billesbølle, Christian B

    2014-01-01

    Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demons...

  14. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    Science.gov (United States)

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  15. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  16. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Science.gov (United States)

    Stockner, Thomas; Montgomery, Therese R; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F; Freissmuth, Michael; Sitte, Harald H

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  17. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    Science.gov (United States)

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  18. Plant High-Affinity Potassium (HKT Transporters Involved in Salinity Tolerance: Structural Insights to Probe Differences in Ion Selectivity

    Directory of Open Access Journals (Sweden)

    Maria Hrmova

    2013-04-01

    Full Text Available High-affinity Potassium Transporters (HKTs belong to an important class of integral membrane proteins (IMPs that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

  19. Plant High-Affinity Potassium (HKT) Transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity.

    Science.gov (United States)

    Waters, Shane; Gilliham, Matthew; Hrmova, Maria

    2013-04-09

    High-affinity Potassium Transporters (HKTs) belong to an important class of integral membrane proteins (IMPs) that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

  20. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Xue-Qiang Zhao; Yong-Guan Zhu; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2007-01-01

    Nitrate is a major nitrogen (N) source for most crops.Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels.Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency.The present study investigated the regulation of the high-affinity nitrate transport system (HATS) by exogenous abscisic acid (ABA) and glutamine (Gin) in wheat (Triticum aestivum L.) roots.Wheat seedlings grown in nutrient solution containing 2 mmollL nitrate as the only nitrogen source for 2 weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h.Treated wheat plants were then divided into two groups.One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L 15 N-labeled nitrate.The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate.Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction.When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced.These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media.Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.

  2. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy.

    Science.gov (United States)

    Kuo, Macus Tien; Fu, Siqing; Savaraj, Niramol; Chen, Helen H W

    2012-09-15

    The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.

  3. Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

    DEFF Research Database (Denmark)

    Cha, Joo Hwan; Zou, Mu-Fa; Adkins, Erika M

    2005-01-01

    Novel fluorescent ligands were synthesized to identify a high-affinity probe that would enable visualization of the dopamine transporter (DAT) in living cells. Fluorescent tags were extended from the N- or 2-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane, using an ethylamino lin...

  4. Monomeric TonB and the Ton box are required for the Formation of a High-Affinity Transporter-TonB Complex†

    Science.gov (United States)

    Freed, Daniel M.; Lukasik, Stephen M.; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S.

    2013-01-01

    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 the present study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB to the outer membrane transporters BtuB, FecA and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high-affinity (tens of nM) to both BtuB and FhuA; however, no high-affinity binding was observed to FecA. 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 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

  5. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    Science.gov (United States)

    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.

  6. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    Science.gov (United States)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

  7. Effect of P Availability on Temporal Dynamics of Carbon Allocation and Glomus intraradices High-Affinity P Transporter Gene Induction in Arbuscular Mycorrhiza

    Science.gov (United States)

    Olsson, Pål Axel; Hansson, Maria C.; Burleigh, Stephen H.

    2006-01-01

    Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment. PMID:16751522

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

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen;

    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 photocrosslin......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...

  9. Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum.

    Science.gov (United States)

    Larson, R J; Pate, J L

    1976-04-01

    Active transport of glucose in prosthecae isolated from cells of Asticcacaulis biprosthecum was stimulated by the non-physiological electron donor N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride. Glucose uptake was mediated by two transport systems; the apparent Km of the high-affinity system was 1.8 muM and that of the low-affinity system was 34 muM. Free glucose accumulated within prosthecae at a concentration 60 to 200 times above that present externally, depending on the Km of the system being observed. The glucose transport system in prosthecae was stereospecific for D-glucose, and neither methyl alpha-D-glucopyranoside nor 2-deoxyglucose was transported. Uptake of glucose was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB), and the inhibition by PCMB but not by NEM was reversed by dithiothreitol. Glucose uptake was also inhibited by the uncoupling agents 5-chloro-3-t-butyl-2'-nitrosalicylanilide (S-13), 5-chloro-3-(p-chlorophenyl)-4'-chlorosalicylanilide (S-6), and carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and by the respiratory inhibitor KCN. Efflux of glucose from preloaded prosthecae was induced by PCMB and KCN, but not by S-13 or CCCP. Glucose uptake was not affected by arsenate or an inhibitor of membrane-bound adenosine triphosphatases, N, N'-dicyclohexylcarbodiimide. The lack of inhibition by these two compounds, combined with the extremely low levels of adenosine 5'-triphosphate present in prosthecae, indicates that adenosine 5'-triphosphate is not involved in the transport of glucose by prosthecae.

  10. Arg-425 of the citrate transporter CitP is responsible for high affinity binding of di- and tricarboxylatese

    NARCIS (Netherlands)

    Bandell, M; Lolkema, JS

    2000-01-01

    The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism

  11. Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates

    NARCIS (Netherlands)

    Bandell, Michael; Lolkema, Juke S.

    2000-01-01

    The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism

  12. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron

    NARCIS (Netherlands)

    Miethke, Marcus; Monteferrante, Carmine G.; Marahiel, Mohamed A.; van Dijl, Jan Maarten

    2013-01-01

    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

  13. Glucose transport in adipose tissue

    NARCIS (Netherlands)

    Schoonen, AJM; Wientjes, KJC

    2005-01-01

    Based on the well-known extraction equation and the histology of subcutaneous adipose tissue, transport of glucose from capillary to microdialysis probe is described. Results are evaluated of previous studies by our group and others. Arguments are presented for a simple scheme in which the mean

  14. The Prostaglandin Transporter: Eicosanoid Reuptake, Control of Signaling, and Development of High-Affinity Inhibitors as Drug Candidates.

    Science.gov (United States)

    Schuster, Victor L; Chi, Yuling; Lu, Run

    2015-01-01

    We discovered the prostaglandin transporter (PGT) and cloned the human cDNA and gene. PGT transports extracellular prostaglandins (PGs) into the cytoplasm for enzymatic inactivation. PGT knockout mice have elevated prostaglandin E2 (PGE2) and neonatal patent ductus arteriosus, which reflects PGT's control over PGE2 signaling at EP1/EP4 cell-surface receptors. Interestingly, rescued PGT knockout pups have a nearly normal phenotype, as do human PGT nulls. Given the benign phenotype of PGT genetic nulls, and because PGs are useful medicines, we have approached PGT as a drug target. Triazine library screening yielded a lead compound of inhibitory constant 50% (IC50) = 3.7 μM, which we developed into a better inhibitor of IC50 378 nM. Further structural improvements have yielded 26 rationally designed derivatives with IC50 < 100 nM. The therapeutic approach of increasing endogenous PGs by inhibiting PGT offers promise in diseases such as pulmonary hypertension and obesity.

  15. The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes.

    Science.gov (United States)

    Teka, Ibrahim A; Kazibwe, Anne J N; El-Sabbagh, Nasser; Al-Salabi, Mohammed I; Ward, Christopher P; Eze, Anthonius A; Munday, Jane C; Mäser, Pascal; Matovu, Enock; Barrett, Michael P; de Koning, Harry P

    2011-07-01

    African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

  16. A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Ramon Wahl

    2010-02-01

    Full Text Available Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1 from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.

  17. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    Science.gov (United States)

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  18. The solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, rs3794087 variant and assessment risk for restless legs syndrome.

    Science.gov (United States)

    Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Martín, Elena; Agúndez, José A G

    2014-02-01

    A glutamatergic dysfunction has been postulated to play a role in restless legs syndrome (RLS) pathophysiology, as glutamate concentrations have been found to increase in the thalamus of RLS patients. The aim of our study was to investigate the possible association between the single nucleotide polymorphism (SNP) rs3794087 in the solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, related with glutamate transport and the risk for RLS. We studied the allelic and genotype frequencies of the SNP rs3794087 in 205 patients with RLS and 328 healthy controls using TaqMan genotyping. The rs3794087 genotype and allelic frequencies did not significantly differ between patients with RLS and controls and were unrelated with the age at onset of RLS, gender, and family history of RLS. The results of our study suggest that the rs3794087 polymorphism is not related to the risk for RLS. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Expression of Brassica napus L. γ-Glutamylcysteine Synthetase and Low-and High-Affinity Sulfate Transporters in Response to Excess Cadmium

    Institute of Scientific and Technical Information of China (English)

    Xin SUN; Xue-Mei SUN; Zhi-Min YANG; Shao-Qiong LI; Jin WANG; Song-Hua WANG

    2005-01-01

    In both the roots and leaves ofBrassica napus L. cv. Youyan No. 8 under treatment with 30 μmol/L Cd, massive production of non-protein thiols (NPT; mainly containing glutathione (GSH) and phytochelatins (PCs)) was induced, together with an increase in γ-glutamylcysteine synthetase (γ-ECS)mRNA transcripts. Because γ-ECS is the key enzyme catalyzing the first step in GSH biosynthesis, which, in turn, is converted to PCs, the Cd-induced increase in γ-ECS expression may be responsible for the observed increase in the production of NPT. Using a quantitative reverse transcription polymerase chain reaction (RT-PCR) approach, the expression of genes encoding a putative low-affinity sulfate transporter (LAST) and a putative high-affinity sulfate transporter (HAST) was determined at the transcriptional level. The RT-PCR analysis of relative transcript amounts indicates that the LAST gene in B. napus leaves showed a constitutive expression, which was hardly affected by Cd treatment. However, treatment with 30 μmol/L Cd for 2 or 3 d induced a marked increase in the expression of LAST in roots. Transcriptional expression of the HAST gene occurred in roots, but not in leaves. The expression of HAST only in the roots suggests that it has a specific function in sulfate uptake from soil and that the putative LAST may be responsible for the transport of sulfate from the roots to the shoots, as well as for the uptake of sulfate from soil. These results indicate that changes in transcriptional expression for sulfate transporters were required for the increased demand for sulfate during Cd stress.

  20. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    Science.gov (United States)

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions.

  1. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

    Directory of Open Access Journals (Sweden)

    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  2. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

    Science.gov (United States)

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-04-23

    AMT1-3 encodes the high affinity NH₄⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  3. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Directory of Open Access Journals (Sweden)

    Aili Bao

    2015-04-01

    Full Text Available AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  4. Time-resolved investigation of molecular components involved in the induction of NO3- high affinity transport system in maize roots

    Directory of Open Access Journals (Sweden)

    Youry Pii

    2016-11-01

    Full Text Available The induction, i.e. the rapid increase of nitrate (NO3- uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3- treatment were characterized in terms of changes in NO3- uptake rate and plasma membrane (PM H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3 and PM H+-ATPase gene families. The behaviour of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼ 150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3- treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A nondenaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  5. Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

    Science.gov (United States)

    Boscari, Alexandre; Van de Sype, Ghislaine; Le Rudulier, Daniel; Mandon, Karine

    2006-08-01

    Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

  6. The Human Carnitine Transporter SLC22A16 Mediates High Affinity Uptake of the Anticancer Polyamine Analogue Bleomycin-A5*

    Science.gov (United States)

    Aouida, Mustapha; Poulin, Richard; Ramotar, Dindial

    2010-01-01

    Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae l-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human l-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, l-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients. PMID:20037140

  7. The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycin-A5.

    Science.gov (United States)

    Aouida, Mustapha; Poulin, Richard; Ramotar, Dindial

    2010-02-26

    Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae L-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human L-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, L-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients.

  8. IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

    Institute of Scientific and Technical Information of China (English)

    章精; 刘志红; 刘栋; 黎磊石

    2001-01-01

    Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

  9. Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3– High Affinity Transport System in Maize Roots

    Science.gov (United States)

    Pii, Youry; Alessandrini, Massimiliano; Dall’Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms. PMID:27877183

  10. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    Science.gov (United States)

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  11. Time-Resolved Investigation of Molecular Components Involved in the Induction of [Formula: see text] High Affinity Transport System in Maize Roots.

    Science.gov (United States)

    Pii, Youry; Alessandrini, Massimiliano; Dall'Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H(+)-ATPase transcripts indicates that those mainly involved in the response to [Formula: see text] treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H(+)-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  12. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    in delivering iron to cells during formation of the tubular epithelial cells of the primordial kidney. No cellular receptor for NGAL has been described. We show here that megalin, a member of the low-density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown...

  13. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    Science.gov (United States)

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( F). For longissimus dorsi, EAAC1 ( W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( adipose, respectively, and not differing ( > 0.45) from omental or mesenchymal adipose. These data demonstrate (1) longissimus dorsi and adipose tissues of steers developing through typical

  14. 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.

    Science.gov (United States)

    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.

  15. Glucose Transporters in Cardiac Metabolism and Hypertrophy

    Science.gov (United States)

    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

  16. Identification of Glucose Transporters in Aspergillus nidulans

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Ploug, T; Stallknecht, B M; Pedersen, O

    1990-01-01

    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...

  19. Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556.

    Science.gov (United States)

    Postma, E; Van den Broek, P J

    1990-01-01

    Regulation of transport of D-glucose and D-fructose was studied in Kluyveromyces marxianus grown in continuous culture. Both substrates could be transported by at least two different transport systems, low-affinity transport and high-affinity proton-sugar symport. The low-affinity transporter, specific for both glucose and fructose, was constitutively present and was apparently not regulated by carbon catabolite repression. Regulation of the activity of the glucose- and fructose-specific proton symport systems appeared to proceed mainly through catabolite repression. Activation of symport did not need the presence of specific inductor molecules in the medium. Nevertheless, the capacities of the proton-sugar symporters varied in cells grown on a wide variety of carbon sources. The possibility that the control of proton symport activity is related to the presence of specific intracellular metabolites is discussed. PMID:2160928

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

    Energy Technology Data Exchange (ETDEWEB)

    Ploug, T.; Stallknecht, B.M.; Pedersen, O.; Kahn, B.B.; Ohkuwa, T.; Vinten, J.; Galbo, H. (Panum Institute, Copenhagen (Denmark))

    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.

  1. Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

    Science.gov (United States)

    Hsieh, I-Shan; Yang, Rong-Sen; Fu, Wen-Mei

    2014-01-01

    Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients. PMID:25310823

  2. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    Science.gov (United States)

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content. © 2015 Authors; published by Portland Press Limited.

  3. Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation.

    Science.gov (United States)

    Milner, Matthew J; Craft, Eric; Yamaji, Naoki; Koyama, Emi; Ma, Jian Feng; Kochian, Leon V

    2012-07-01

    • In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. • We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. • Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. • In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem. No claim to original US government works. New Phytologist © 2012 New Phytologist Trust.

  4. Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ladefoged, Lucy Kate; Kristensen, Trine N. Bjerre

    2016-01-01

    The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still...

  5. [Glucose transporter type 1 (GLUT-1) deficiency].

    Science.gov (United States)

    Cano, A; Ticus, I; Chabrol, B

    2008-11-01

    Impaired glucose transport across the blood brain barrier results in glucose transporter type 1 (GLUT-1) deficiency syndrome, first described in 1991. It is characterized by infantile seizures refractory to anticonvulsive treatments, microcephaly, delays in mental and motor development, spasticity, ataxia, dysarthria and other paroxysmal neurologic phenomena, often occurring prior to meals. Affected infants are normal at birth following an uneventful pregnancy and delivery. Seizures usually begin between the age of one and four months and can be preceded by apneic episodes or abnormal eyes movements. Patients with atypical presentations such as mental retardation and intermittent ataxia without seizures, or movement disorders characterized by choreoathetosis and dystonia, have also been described. Glucose is the principal fuel source for the brain and GLUT-1 is the only vehicle by which glucose enters the brain. In case of GLUT-1 deficiency, the risk of clinical manifestations is increased in infancy and childhood, when the brain glucose demand is maximal. The hallmark of the disease is a low glucose concentration in the cerebrospinal fluid in a presence of normoglycemia (cerebrospinal fluid/blood glucose ratio less than 0.4). The GLUT-1 defect can be confirmed by molecular analysis of the SCL2A1 gene or in erythrocytes by glucose uptake studies and GLUT-1 immunoreactivity. Several heterozygous mutations, with a majority of de novo mutations, resulting in GLUT-1 haploinsufficiency, have been described. Cases with an autosomal dominant transmission have been established and adults can exhibit symptoms of this deficiency. Ketogenic diet is an effective treatment of epileptic manifestations as ketone bodies serve as an alternative fuel for the developing brain. However, this diet is not effective on cognitive impairment and other treatments are being evaluated. The physiopathology of this disorder is partially unclear and its understanding could explain the clinical

  6. Characterisation of [11C]PR04.MZ in Papio anubis baboon: A selective high-affinity radioligand for quantitative imaging of the dopamine transporter

    Energy Technology Data Exchange (ETDEWEB)

    Riss P. J.; Fowler J.; Riss, P.J.; Hooker, J.M.; Shea, C.; Xu, Y.; Carter, P.; Warner, D.; Ferrari V.; Kim, S.W.; Aigbirhio, F.I.; Fowler, J.S.; Roesch, F.

    2011-10-25

    N-(4-fluorobut-2-yn-1-yl)-2{beta}-carbomethoxy-3{beta}-(4{prime}-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [{sup 11}C]PR04.MZ ([{sup 11}C]-1) has been developed using GMP compliant equipment. An adult female Papioanubis baboon was studied using a test-retest protocol with [{sup 11}C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (fP), plasma input functions and metabolic degradation of the radiotracer [{sup 11}C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (VT) and non-displaceable binding potentials (BPND) for various brain regions and the blood were obtained from kinetic modelling. [{sup 11}C]-1 shows promising results as aselective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.

  7. Characterisation of [¹¹C]PR04.MZ in Papio anubis baboon: a selective high-affinity radioligand for quantitative imaging of the dopamine transporter.

    Science.gov (United States)

    Riss, Patrick J; Hooker, Jacob M; Shea, Colleen; Xu, Youwen; Carter, Pauline; Warner, Donald; Ferrari, Valentina; Kim, Sung-Won; Aigbirhio, Franklin I; Fowler, Joanna S; Roesch, Frank

    2012-01-01

    N-(4-fluorobut-2-yn-1-yl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [(11)C]PR04.MZ ([(11)C]-1) has been developed using GMP compliant equipment. An adult female Papio anubis baboon was studied using a test-retest protocol with [(11)C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (f(P)), plasma input functions and metabolic degradation of the radiotracer [(11)C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (V(T)) and non-displaceable binding potentials (BP(ND)) for various brain regions and the blood were obtained from kinetic modelling. [(11)C]-1 shows promising results as a selective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. The expression and regulation of glucose transporters in tumor cells

    Directory of Open Access Journals (Sweden)

    Pengfei Zhao

    2016-12-01

    Full Text Available Glucose transporter proteins are involved in many physiological and biochemical processes. In particular, the high expressions of sodium-glucose cotransporter and glucose transporter proteins in tumor cells show that these two transporters play a key role in tumor cell metabolism. Studying the crystal structure and conformation of human glucose transporter proteins has enabled the development of drugs based on specific binding sites, opening up a new path towards more effective cancer treatments. This mini review serves to summarize our existing understanding of the metabolic pathways of tumor cells, focusing on the roles of glucose transporter proteins.

  9. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

    Science.gov (United States)

    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  10. SbHKT1;4, a member of the high-affinity potassium transporter gene family from Sorghum bicolor, functions to maintain optimal Na+/K+ balance under Na+ stress

    Institute of Scientific and Technical Information of China (English)

    Tian-Tian Wang; Zhi-Jie Ren; Zhi-Quan Liu; Xue Feng; Rui-Qi Guo; Bao-Guo Li; Le-Gong Li; HaiChun Jing

    2014-01-01

    In halophytic plants, the high-affinity potassium transporter HKT gene family can selectively uptake Kþ in the presence of toxic concentrations of Naþ. This has so far not been well examined in glycophytic crops. Here, we report the characterization of SbHKT1;4, a member of the HKT gene family from Sorghum bicolor. Upon Naþ stress, SbHKT1;4 expression was more strongly upregulated in salt-tolerant sorghum accession, correlating with a better balanced Naþ/Kþ ratio and enhanced plant growth. Heterogeneous expression analyses in mutants of Saccharomyces cerevisiae and Arabidopsis thaliana indicated that overexpressing SbHKT1;4 resulted in hypersensitivity to Naþ stress, and such hypersensitivity could be alleviated with the supply of elevated levels of Kþ, implicating that SbHKT1;4 may mediate Kþ uptake in the presence of excessive Naþ. Further electrophysiological evidence demonstrated that SbHKT1;4 could transport Naþ and Kþ when expressed in Xenopus laevis oocytes. The relevance of the finding that SbHKT1;4 functions to maintain optimal Naþ/Kþ balance under Naþ stress to the breeding of salt-tolerant glycophytic crops is discussed.

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

    Science.gov (United States)

    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.

  12. Zinc Oxide Nanostructured Biosensor for Glucose Detection

    Institute of Scientific and Technical Information of China (English)

    X. W.Sun; J.X. Wang; A. Wei

    2008-01-01

    Zinc oxide (ZnO) nanocombs were fabricated by vapor phase transport, and nanorods and hierarchical nanodisk structures by aqueous thermal decomposition. Glucose biosensors were constructed using these ZnO nanostructures as supporting materials for glucose oxidase (GOx) loading. These ZnO glucose biosensors showed a high sensitivity for glucose detection and high affinity of GOx to glucose as well as the low detection limit. The results demonstrate that ZnO nanostructures have potential applications in biosensors.

  13. Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.

    Science.gov (United States)

    Liu, Xiaoqin; Huang, Daimin; Tao, Jinyuan; Miller, Anthony J; Fan, Xiaorong; Xu, Guohua

    2014-10-01

    A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.

  14. Glucose sensing and signalling; regulation of intestinal glucose transport.

    Science.gov (United States)

    Shirazi-Beechey, S P; Moran, A W; Batchelor, D J; Daly, K; Al-Rammahi, M

    2011-05-01

    Epithelial cells lining the inner surface of the intestinal epithelium are in direct contact with a lumenal environment that varies dramatically with diet. It has long been suggested that the intestinal epithelium can sense the nutrient composition of lumenal contents. It is only recently that the nature of intestinal nutrient-sensing molecules and underlying mechanisms have been elucidated. There are a number of nutrient sensors expressed on the luminal membrane of endocrine cells that are activated by various dietary nutrients. We showed that the intestinal glucose sensor, T1R2+T1R3 and the G-protein, gustducin are expressed in endocrine cells. Eliminating sweet transduction in mice in vivo by deletion of either gustducin or T1R3 prevented dietary monosaccharide- and artificial sweetener-induced up-regulation of the Na+/glucose cotransporter, SGLT1 observed in wild-type mice. Transgenic mice, lacking gustducin or T1R3 had deficiencies in secretion of glucagon-like peptide 1 (GLP-1) and, glucose-dependent insulinotrophic peptide (GIP). Furthermore, they had an abnormal insulin profile and prolonged elevation of postprandial blood glucose in response to orally ingested carbohydrates. GIP and GLP-1 increase insulin secretion, while glucagon-like peptide 2 (GLP-2) modulates intestinal growth, blood flow and expression of SGLT1. The receptor for GLP-2 resides in enteric neurons and not in any surface epithelial cells, suggesting the involvement of the enteric nervous system in SGLT1 up-regulation. The accessibility of the glucose sensor and the important role that it plays in regulation of intestinal glucose absorption and glucose homeostasis makes it an attractive nutritional and therapeutic target for manipulation.

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

    Science.gov (United States)

    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.

  16. Glucose transporter 1 localisation throughout pregnancy in the carnivore placenta

    DEFF Research Database (Denmark)

    Wooding, F.B.P.; Dantzer, Vibeke; Klisch, K.

    2007-01-01

    Glucose is one of the major fetal nutrients. Maternofetal transfer requires transport across the several placental membranes. This transfer is mediated by one or more of the fourteen known isoforms of glucose transporter. So far only Glucose Transporters 1 and 3 (GT1, GT3) have been shown...... to be located in placental membranes. GT1 may be the only one on the syncytiotrophoblast (human) or both may be present on the same membrane (rodents) or be required in sequence (ruminants, horses and elephant). This paper shows GT1 to be the only transporter demonstrable by immunocytochemistry in carnivore...

  17. Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake

    DEFF Research Database (Denmark)

    Richter, Erik; Hansen, B F; Hansen, S A

    1988-01-01

    in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure......, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.......The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing...

  18. El receptor de la hormona de crecimiento humana (hGH y la proteína de transporte de alta afinidad de la hGH Human Growth Hormone (GH Receptor and the High Affinity GH-Binding Protein

    Directory of Open Access Journals (Sweden)

    María Gabriela Ballerini

    2008-03-01

    Full Text Available La hormona de crecimiento humana (hGH circula parcialmente unida a su proteína de transporte de alta afinidad (GHBP la cual resulta del clivaje proteolítico del dominio extracelular del receptor de GH. Recientemente la enzima TACE se identificó como la metaloproteasa responsable del clivaje y liberación de GHBP a circulación. Aunque aún se desconoce la función específica de esta proteína de transporte, distintos trabajos en la literatura demuestran efectos que potencian y efectos inhibitorios sobre la acción de GH. Por otro lado, existen evidencias que demuestran una fuerte relación entre la GHBP y el nivel de receptor de GH en el hígado en situaciones fisiológicas y patológicas. Esto permitió proponer a la determinación de GHBP en suero como un marcador periférico de la abundancia del receptor de GH en los tejidos. La determinación de la concentración de GHBP sería de especial interés para evaluar pacientes con diagnóstico probable de insensibilidad a la acción de GH y orientar el posterior estudio de anormalidades en el gen del receptor de GH. En la presente revisión, también se abordan dificultades metodológicas relacionadas a la medición de GHBP sérica.Human circulating growth hormone (GH is partly bound to a high-affinity binding protein (GHBP which is derived from proteolytical cleavage of the extracellular domain of the GH receptor. Recently, the metalloproteinase TACE has been identified as an important enzyme responsive for inducing GHBP shedding. Although the specific function of GHBP is not fully known, both enhancing and inhibitory roles of this binding protein on GH action have been proposed. Many reports have demonstrated a close relationship between GHBP and the liver GH receptor status in physiological conditions and diseases. Moreover, serum GHBP measurement has been proposed as an useful peripheral index of the GH receptor abundance. Related to the latter, circulating GHBP concentration would be of

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

    Directory of Open Access Journals (Sweden)

    Lihong Chen

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    Chen, Lihong; Tuo, Biguang; Dong, Hui

    2016-01-14

    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 ([Ca(2+)]cyt) can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca(2+) in IEC are regulated by cation channels and transporters, such as Ca(2+) channels, K⁺ channels, Na⁺/Ca(2+) 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.

  1. Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes.

    Science.gov (United States)

    Lewko, Barbara; Bryl, Ewa; Witkowski, Jacek M; Latawiec, Elzbieta; Angielski, Stefan; Stepinski, Jan

    2005-02-01

    Recent studies show that mechanical stress modifies both morphology and protein expression in podocytes. Ambient glucose is another factor modulating protein synthesis in these cells. In diabetes, podocytes experience elevated glucose concentrations as well as mechanical strain generated by high intracapillary pressures. Both these factors are responsible for podocyte injury, leading to impairment of kidney glomerular function. In the present study, we examined the effects of glucose concentration and mechanical stress on glucose uptake in podocytes. Following a 24 h pre-incubation in low (2.5 mM, LG), normal (5.6 mM, NG) or high (30 mM, HG) glucose media, cultured rat podocytes were exposed to 4 h mechanical stress. We used the labelled glucose analogue, [3H]2-deoxy-D-glucose, to measure glucose uptake. The distribution of facilitative glucose transporters GLUT2 and GLUT4 was assessed by flow cytometry. In the control (static) cells, glucose uptake was similar in the three glucose groups. In mechanically stressed podocytes, glucose uptake increased 2-fold in the LG and NG groups but increased 3-fold in the HG group. In the NG cells, mechanical load increased the membrane expression of GLUT2 and reduced the membrane-bound GLUT4. In stretched HG cells, the membrane expression of both GLUT2 and GLUT4 was decreased. High glucose decreased the plasma membrane GLUT2 content in the stretched cells, whereas both static and stretched podocytes showed an elevation in GLUT4. Mechanical stress potentiated glucose uptake in podocytes and this effect was enhanced by high ambient glucose. The decreased expression of GLUT2 and GLUT4 on the surface of stretched cells suggests that the activity of other glucose transporters may be regulated by mechanical stress in podocytes.

  2. Biological evaluation of two iodine-123-labeled D-glucose acetals prepared as glucose transporter radioligands

    Energy Technology Data Exchange (ETDEWEB)

    Brunet-Desruet, Marie-Dominique; Ghezzi, Catherine; Morin, Christophe; Comet, Michel; Fagret, Daniel

    1998-07-01

    Two iodinated acetals of D-glucose, 4,6-(R)-O-(2'-iodoethylidene)-{alpha}, {beta}-D-glucose and 4,6-(R)-O-(4'-iodobenzylidene)-{alpha}, {beta}-D-glucose , were prepared and their potential as suitable SPECT radioligands for imaging of glucose transporters was studied. Both are analogs of acetal D-glucose derivatives, which are known to bind to the exofacial sites of the glucose transport protein (GluT). To assess whether iodinated acetals 1 and 2 interacted with the glucose transporter, they were tested in vitro in human erythrocytes (GluT1) and neonatal rat cardiomyocytes (GluT4). The results indicated that 1 and 2 had a very low affinity for the glucose transporter and probably accumulated in cells. Study of their tissue distribution was carried out in the mouse in vivo: Both compounds showed fast tissue clearance with preferential renal elimination. It is concluded that iodinated acetals of D-glucose 1 and 2 are not suitable for GluT targeting in vivo.

  3. Dyes with high affinity for polylactide

    Institute of Scientific and Technical Information of China (English)

    Liang He; Shu Fen Zhang; Bing Tao Tang; Li Li Wang; Jin Zong Yang

    2007-01-01

    Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes.The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.

  4. Peritoneal transport characteristics with glucose polymer based dialysate.

    Science.gov (United States)

    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

    /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.

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

    Science.gov (United States)

    Naftalin, Richard J; Afzal, Iram; Cunningham, Philip; Halai, Mansur; Ross, Clare; Salleh, Naguib; Milligan, Stuart R

    2003-01-01

    This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea. Testosterone, 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA) and DHEA-3-acetate inhibit glucose exit from human erythrocytes with half-maximal inhibitions (Ki) of 39.2±8.9, 29.6±3.7, 48.1±10.2 and 4.8±0.98 μM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR). Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3-gallate (ECG) has a Ki ECG of 0.14±0.01 μM, and epigallocatechin 3-gallate (EGCG) has a Ki EGCG of 0.97±0.13 μM. Flutamide reverses these effects. Androgen-screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action. There are sequence homologies between GLUT1 and the ligand-binding domain (LBD) of hAR containing the amino-acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3-keto and 17-β OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1. PMID:12970085

  6. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

    Energy Technology Data Exchange (ETDEWEB)

    Morris, D.I.; Robbins, J.D.; Ruoho, A.E.; Sutkowski, E.M.; Seamon, K.B. (Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, MD (USA))

    1991-07-15

    Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

  7. The role of cysteine residues in glucose-transporter-GLUT1-mediated transport and transport inhibition.

    Science.gov (United States)

    Wellner, M; Monden, I; Keller, K

    1994-01-01

    The role of cysteine residues in transport function of the glucose transporter GLUT1 was investigated by a mutagenesis-expression strategy. Each of the six cysteine residues was individually replaced by site-directed mutagenesis. Expression of the heterologous wild-type or mutant glucose transporters and transport measurements at two hexose concentrations (50 microM and 5 mM) were undertaken in Xenopus oocytes. The catalytic activity of GLUT1 was retained, despite substitution of each single cysteine residue, which indicated that no individual residue is essential for hexose transport. This finding questions the involvement of oligomerization or intramolecular stabilization by a single disulphide bond as a prerequisite for transporter activation under basal conditions. Application of the impermeant mercurial thiol-group-reactive reagent p-chloromercuribenzenesulphonate (pCMBS) to the external or internal surface of plasma membrane demonstrated that cysteine-429, within the sixth external loop, and cysteine-207, at the beginning of the large intracellular loop which connects transmembrane segments 6 and 7, are the residues which are involved in transport inhibition by impermeant thiol-group-reactive reagents from either side of the cell. These data support the predicted membrane topology of the transport protein by transport measurements. If residues other than the cysteines at positions 429 or 207 are exposed to either side of the plasma membrane by conformational changes, they do not contribute to the transport inhibition by pCMBS. Application of pCMBS to one side of the plasma membrane also inhibited transport from the opposite direction, most likely due to the hindrance of sugar-induced interconversion of transporter conformation. PMID:8192671

  8. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  10. Alternating Carrier Models of Asymmetric Glucose Transport Violate the Energy Conservation Laws

    Science.gov (United States)

    Naftalin, Richard J

    2008-01-01

    Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric “carrier” (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\propto}1/K_{{\\mathrm{D}}}^{{\\mathrm{in}}}\\end{equation*}\\end{document}) and slower unliganded “free” carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, ΔGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}K_{{\\mathrm{D}}}^{{\\mathrm{in}}}/K_{{\\mathrm{D}}}^{{\\mathrm{out}}}\\end{equation*}\\end{document}), where R is the universal gas constant (8.314 Joules/M/K°), and T is the temperature, assumed here to be 300 K°, sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly

  11. Regulation of endogenous glucose production in glucose transporter 4 over-expressing mice.

    Directory of Open Access Journals (Sweden)

    Eric D Berglund

    Full Text Available Strategies to amplify whole-body glucose disposal are key therapies to treat type 2 diabetes. Mice that over-express glucose transporter 4 (Glut4 in skeletal muscle, heart, and adipose tissue (G4Tg exhibit increased fasting glucose disposal and thus lowered blood glucose. Intriguingly, G4Tg mice also exhibit improved insulin-stimulated suppression of endogenous glucose production even though Glut4 is not present in the liver. It is unclear, however, if hepatic gluco-regulation is altered in G4Tg mice in the basal, non-insulin-stimulated state. The current studies were performed to examine fasting hepatic glucose metabolism in G4Tg mice and to determine whether gluco-regulatory adaptations exist in the non-insulin-stimulated condition. To test this question, phloridzin-glucose clamps were used to match blood glucose and pancreatic hormone levels while tracer dilution techniques were used to measure glucose flux. These techniques were performed in chronically-catheterized, conscious, and un-stressed 5h-fasted G4Tg and wild-type (WT littermates. Results show reduced blood glucose, hepatic glycogen content, and hepatic glucokinase (GK activity/expression as well as higher endogenous glucose production, glucose disposal, arterial glucagon, and hepatic glucose-6-phosphatase (G6Pase activity/expression in G4Tg mice versus WT controls. Clamping blood glucose for 90 min at ~115 mg/dLin G4Tg and WT mice normalized nearly all variables. Notably, however, net hepatic glycogen synthetic rates were disproportionately elevated compared to changes in blood glucose. In conclusion, these studies demonstrate that basal improvements in glucose tolerance due to increased uptake in extra-hepatic sites provoke important gluco-regulatory adaptations in the liver. Although changes in blood glucose underlie the majority of these adaptations, net hepatic glycogen synthesis is sensitized. These data emphasize that anti-diabetic therapies that target skeletal muscle, heart

  12. Berberine stimulates glucose transport through a mechanism distinct from insulin.

    Science.gov (United States)

    Zhou, Libin; Yang, Ying; Wang, Xiao; Liu, Shangquan; Shang, Wenbin; Yuan, Guoyue; Li, Fengying; Tang, Jinfeng; Chen, Mingdao; Chen, Jialun

    2007-03-01

    Berberine exerts a hypoglycemic effect, but the mechanism remains unknown. In the present study, the effect of berberine on glucose uptake was characterized in 3T3-L1 adipocytes. It was revealed that berberine stimulated glucose uptake in 3T3-L1 adipocytes in a dose- and time-dependent manner with the maximal effect at 12 hours. Glucose uptake was increased by berberine in 3T3-L1 preadipocytes as well. Berberine-stimulated glucose uptake was additive to that of insulin in 3T3-L1 adipocytes, even at the maximal effective concentrations of both components. Unlike insulin, the effect of berberine on glucose uptake was insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and SB203580, an inhibitor of p38 mitogen-activated protein kinase. Berberine activated extracellular signal-regulated kinase (ERK) 1/2, but PD98059, an ERK kinase inhibitor, only decreased berberine-stimulated glucose uptake by 32%. Berberine did not induce Ser473 phosphorylation of Akt nor enhance insulin-induced phosphorylation of Akt. Meanwhile, the expression and cellular localization of glucose transporter 4 (GLUT4) were not altered by berberine. Berberine did not increase GLUT1 gene expression. However, genistein, a tyrosine kinase inhibitor, completely blocked berberine-stimulated glucose uptake in 3T3-L1 adipocytes and preadipocytes, suggesting that berberine may induce glucose transport via increasing GLUT1 activity. In addition, berberine increased adenosine monophosphate-activated protein kinase and acetyl-coenzyme A carboxylase phosphorylation. These findings suggest that berberine increases glucose uptake through a mechanism distinct from insulin, and activated adenosine monophosphate-activated protein kinase seems to be involved in the metabolic effect of berberine.

  13. Mechanisms of expression and translocation of major fission yeast glucose transporters regulated by CaMKK/phosphatases, nuclear shuttling, and TOR.

    Science.gov (United States)

    Saitoh, Shigeaki; Mori, Ayaka; Uehara, Lisa; Masuda, Fumie; Soejima, Saeko; Yanagida, Mitsuhiro

    2015-01-15

    Hexose transporters are required for cellular glucose uptake; thus they play a pivotal role in glucose homeostasis in multicellular organisms. Using fission yeast, we explored hexose transporter regulation in response to extracellular glucose concentrations. The high-affinity transporter Ght5 is regulated with regard to transcription and localization, much like the human GLUT transporters, which are implicated in diabetes. When restricted to a glucose concentration equivalent to that of human blood, the fission yeast transcriptional regulator Scr1, which represses Ght5 transcription in the presence of high glucose, is displaced from the nucleus. Its displacement is dependent on Ca(2+)/calmodulin-dependent kinase kinase, Ssp1, and Sds23 inhibition of PP2A/PP6-like protein phosphatases. Newly synthesized Ght5 locates preferentially at the cell tips with the aid of the target of rapamycin (TOR) complex 2 signaling. These results clarify the evolutionarily conserved molecular mechanisms underlying glucose homeostasis, which are essential for preventing hyperglycemia in humans.

  14. Kinetics of glucose transport in rat muscle

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, H; Vinten, J

    1987-01-01

    -MG concentration exhibited Michaelis-Menten kinetics. Uptake by simple diffusion could not be detected. The maximum 3-O-MG transport velocity (Vmax) was increased more by maximum isometric contractions (10- to 40-fold, depending on fiber type) than by insulin (20,000 microU/ml; 3- to 20-fold) in both red and white...

  15. Brain Glucose Transporter (Glut3) Haploinsufficiency Does Not Impair Mouse Brain Glucose Uptake

    OpenAIRE

    Stuart, Charles A.; Ross, Ian R.; Howell, Mary E. A.; McCurry, Melanie P.; Wood, Thomas G.; Ceci, Jeffrey D.; Kennel, Stephen J.; Wall, Jonathan

    2011-01-01

    Mouse brain expresses three principle glucose transporters. Glut1 is an endothelial marker and is the principal glucose transporter of the blood-brain barrier. Glut3 and Glut6 are expressed in glial cells and neural cells. A mouse line with a null allele for Glut3 has been developed. The Glut3−/− genotype is intrauterine lethal by seven days post-coitis, but the heterozygous (Glut3+/−) littermate survives, exhibiting rapid post-natal weight gain, but no seizures or other behavioral aberration...

  16. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    Science.gov (United States)

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  17. Characterization of 6-deoxy-6-iodo-D-glucose: A potential new tool to assess glucose transport

    Energy Technology Data Exchange (ETDEWEB)

    Henry, Christelle; Tanti, Jean-Francois; Gremeaux, Thierry; Morin, Christophe; Van Obberghen, Emmanuel; Comet, Michel; Le Marchand-Brustel, Yannick

    1997-01-01

    6-deoxy-6-iodo-D-glucose (6-DIG) was rapidly taken up by adipocytes. Insulin increased 6-DIG transport in adipocytes isolated from both rats and mice. This stimulation was more important in rat than in mouse adipocytes, in agreement with their respective amount of Glut 4 transporters. In two insulin-resistant states, the biological behavior of 6-DIG and 3-O-methyl-D-glucose was similar. These results indicated that 6-DIG, which was transported into the cells via the glucose transporters, could be potentially useful to measure modifications of glucose transport.

  18. Effect of aspirin on glucose-D transport in intestine of rat

    Institute of Scientific and Technical Information of China (English)

    Mazhar Mushtaq; Farah Deeba Khan; M. Naeem Akhtar; Saghir Ahmad Jafri; Mehboob Bari

    2009-01-01

    Objective: The present study was designed to evaluate the effect a commonly prescribed Non Steroidal Anti In-flammatory Drug (NSAID) i.e. aspirin on brush border membrane in terms of changes in the intestinal transport level of glucose which is monosaccharide with absolute requirement in the body and hence its absorption is directly proportional on the morphology of the intestinal mucosa. Method: Albino rats (Rattus Norvegicus) were divided into two different groups, Group Ⅰ (Control), Group Ⅱ ( aspirin-treated, 50 mg aspirin/kg of body weight). The treatment was continued for 28 days. On the 29th day after o-, vernight fasting, intestine was removed from animals of both groups. Changes in transport of glucose-D in intestine were studied. Result: The results indicated a significant decrease in the transport of glucose-D in aspirin treated group as compared to the con-trol group. Conclusion: Cautious use of NSAID is recommended in commonly observed symptom such as headache and to those patients who are given as a prophylaxis for thrombosis.

  19. In vitro characterization of luseogliflozin, a potent and competitive sodium glucose co-transporter 2 inhibitor: Inhibition kinetics and binding studies.

    Science.gov (United States)

    Uchida, Saeko; Mitani, Akiko; Gunji, Emi; Takahashi, Teisuke; Yamamoto, Koji

    2015-05-01

    In this study, we evaluated an inhibition model of luseogliflozin on sodium glucose co-transporter 2 (SGLT2). We also analyzed the binding kinetics of the drug to SGLT2 protein using [(3)H]-luseogliflozin. Luseogliflozin competitively inhibited human SGLT2 (hSGLT2)-mediated glucose uptake with a Ki value of 1.10 nM. In the absence of glucose, [(3)H]-luseogliflozin exhibited a high affinity for hSGLT2 with a Kd value of 1.3 nM. The dissociation half-time was 7 h, suggesting that luseogliflozin dissociates rather slowly from hSGLT2. These profiles of luseogliflozin might contribute to the long duration of action of this drug.

  20. Berberine acutely activates the glucose transport activity of GLUT1.

    Science.gov (United States)

    Cok, Alexandra; Plaisier, Christina; Salie, Matthew J; Oram, Daniel S; Chenge, Jude; Louters, Larry L

    2011-07-01

    Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 μM. Significant activation (P berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K(m) of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V(max) of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

  1. Hepatic expression and cellular distribution of the glucose transporter family

    Institute of Scientific and Technical Information of China (English)

    Sumera Karim; David H Adams; Patricia F Lalor

    2012-01-01

    Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules.To date 14 members of this family,also called the solute carrier 2A proteins have been identified which are divided on the basis of transport characteristics and sequence similarities into several families (Classes 1 to 3).The expression of these different receptor subtypes varies between different species,tissues and cellular subtypes and each has differential sensitivities to stimuli such as insulin.The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis,storage and redistribution of carbohydrates.Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure,confirming the importance of understanding the molecular regulation of hepatic glucose homeostasis.There is a considerable literature describing the expression and function of receptors that regulate glucose uptake and release by hepatocytes,the most import cells in glucose regulation and glycogen storage.However there is less appreciation of the roles of GLUTs expressed by non parenchymal cell types within the liver,all of which require carbohydrate to function.A better understanding of the detailed cellular distribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis.This review summarises the available literature on hepatocellular expression of GLUTs in health and disease and highlights areas where further investigation is required.

  2. Enhancement of glucose transport by selected plant foods in muscle cell line L6.

    Science.gov (United States)

    Noipha, K; Ratanachaiyavong, S; Ninla-Aesong, P

    2010-08-01

    Glucose uptake activity of 11 plant foods was assessed in L6 myotubes. Among them onion and ginger showed potent enhancement of glucose transport. This effect required new protein synthesis of glucose transporters. In addition, onion-induced glucose uptake in L6 myotubes was mediated through activation of phosphoinositide 3-kinase.

  3. Altered DNA methylation of glucose transporter 1 and glucose transporter 4 in patients with major depressive disorder.

    Science.gov (United States)

    Kahl, Kai G; Georgi, Karsten; Bleich, Stefan; Muschler, Marc; Hillemacher, Thomas; Hilfiker-Kleinert, Denise; Schweiger, Ulrich; Ding, Xiaoqi; Kotsiari, Alexandra; Frieling, Helge

    2016-05-01

    Alterations in brain glucose metabolism and in peripheral glucose metabolism have frequently been observed in major depressive disorder (MDD). The insulin independent glucose transporter 1 (GLUT1) plays a key role in brain metabolism while the insulin-dependent GLUT4 is the major glucose transporter for skeletal and cardiac muscle. We therefore examined methylation of GLUT1 and GLUT4 in fifty-two depressed inpatients and compared data to eighteen healthy comparison subjects. DNA methylation of the core promoter regions of GLUT1 and GLUT4 was assessed by bisulfite sequencing. Further factors determined were fasting glucose, cortisol, insulin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). We found significantly increased methylation of the GLUT1 in depressed inpatients compared to healthy comparison subjects (CG). Further findings comprise increased concentrations of fasting cortisol, glucose, insulin, and increased IL-6 and TNF-α. After six weeks of inpatient treatment, significantly lower GLUT1 methylation was observed in remitted patients compared to non-remitters. GLUT4 methylation was not different between depressed patients and CG, and did not differ between remitted and non-remitted patients. Although preliminary we conclude from our results that the acute phase of major depressive disorder is associated with increased GLUT1 methylation and mild insulin resistance. The successful treatment of depression is associated with normalization of GLUT1 methylation in remitters, indicating that this condition may be reversible. Failure of normalization of GLUT1 methylation in non-remitters may point to a possible role of impeded brain glucose metabolism in the maintenance of MDD.

  4. Glucose transport by epithelia prepared from harvested enterocytes

    DEFF Research Database (Denmark)

    Kimura, Yasuhiro; van der Merwe, Marie; Bering, Stine Brandt

    2015-01-01

    , 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...... 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...

  5. Crystal structure of the human glucose transporter GLUT1

    Science.gov (United States)

    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.

  6. Molecular Dynamics Simulations of the Human Glucose Transporter GLUT1.

    Directory of Open Access Journals (Sweden)

    Min-Sun Park

    Full Text Available 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 cancer. 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.

  7. Contribution of Glucose Transport to the Control of the Glycolytic Flux in Trypanosoma brucei

    Science.gov (United States)

    Bakker, Barbara M.; Walsh, Michael C.; Ter Kuile, Benno H.; Mensonides, Femke I. C.; Michels, Paul A. M.; Opperdoes, Fred R.; Westerhoff, Hans V.

    1999-08-01

    The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.

  8. Is contraction-stimulated glucose transport feedforward regulated by Ca2+?

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Angin, Yeliz; Sylow, Lykke

    2014-01-01

    feedforward regulator of the translocation of glucose transporter 4 to the cell surface to facilitate transmembrane glucose transport. This review summarizes the evidence supporting the Ca(2+) feedforward model and its proposed signalling links to regulation of glucose transport in skeletal muscle and other......-stimulated glucose transport. A revised working model is proposed, in which muscle glucose transport during contraction is not directly regulated by SR Ca(2+) release but rather responds exclusively to feedback signals activated secondary to cross-bridge cycling and tension development....

  9. Adipocyte glucose transport regulation by eicosanoid precursors and inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    Directory of Open Access Journals (Sweden)

    Fernando eAleman

    2014-09-01

    Full Text Available Potassium (K+ is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter.

  11. Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol

    Energy Technology Data Exchange (ETDEWEB)

    Singh, S.P.; Pullen, G.L.; Srivenugopal, K.S.; Yuan Xiaohua; Snyder, A.K. (Veterans Affairs Medical Center, North Chicago, IL (United States) Chicago Medical School, North Chicago, IL (United States))

    1992-01-01

    Using pregnant rats fed equicaloric liquid diets (AF, ad libitum-fed controls; PF, pair-fed controls; EF, ethanol-fed), the authors have previously shown that maternal alcoholism produces a specific and significant decrease of glucose in the fetal brain, which is accompanied by growth retardation. To further define the mechanisms of ethanol-induced perturbations in fetal fuel supply, they have examined (I) the uptake of 2-deoxyglucose (2-DG) by dissociated brain cells from fetal rats that were exposed to ethanol in utero and (II) the steady-state levels of the glucose transporter-1 (GT-1) mRNA. A 9% decrease in brain weight and a 54.8% reduction in 2-DG uptake into brain cells were found in offspring of EF mothers compared to the AF group. Brain weight correlated with the rate of 2-DG uptake. Northern blot analysis showed a 50% reduction of GT-1 mRNA in EF brain relative to that in the AF and PF groups. They conclude that glucose transport into the brain is an important parameter altered by maternal ethanol ingestion.

  12. Implications of Glucose Transporter Protein Type 1 (GLUT1)-Haplodeficiency in Embryonic Stem Cells for Their Survival in Response to Hypoxic Stress

    Science.gov (United States)

    Heilig, Charles; Brosius, Frank; Siu, Brian; Concepcion, Luis; Mortensen, Richard; Heilig, Kathleen; Zhu, Min; Weldon, Richard; Wu, Guimei; Conner, David

    2003-01-01

    Glucose transporter protein type 1 (GLUT1) is a major glucose transporter of the fertilized egg and preimplantation embryo. Haploinsufficiency for GLUT1 causes the GLUT1 deficiency syndrome in humans, however the embryo appears unaffected. Therefore, here we produced heterozygous GLUT1 knockout murine embryonic stem cells (GT1+/−) to study the role of GLUT1 deficiency in their growth, glucose metabolism, and survival in response to hypoxic stress. GT1(−/−) cells were determined to be nonviable. Both the GLUT1 and GLUT3 high-affinity, facilitative glucose transporters were expressed in GT1(+/+) and GT1(+/−) embryonic stem cells. GT1(+/−) demonstrated 49 ± 4% reduction of GLUT1 mRNA. This induced a posttranscriptional, GLUT1 compensatory response resulting in 24 ± 4% reduction of GLUT1 protein. GLUT3 was unchanged. GLUT8 and GLUT12 were also expressed and unchanged in GT1(+/−). Stimulation of glycolysis by azide inhibition of oxidative phosphorylation was impaired by 44% in GT1(+/−), with impaired up-regulation of GLUT1 protein. Hypoxia for up to 4 hours led to 201% more apoptosis in GT1(+/−) than in GT1(+/+) controls. Caspase-3 activity was 76% higher in GT1(+/−) versus GT1(+/+) at 2 hours. Heterozygous knockout of GLUT1 led to a partial GLUT1 compensatory response protecting nonstressed cells. However, inhibition of oxidative phosphorylation and hypoxia both exposed their increased susceptibility to these stresses. PMID:14578187

  13. Glucose transport by epithelia prepared from harvested enterocytes

    DEFF Research Database (Denmark)

    Kimura, Yasuhiro; van der Merwe, Marie; Bering, Stine Brandt;

    2015-01-01

    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......, 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...

  14. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    Science.gov (United States)

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  15. Sweet talk: insights into the nature and importance of glucose transport in lung epithelium.

    Science.gov (United States)

    Garnett, James P; Baker, Emma H; Baines, Deborah L

    2012-11-01

    For over 50 years, glucose has been recognised to cross the lung epithelial barrier and be transported by lung epithelial cells. However, until recently, research into these processes focused on their effects on lung liquid volume. Here, we consider a newly identified role for pulmonary glucose transport in maintaining low airway surface liquid (ASL) glucose concentrations and propose that this contributes to lung defence against infection. Glucose diffuses into ASL via paracellular pathways at a rate determined by paracellular permeability and the transepithelial glucose gradient. Glucose is removed from ASL in proximal airways via facilitative glucose transporters, down a concentration gradient generated by intracellular glucose metabolism. In the distal lung, glucose transport via sodium-coupled glucose transporters predominates. These processes vary between species but universally maintain ASL glucose at 3-20-fold lower concentrations than plasma. ASL glucose concentrations are increased in respiratory disease and by hyperglycaemia. Elevated ASL glucose in intensive care patients was associated with increased Staphylococcus aureus infection. Diabetic patients with and without chronic lung disease are at increased risk of respiratory infection. Understanding of mechanisms underlying lung glucose homeostasis could identify new therapeutic targets for control of ASL glucose and prevention and treatment of lung infection.

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

    Science.gov (United States)

    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.

  17. Sodium glucose transporter 2 (SGLT2 inhibition and ketogenesis

    Directory of Open Access Journals (Sweden)

    Sanjay Kalra

    2015-01-01

    Full Text Available Sodium glucose transporter 2 (SGLT2 inhibitors are a recently developed class of drug that have been approved for use in type 2 diabetes. Their unique extra-pancreatic glucuretic mode of action has encouraged their usage in type 1 diabetes as well. At the same time, reports of pseudo ketoacidosis and ketoacidosis related to their use have been published. No clear mechanism for this phenomenon has been demonstrated so far. This communication delves into the biochemical effects of SGLT2 inhibition, discusses the utility of these drugs and proposes steps to maximize safe usage of the molecules.

  18. Screening for Inhibitors of Essential Leishmania Glucose Transporters

    Science.gov (United States)

    2013-07-01

    600 are natural products , and ~400 are other bioactive components. The library was constructed to have a wide range of biological activities and...within each plate. Each well contained 20 µl of ∆lmxgt parasites, complemented with either LmxGT2 or GLUT1, suspended in DME -L medium (7) at an initial...expressing the LmGT2 transporter was grown for 72 hr in DME -L medium containing 5 mM glucose in the presence of compounds (2000) from the MicroSource

  19. Sodium glucose transporter 2 (SGLT2) inhibition and ketogenesis.

    Science.gov (United States)

    Kalra, Sanjay; Sahay, Rakesh; Gupta, Yashdeep

    2015-01-01

    Sodium glucose transporter 2 (SGLT2) inhibitors are a recently developed class of drug that have been approved for use in type 2 diabetes. Their unique extra-pancreatic glucuretic mode of action has encouraged their usage in type 1 diabetes as well. At the same time, reports of pseudo ketoacidosis and ketoacidosis related to their use have been published. No clear mechanism for this phenomenon has been demonstrated so far. This communication delves into the biochemical effects of SGLT2 inhibition, discusses the utility of these drugs and proposes steps to maximize safe usage of the molecules.

  20. Steviol Glycosides Modulate Glucose Transport in Different Cell Types

    Science.gov (United States)

    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

  1. Steviol Glycosides Modulate Glucose Transport in Different Cell Types

    Directory of Open Access Journals (Sweden)

    Benedetta Rizzo

    2013-01-01

    Full Text Available 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.

  2. Steviol glycosides modulate glucose transport in different cell types.

    Science.gov (United States)

    Rizzo, Benedetta; Zambonin, Laura; Angeloni, Cristina; Leoncini, Emanuela; Dalla Sega, Francesco Vieceli; 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.

  3. Domain assembly of the GLUT1 glucose transporter.

    Science.gov (United States)

    Cope, D L; Holman, G D; Baldwin, S A; Wolstenholme, A J

    1994-01-01

    A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation. Images Figure 1 PMID:8002929

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

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

    2016-03-01

    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.

  7. Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes

    DEFF Research Database (Denmark)

    Stallknecht, B; Andersen, P H; Vinten, J

    1993-01-01

    Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  8. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  9. Glucose transporter expression in human skeletal muscle fibers

    DEFF Research Database (Denmark)

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

    2000-01-01

    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...

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

    Science.gov (United States)

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

    2016-08-12

    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.

  11. Influence of preovulatory estradiol on conceptus survival and uterine glucose transporter expression

    Science.gov (United States)

    Glucose is an essential component of uterine secretions, and is delivered into the uterine lumen by glucose transporters. We have previously reported increased concentrations of glucose in uterine flushes of cows that exhibited estrus. Our objective in the present study was to determine the effects...

  12. The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

    Directory of Open Access Journals (Sweden)

    Pia V Röder

    Full Text Available Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.

  13. Effects of noradrenaline on the cell-surface glucose transporters in cultured brown adipocytes: novel mechanism for selective activation of GLUT1 glucose transporters.

    Science.gov (United States)

    Shimizu, Y; Satoh, S; Yano, H; Minokoshi, Y; Cushman, S W; Shimazu, T

    1998-01-01

    Glucose transport into rat brown adipocytes has been shown to be stimulated directly by the sympathetic neurotransmitter, noradrenaline, without a significant increase in the protein content of either GLUT1 or GLUT4 glucose transporter in the plasma membrane [Shimizu, Kielar, Minokoshi and Shimazu (1996) Biochem. J. 314, 485-490]. In the present study, we labelled the exofacial glucose-binding sites of GLUT1 and GLUT4 with a membrane-impermeant photoaffinity reagent, 2-N-[4-(1-azitrifluoroethyl)benzoyl]-[2-3H]1,3-bis- (D-mannos-4-yloxy)-2-propylamine (ATB-[3H]BMPA), to determine which isoform is responsible for the noradrenaline-induced increase in glucose transport into intact brown adipocytes in culture. Insulin stimulated the rate of hexose transport by increasing ATB-[3H]BMPA-labelled cell-surface GLUT4. In contrast, the noradrenaline-induced increase in glucose transport was not accompanied by an increased ATB-[3H]BMPA labelling of GLUT4, nor with an increased amount of GLUT4 in the plasma membrane fraction as assessed by Western blotting, indicating that noradrenaline does not promote the translocation of GLUT4. However, noradrenaline induced an increase in photoaffinity labelling of cell-surface GLUT1 without an apparent increase in the immunoreactive GLUT1 protein in the plasma membrane. This is suggestive of an increased affinity of GLUT1 for the ligand. In fact, the Ki value of non-radioactive ATB-BMPA for 2-deoxy-D-glucose uptake was significantly decreased after treatment of the cells with noradrenaline. The increased photoaffinity labelling of GLUT1 and increased glucose transport caused by noradrenaline were inhibited by a cAMP antagonist, cAMP-S Rp-isomer. These results demonstrate that noradrenaline stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 through a cAMP-dependent mechanism. PMID:9461536

  14. Glucose transporter 8 (GLUT8) regulates enterocyte fructose transport and global mammalian fructose utilization.

    Science.gov (United States)

    DeBosch, Brian J; Chi, Maggie; Moley, Kelle H

    2012-09-01

    Enterocyte fructose absorption is a tightly regulated process that precedes the deleterious effects of excess dietary fructose in mammals. Glucose transporter (GLUT)8 is a glucose/fructose transporter previously shown to be expressed in murine intestine. The in vivo function of GLUT8, however, remains unclear. Here, we demonstrate enhanced fructose-induced fructose transport in both in vitro and in vivo models of enterocyte GLUT8 deficiency. Fructose exposure stimulated [(14)C]-fructose uptake and decreased GLUT8 protein abundance in Caco2 colonocytes, whereas direct short hairpin RNA-mediated GLUT8 knockdown also stimulated fructose uptake. To assess GLUT8 function in vivo, we generated GLUT8-deficient (GLUT8KO) mice. GLUT8KO mice exhibited significantly greater jejunal fructose uptake at baseline and after high-fructose diet (HFrD) feeding vs. wild-type mice. Strikingly, long-term HFrD feeding in GLUT8KO mice exacerbated fructose-induced increases in blood pressure, serum insulin, low-density lipoprotein and total cholesterol vs. wild-type controls. Enhanced fructose uptake paralleled with increased abundance of the fructose and glucose transporter, GLUT12, in HFrD-fed GLUT8KO mouse enterocytes and in Caco2 cultures exposed to high-fructose medium. We conclude that GLUT8 regulates enterocyte fructose transport by regulating GLUT12, and that disrupted GLUT8 function has deleterious long-term metabolic sequelae. GLUT8 may thus represent a modifiable target in the prevention and treatment of malnutrition or the metabolic syndrome.

  15. Glucose transporter-1 deficiency syndrome: The expanding clinical and genetic spectrum of a treatable disorder

    NARCIS (Netherlands)

    W.G. Leen (Wilhelmina); J. Klepper (Joerg); M.M. Verbeek (Marcel); M. Leferink (Maike); T. Hofste (Tom); B.G.M. van Engelen (Baziel); R.A. Wevers (Ron); T. Arthur (Todd); N. Bahi-Buisson (Nadia); D. Ballhausen (Diana); J. Bekhof (Jolita); P. van Bogaert (Patrick); I. Carrilho (Inês); B. Chabrol (Brigitte); M.P. Champion (Michael); J. Coldwell (James); P. Clayton (Peter); E. Donner (Elizabeth); A. Evangeliou (Athanasios); F. Ebinger (Friedrich); K. Farrell (Kevin); R.J. Forsyth (Rob); C.G.E.L. de Goede (Christian); S. Gross (Stephanie); S. Grünewald (Sonja); H. Holthausen (Hans); S. Jayawant (Sandeep); K. Lachlan (Katherine); V. Laugel (Vincent); K. Leppig (Kathy); M.J. Lim (Ming); G.M.S. Mancini (Grazia); A.D. Marina; L. Martorell (Loreto); J. McMenamin (Joe); M.E.C. Meuwissen (Marije); H. Mundy (Helen); N.O. Nilsson (Nils); A. Panzer (Axel); B.T. Poll-The; C. Rauscher (Christian); C.M.R. Rouselle (Christophe); I. Sandvig (Inger); T. Scheffner (Thomas); E. Sheridan (Eamonn); N. Simpson (Neil); P. Sykora (Parol); R. Tomlinson (Richard); J. Trounce (John); D.W.M. Webb (David); B. Weschke (Bernhard); H. Scheffer (Hans); M.A. Willemsen (Michél)

    2010-01-01

    textabstractGlucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing an

  16. Glucose transporter-1 deficiency syndrome : the expanding clinical and genetic spectrum of a treatable disorder

    NARCIS (Netherlands)

    Leen, Wilhelmina G.; Klepper, Joerg; Verbeek, Marcel M.; Leferink, Maike; Hofste, Tom; van Engelen, Baziel G.; Wevers, Ron A.; Arthur, Todd; Bahi-Buisson, Nadia; Ballhausen, Diana; Bekhof, Jolita; van Bogaert, Patrick; Carrilho, Ines; Chabrol, Brigitte; Champion, Michael P.; Coldwell, James; Clayton, Peter; Donner, Elizabeth; Evangeliou, Athanasios; Ebinger, Friedrich; Farrell, Kevin; Forsyth, Rob J.; de Goede, Christian G. E. L.; Gross, Stephanie; Grunewald, Stephanie; Holthausen, Hans; Jayawant, Sandeep; Lachlan, Katherine; Laugel, Vincent; Leppig, Kathy; Lim, Ming J.; Mancini, Grazia; Della Marina, Adela; Martorell, Loreto; McMenamin, Joe; Meuwissen, Marije E. C.; Mundy, Helen; Nilsson, Nils O.; Panzer, Axel; Poll-The, Bwee T.; Rauscher, Christian; Rouselle, Christophe M. R.; Sandvig, Inger; Scheffner, Thomas; Sheridan, Eamonn; Simpson, Neil; Sykora, Parol; Tomlinson, Richard; Trounce, John; Webb, David; Weschke, Bernhard; Scheffer, Hans; Willemsen, Michel A.

    2010-01-01

    Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex

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

    Science.gov (United States)

    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…

  18. Role of the AMPKgamma3 isoform in hypoxia-stimulated glucose transport in glycolytic skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Glund, Stephan; Tom, Robby Z

    2009-01-01

    Skeletal muscle glucose transport is regulated via the canonical insulin-signaling cascade as well as by energy-sensing signals. 5'-AMP-activated protein kinase (AMPK) has been implicated in the energy status regulation of glucose transport. We determined the role of the AMPKgamma3 isoform...

  19. [High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1].

    Science.gov (United States)

    Hong, Mengqi; Nie, Zhenyu; Chen, Zhengyue; Yu, Xiongwei; Bao, Beiyan

    2016-05-25

    Objective: To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis. Methods: Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-β1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF in peritoneal membrane and HPECs. Results:In vivo, compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly increased (all P<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-β1 and CTGF (all P<0.05). In vitro, the mRNA and protein expressions of GLUT1, SGLT1, TGF-β1

  20. Metformin-induced regulation of the intestinal D-glucose transporters.

    OpenAIRE

    Sakar, Yassine; Meddah, Bouchra; El Abbes Faouzi, Moulay; Cherrah, Yahia; Bado, André; Ducroc, Robert

    2010-01-01

    International audience; Metformin is an orally administered drug that lowers blood glucose and improves insulin sensitivity in patients with non insulin-dependent diabetes. Although the antihyperglycemic effect of metformin has been extensively studied, its cellular mechanism(s) of action (including the effect on enterocyte) remains to be defined. This study was designed to examine the effect of metformin on glucose transporters in enterocyte. Na(+)-dependent glucose transporter-1 (SGLT-1) ac...

  1. Adaptive evolution for fast growth on glucose and the effects on the regulation of glucose transport system in Clostridium tyrobutyricum.

    Science.gov (United States)

    Jiang, Ling; Li, Shuang; Hu, Yi; Xu, Qing; Huang, He

    2012-03-01

    Laboratory adaptive evolution of microorganisms offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. By combining a fibrous-bed bioreactor, we successfully developed a simple and valuable adaptive evolution strategy in repeated-batch fermentation mode with high initial substrate concentration and evolved Clostridium tyrobutyricum mutant with significantly improved butyric acid volumetric productivity up to 2.25 g/(L h), which is the highest value in batch fermentation reported so far. Further experiments were conducted to pay attention to glucose transport system in consideration of the high glucose consumption rate resulted from evolution. Complete characterization and comparison of the glucose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) were carried out in the form of toluene-treated cells and cell-free extracts derived from both C. tyrobutyricum wide-type and mutant, while an alternative glucose transport route that requires glucokinase was confirmed by the phenomena of resistance to the glucose analogue 2-deoxyglucose and ATP-dependent glucose phosphorylation. Our results suggest that C. tyrobutyricum mutant is defective in PTS activity and compensates for this defect with enhanced glucokinase activity, resulting in the efficient uptake and consumption of glucose during the whole metabolism.

  2. Stretch-stimulated glucose transport in skeletal muscle is regulated by Rac1

    DEFF Research Database (Denmark)

    Sylow, Lykke; Møller, Lisbeth L V; Kleinert, Maximilian

    2015-01-01

    Alternatives to the canonical insulin signaling pathway for glucose transport are muscle contraction/exercise. Mechanical stress is an integrated part of the muscle contraction/relaxation cycle and passive stretch has been shown to increase muscle glucose transport. However, the signaling mechanism...... regulating stretch-stimulated glucose transport is not well understood. We recently reported that the actin cytoskeleton regulating GTPase, Rac1 was activated in mouse muscle in response to stretching. Rac1 is a regulator of contraction- and insulin-stimulated glucose transport but its role in stretch...... part of the mechanical stress-component of the contraction-stimulus to glucose transport in skeletal muscle. This article is protected by copyright. All rights reserved....

  3. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    Science.gov (United States)

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  4. A novel strategy for the treatment of diabetes mellitus - sodium glucose co-transport inhibitors.

    Science.gov (United States)

    Niazi, Asfandyar Khan; Niazi, Saad Hameed

    2010-12-01

    Diabetes is one of the most common chronic diseases, affecting almost 3 million in Canada alone and is characterized by increased blood glucose levels. Treatment varies from lifestyle changes to oral anti-diabetics and/or insulin. Sodium glucose co-transport inhibitors may offer promising treatment for patients suffering from diabetes. The inhibitors act by increasing the loss of glucose in urine by decreasing the reabsorption of glucose from the proximal tubules of nephrons. The aim of this review was to assess the efficacy of sodium glucose co-transport inhibitors in the treatment of diabetes as well as any adverse effects. Databases such as MEDLINE, COCHRANE and EMBASE were systematically searched for literature on the efficacy of sodium glucose co-transport inhibitors in improving the glycemic control of patients with diabetes. Research showed that sodium glucose co-transport inhibitors significantly decreased blood glucose levels by increasing glucosuria. Due to the diuretic effects of these inhibitors, diabetic patients who were suffering from hypertension showed a decrease in blood pressure. The caloric loss associated with these inhibitors resulted in weight loss as well. The most common adverse effect seen in patients on these medications was mycotic infection of the urinary or genital tract. Sodium glucose co-transport inhibitors may be an effective line of treatment for diabetes. Although short-term research has shown these drugs to be safe and well-tolerated, studies should be conducted to assess the long-term effects of these drugs.

  5. AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle.

    Science.gov (United States)

    de Laat, M A; Robinson, M A; Gruntmeir, K J; Liu, Y; Soma, L R; Lacombe, V A

    2015-09-01

    Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P managing IR requires investigation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Junliang Li; Xinke Xu; Shanyi Zhang; Meiguang Zheng; Zhonghua Wu; Yinlun Weng; Leping Ouyang; Jian Yu; Fangcheng Li

    2012-01-01

    In this study, we investigated the effects of adenovirus-mediated transfection of PC12 cells with glucose transporter 3 after ischemic injury. The results of flow cytometry and TUNEL showed that exogenous glucose transporter 3 significantly suppressed PC12 cell apoptosis induced by ischemic injury. The results of isotopic scintiscan and western blot assays showed that, the glucose uptake rate was significantly increased and nuclear factor kappaB expression was significantly decreased after adenovirus-mediated transfection of ischemic PC12 cells with glucose transporter 3. These results suggest that adenovirus-mediated transfection of cells with glucose transporter 3 elevates the energy metabolism of PC12 cells with ischemic injury, and inhibits cell apoptosis.

  7. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    Science.gov (United States)

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  8. High affinity ligands for 'diazepam-insensitive' benzodiazepine receptors.

    Science.gov (United States)

    Wong, G; Skolnick, P

    1992-01-14

    Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their 'diazepam-sensitive' (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the 'diazepam-insensitive' (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (Ki approximately 2.6-20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio approximately 1; other substances had ratios ranging from 13 to greater than 1000. Ligands with high to moderate affinities at DI were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as 'GABA neutral' (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.

  9. Disruption of microtubules in rat skeletal muscle does not inhibit insulin- or contraction-stimulated glucose transport

    DEFF Research Database (Denmark)

    Ai, Hua; Ralston, Evelyn; Lauritzen, Hans P M M

    2003-01-01

    or epitrochlearis muscles. In contrast, nocodazole, another microtubule-disrupting drug, rapidly and dose dependently blocked insulin- and contraction-stimulated glucose transport. A similar discrepancy between colchicine and nocodazole was also found in their ability to block glucose transport in muscle giant...... "ghost" vesicles. This suggests that the ability of insulin and contractions to stimulate glucose transport in muscle does not require an intact microtubule network and that nocodazole inhibits glucose transport independently of its microtubule-disrupting effect....

  10. GLP-1 analog raises glucose transport capacity of blood-brain barrier in Alzheimer's disease

    DEFF Research Database (Denmark)

    Gejl, M.; Brock, B.; Egefjord, L.

    2017-01-01

    Objectives: Glucose enters the brain tissue from plasma by facilitated diffusion across the two membranes of the endothelium of the blood-brain barrier (BBB), mediated by the glucose transporter 1 (GLUT1). There is evidence in Alzheimer's disease (AD) of reduction of glucose transport across...... claim that the GLP-1 analog liraglutide may prevent the decline of blood-brain glucose transfer in AD. Methods: In this 26-week test of the hypothesis, we randomized 38 patients with AD to treatment with the GLP-1 analog liraglutide (n = 18) or placebo (n = 20). We determined blood-brain glucose...... transport capacity (Tmax) with [18F]FDG (FDG) (ClinicalTrials.gov NCT01469351). Results: In both groups, the Tmax estimates declined in proportion to the duration of AD. The GLP-1 analog treatment very significantly (P 

  11. Expression of hexokinases and glucose transporters in treated and untreated oesophageal adenocarcinoma

    NARCIS (Netherlands)

    Fonteyne, Philippe; Casneuf, Veerle; Pauwels, Patrick; Van Damme, Nancy; Peeters, Marc; Dierckx, Rudi; Van de Wiele, Christophe

    2009-01-01

    The aim of this study was to assess the expression pattern of the high glucose affinity glucose transporters GLUT 1, 2, 3, 4, 8 and 9 and of hexokinases I, II and III in newly diagnosed oesophageal adenocarcinoma by means of immunohistochemistry. Twenty patients eligible to undergo primary surgery a

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

    Science.gov (United States)

    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…

  13. Expression of hexokinases and glucose transporters in treated and untreated oesophageal adenocarcinoma

    NARCIS (Netherlands)

    Fonteyne, Philippe; Casneuf, Veerle; Pauwels, Patrick; Van Damme, Nancy; Peeters, Marc; Dierckx, Rudi; Van de Wiele, Christophe

    2009-01-01

    The aim of this study was to assess the expression pattern of the high glucose affinity glucose transporters GLUT 1, 2, 3, 4, 8 and 9 and of hexokinases I, II and III in newly diagnosed oesophageal adenocarcinoma by means of immunohistochemistry. Twenty patients eligible to undergo primary surgery a

  14. Peritoneal transport characteristics with glucose polymer-based dialysis fluid in children.

    NARCIS (Netherlands)

    Rusthoven, E.; Krediet, R.T.; Willems, J.L.; Monnens, L.A.H.; Schröder, C.H.

    2004-01-01

    Scarce data are available on the use of glucose polymer-based dialysate in children. The effects of glucose polymer-based dialysate on peritoneal fluid kinetics and solute transport were studied in pediatric patients who were on chronic peritoneal dialysis, and a comparison was made with previously

  15. Reversible white matter lesions during ketogenic diet therapy in glucose transporter 1 deficiency syndrome.

    Science.gov (United States)

    Shiohama, Tadashi; Fujii, Katsunori; Takahashi, Satoru; Nakamura, Fumito; Kohno, Yoichi

    2013-12-01

    Glucose transporter type 1 deficiency syndrome is caused by brain energy failure resulting from a disturbance in glucose transport. We describe a 4-year-old boy with classical type glucose transporter type 1 deficiency syndrome with a heterozygous splice acceptor site mutation (c.517-2A>G) in the SLCA2A1 gene. We initiated a ketogenic diet at 4 months of age. However, even though his condition was good during ketogenic diet therapy, multiple cerebral white matter and right cerebellum lesions appeared at 9 months of age. The lesions in the cerebral white matter subsequently disappeared, indicating that white matter lesions during diet therapy may be reversible and independent of the ketogenic diet. This is the first report of reversible white matter lesions during ketogenic diet therapy in glucose transporter type 1 deficiency syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae.

    Science.gov (United States)

    Aouida, Mustapha; Leduc, Anick; Poulin, Richard; Ramotar, Dindial

    2005-06-24

    Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

  17. Oxidant stress and skeletal muscle glucose transport: roles of insulin signaling and p38 MAPK.

    Science.gov (United States)

    Kim, John S; Saengsirisuwan, Vitoon; Sloniger, Julie A; Teachey, Mary K; Henriksen, Erik J

    2006-09-01

    Oxidative stress can impact the regulation of glucose transport activity in a variety of cell lines. In the present study, we assessed the direct effects of an oxidant stress on the glucose transport system in intact mammalian skeletal muscle preparations. Type IIb (epitrochlearis) and type I (soleus) muscles from insulin-sensitive lean Zucker rats were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase to produce the oxidant hydrogen peroxide (H(2)O(2)) (60-90 microM). Glucose transport, glycogen synthase activity, and metabolic signaling factors were then assessed. H(2)O(2) significantly (p oxidant stress was prevented by the PI3-kinase inhibitor wortmannin. The oxidant stress also significantly increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and 5'-AMP-dependent protein kinase. Interestingly, selective inhibition of p38 MAPK using A304000 substantially reduced the activation of glucose transport induced by the oxidant stress. These results support a direct role for oxidative stress in the activation of the glucose transport system in mammalian skeletal muscle and indicate that this process involves engagement of and possible interactions between the PI3-kinase-dependent signaling pathway and activation of p38 MAPK.

  18. A glucose transporter can mediate ribose uptake: definition of residues that confer substrate specificity in a sugar transporter.

    Science.gov (United States)

    Naula, Christina M; Logan, Flora J; Logan, Flora M; Wong, Pui Ee; Barrett, Michael P; Burchmore, Richard J

    2010-09-24

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

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

    Science.gov (United States)

    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. A karyopherin alpha2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells.

    Science.gov (United States)

    Cassany, Aurélia; Guillemain, Ghislaine; Klein, Christophe; Dalet, Véronique; Brot-Laroche, Edith; Leturque, Armelle

    2004-01-01

    We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

  1. The insulin-like growth factor I receptor regulates glucose transport by astrocytes.

    Science.gov (United States)

    Hernandez-Garzón, Edwin; Fernandez, Ana M; Perez-Alvarez, Alberto; Genis, Laura; Bascuñana, Pablo; Fernandez de la Rosa, Ruben; Delgado, Mercedes; Angel Pozo, Miguel; Moreno, Estefania; McCormick, Peter J; Santi, Andrea; Trueba-Saiz, Angel; Garcia-Caceres, Cristina; Tschöp, Matthias H; Araque, Alfonso; Martin, Eduardo D; Torres Aleman, Ignacio

    2016-11-01

    Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

  2. Insulin stimulated-glucose transporter Glut 4 is expressed in the retina.

    Directory of Open Access Journals (Sweden)

    Gustavo Sánchez-Chávez

    Full Text Available The vertebrate retina is a very metabolically active tissue whose energy demands are normally met through the uptake of glucose and oxygen. Glucose metabolism in this tissue relies upon adequate glucose delivery from the systemic circulation. Therefore, glucose transport depends on the expression of glucose transporters. Here, we show retinal expression of the Glut 4 glucose transporter in frog and rat retinas. Immunohistochemistry and in situ hybridization studies showed Glut 4 expression in the three nuclear layers of the retina: the photoreceptor, inner nuclear and ganglionar cell layers. In the rat retina immunoprecipitation and Western blot analysis revealed a protein with an apparent molecular mass of 45 kDa. ¹⁴C-glucose accumulation by isolated rat retinas was significantly enhanced by physiological concentrations of insulin, an effect blocked by inhibitors of phosphatidyl-inositol 3-kinase (PI3K, a key enzyme in the insulin-signaling pathway in other tissues. Also, we observed an increase in ³H-cytochalasin binding sites in the presence of insulin, suggesting an increase in transporter recruitment at the cell surface. Besides, insulin induced phosphorylation of Akt, an effect also blocked by PI3K inhibition. Expression of Glut 4 was not modified in retinas of a type 1 diabetic rat model. To our knowledge, our results provide the first evidence of Glut4 expression in the retina, suggesting it as an insulin- responsive tissue.

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

    Science.gov (United States)

    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.

  4. Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters.

    Science.gov (United States)

    Bird, T A; Davies, A; Baldwin, S A; Saklatvala, J

    1990-08-15

    Exposure of quiescent cultures of human gingival fibroblasts (HuGi) and porcine synovicocytes (PSF) to human recombinant interleukin 1 alpha or -beta (IL1 alpha and -beta) enhanced the rate of glycolysis as judged by increased lactate production. The cytokines also increased uptake of [3H]2-deoxyglucose (DG) in a time- and dose-dependent manner. Stimulation of DG uptake was first evident 6-8 h following addition of IL1 and was maximal by 24-30 h. IL1 alpha and -beta were equipotent. Half-maximal stimulation occurred at approximately 1 pM IL1; maximal stimulation (2.5-4.5-fold in HuGi, 3-7-fold in PSF) was obtained with approximately 80 pM IL1. The dose-response curves for lactate production and DG uptake were similar. Increased DG uptake was blocked by specific antisera to IL1 and by inhibitors of protein and RNA synthesis but not by indomethacin, an inhibitor of prostaglandin production. DG uptake was enhanced by IL1 in serum-starved cells in the presence of neutralizing anti-platelet-derived growth factor serum. The effect was therefore not secondary to prostaglandin or platelet-derived growth factor production. No increase in cell cycling was detected in IL1-treated cells under the experimental conditions. Kinetic analysis revealed that the Vmax for DG uptake was increased by IL1 (from 36 to 144 pmol/min/mg of cell protein), whereas the Km was unchanged. HuGi cells were pulse-labeled with [35S]methionine following exposure to IL1. Cell lysates were immunoprecipitated using a specific antiserum raised against human erythrocyte glucose transporter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography of these immunoprecipitates revealed dose- and time-dependent increases in the net rate of glucose transporter synthesis which mirrored the changes in DG uptake.

  5. Decreased muscle GLUT-4 and contraction-induced glucose transport after eccentric contractions

    DEFF Research Database (Denmark)

    Kristiansen, S; Asp, Svend; Richter, Erik

    1996-01-01

    Eccentric exercise causes muscle damage and decreased muscle glycogen and glucose transporter isoform (GLUT-4) protein content. We investigated whether the contraction-induced increase in skeletal muscle glucose transport and muscle performance is affected by prior eccentric contractions. The calf...... than in CT rats. In the GW and GR muscle, prior eccentric exercise decreased contraction-induced stimulation of glucose transport compared with CT, ST, and CC rats despite no difference in tension development and oxygen uptake among the groups. There was no change in total GLUT-4 content and glucose...... muscles from rats were stimulated for eccentric (EC) or concentric (CC) contractions or were passively stretched (ST). Muscles from unstimulated control (CT) rats were also studied. Two days later, all rats had their isolated hindlimbs perfused either at rest or during 15 min of isometric muscle...

  6. Glucose transporter-8 (GLUT8) mediates glucose intolerance and dyslipidemia in high-fructose diet-fed male mice.

    Science.gov (United States)

    DeBosch, Brian J; Chen, Zhouji; Finck, Brian N; Chi, Maggie; Moley, Kelle H

    2013-11-01

    Members of the glucose transporter (GLUT) family of membrane-spanning hexose transporters are subjects of intensive investigation for their potential as modifiable targets to treat or prevent obesity, metabolic syndrome, and type 2 diabetes mellitus. Mounting evidence suggests that the ubiquitously expressed class III dual-specificity glucose and fructose transporter, GLUT8, has important metabolic homeostatic functions. We therefore tested the hypothesis that GLUT8 mediates the deleterious metabolic effects of chronic high-fructose diet exposure. Here we demonstrate resistance to high-fructose diet-induced glucose intolerance and dyslipidemia concomitant with enhanced oxygen consumption and thermogenesis in GLUT8-deficient male mice. Independent of diet, significantly lower systolic blood pressure both at baseline and after high-fructose diet feeding was also observed by tail-cuff plethysmography in GLUT8-deficient mice vs wild-type controls. Resistance to fructose-induced metabolic dysregulation occurred in the context of enhanced hepatic peroxisome proliferator antigen receptor-γ (PPARγ) protein abundance, whereas in vivo hepatic adenoviral GLUT8 overexpression suppressed hepatic PPARγ expression. Taken together, these findings suggest that GLUT8 blockade prevents fructose-induced metabolic dysregulation, potentially by enhancing hepatic fatty acid metabolism through PPARγ and its downstream targets. We thus establish GLUT8 as a promising target in the prevention of diet-induced obesity, metabolic syndrome, and type 2 diabetes mellitus in males.

  7. Family of Ricinus communis Monosaccharide Transporters and RcSTP1 in Promoting the Uptake of a Glucose-Fipronil Conjugate.

    Science.gov (United States)

    Mao, Gen-Lin; Yan, Yin; Chen, Yan; Wang, Bing-Feng; Xu, Fei-Fei; Zhang, Zhi-Xiang; Lin, Fei; Xu, Han-Hong

    2017-08-02

    Enhancing the systemic distribution of a bioactive compound by exploiting the vascular transport system of a plant presents a means of reducing both the volume and frequency of pesticide/fungicide application. The foliar uptake of the glucose-fipronil conjugate N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-d-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF) achieved in castor bean (Ricinus communis) and its transport via the phloem are known to be mediated by monosaccharide transporter(s) [MST(s)], although neither the identity of the key MST(s) involved nor the mechanistic basis of its movement have yet to be described. On the basis of homology with Arabidopsis thaliana sugar transporters, the castor bean genome was concluded to harbor 53 genes encoding a sugar transporter, falling into the eight previously defined subfamilies INT, PMT, VGT, STP, ERD6, pGlucT, TMT, and SUT. Transcriptional profiling identified the product of RcSTP1 as a candidate for mediating GTF uptake, because this gene was induced by exposure of the plant to GTF. When RcSTP1 was transiently expressed in onion epidermis cells, the site of RcSTP1 deposition was shown to be the plasma membrane. A functional analysis based on RcSTP1 expression in Xenopus laevis oocytes demonstrated that its product has a high affinity for GTF. The long-distance root-to-shoot transport of GTF was enhanced in a transgenic soybean chimera constitutively expressing RcSTP1.

  8. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    Science.gov (United States)

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (PPiracetam and TRH have no direct effects on net glucose transport, but competitively antagonise hypnotic drug inhibition of glucose transport. Other nootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis. PMID:15148255

  9. Expression and Localization of Glucose Transporters in Rodent Submandibular Salivary Glands

    Directory of Open Access Journals (Sweden)

    Sibel Cetik

    2014-04-01

    Full Text Available Background/Aim: The submandibular gland is one of the three major salivary glands, producing a mixed secretion; this saliva is hypotonic compared to plasma. It also secretes glucose, but the mechanisms responsible for this process are poorly understood. Our study addressed the question whether glucose transporters are expressed and how are they localized within specific rodent submandibular cells, in order to estimate a possible implication in salivary glucose disposal. Methods: Immunohistochemistry, RT-qPCR and Western blotting were performed to determine the presence/localization of glucose transporters in rodent submandibular glands. Results: GLUT4 was identified in the submandibular salivary gland at both mRNA and protein level. The immunohistochemical analysis revealed its localization preponderantly in the ductal cells of the gland, near to the basolateral. SGLT1 and GLUT1 were highly expressed in submandibular tissues in both acinar and ductal cells, but not GLUT2. These results were confirmed by RT-qPCR. It was also documented that insulin stimulates the net uptake of D-glucose by ductal rings prepared from submandibulary salivary glands, the relative magnitude of such an enhancing action being comparable to that found in hemidiaphragms. Conclusion: At least three major glucose transporters are expressed in the rodent submandibular glands, of which GLUT4 is specifically localized near the basolateral side of ductal structures. This points-out its possible role in regulating glucose uptake from the bloodstream, most likely to sustain ductal cellular metabolism.

  10. Characteristics of glucose transport across the microvillous membranes of human term placenta

    Directory of Open Access Journals (Sweden)

    Ravinderjit Kaur Anand

    Full Text Available Transport characteristics of D-glucose were studied in the microvillous vesicles isolated from the human term placenta. Transport occurred by selective and rapid facilitated diffusion system which was inhibitable by phloretin and HgCl2. The transport was dependent on a transmembrane. Na+-gradient indicating a "secondary active transport" system operating. The transport influx was saturable and the kinetic analysis based on Hanes-Woolf plot produced a kt and Jmax value of 1.2 mM and 34 nmoles. mgprotein-1.min-1, respectively. The efflux of D-glucose from the membrane vesicles in a pre-equilibrated assay conditions showed a distinct biphasic pattern differing significantly in the half time efflux. The t1/2 of the fast and slow components was found to be 15 sec and 660 sec, respectively. The transport showed distinct sensitivity to temperature and the Ea values both below and above the transition temperature of 37 ºC, as calculated from the Arrhenius plot were found to be 7600 and 5472 kCa1.mol-1, respectively. Inhibition studies with a number of sugars for hexose transport pathway showed that the glucose epimers, phosphorylated sugars, and even the disaccharides and the pentose sugars competed effectively with D-glucose. The influx was also inhibited by a number of steroids such as progesterone, 17α-hydroxyprogesterone, testosterone and estrogen. Insulin was found to increase glucose transport in a dose- dependent fashion at a concentration of 0.2-1 unit.ml-1. Ouabain, dinitrophenoi and nicotine strongly inhibited D-glucose uptake in the membrane vesicles.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Asymmetric subcellular distribution of glucose transporters in the endothelium of small contractile arteries.

    Science.gov (United States)

    Gaudreault, N; Scriven, D R L; Moore, E D W

    2006-01-01

    The authors have recently reported the presence and asymmetric distribution of the glucose transporters GLUT-1 to -5 and SGLT-1 in the endothelium of rat coronary artery (Gaudreault et al. 2004, Diabetologica, 47, 2081-2092). In the present study the authors investigate and compare the presence and subcellular distribution of the classic glucose transporter isoforms in endothelial cells of cerebral, renal, and mesenteric arteries. The GLUTs and SGLT-1 were examined with immunohistochemistry and wide-field fluorescence microscopy coupled to deconvolution in en face preparation of intact artery. We identified GLUT-1 to -5 and SGLT-1 in the endothelial cells of all three vascular beds. The relative level of expression for each isoform was found comparable amongst arteries. Clusters of the glucose transporter isoforms were found at a high density in proximity to the cell-to-cell junctions. In addition, a consistent asymmetric distribution of GLUT-1 to -5 was found, predominantly located on the abluminal side of the endothelium in all three vascular beds examined (ranging from 68% to 91%, p<.05). The authors conclude that the expression and subcellular distribution of glucose transporters are similar in endothelial cells from vascular beds of comparable diameter and suggest that their subcellular organization may facilitate transendothelial transport of glucose in small contractile arteries.

  13. Role of vitamin D on the expression of glucose transporters in L6 myotubes

    Directory of Open Access Journals (Sweden)

    Bubblu Tamilselvan

    2013-01-01

    Full Text Available Altered expression of glucose transporters is a major characteristic of diabetes. Vitamin D has evolved widespread interest in the pathogenesis and prevention of diabetes. The present study was designed to investigate the effect of vitamin D in the overall regulation of muscle cell glucose transporter expression. L6 cells were exposed to type 1 and type 2 diabetic conditions and the effect of calcitriol (1,25, dihydroxy cholicalciferol on the expression of glucose transporters was studied by real time polymerase chain reaction (RT-PCR. There was a significant decrease in glucose transporter type 1 (GLUT1, GLUT4, vitamin D receptor (VDR, and IR expression in type 1 and 2 diabetic model compared to control group. Treatment of myoblasts with 10-7 M calcitriol for 24 h showed a significant increase in GLUT1, GLUT4, VDR, and insulin receptor (IR expression. The results indicate a potential antidiabetic function of vitamin D on GLUT1, GLUT4, VDR, and IR by improving receptor gene expression suggesting a role for vitamin D in regulation of expression of the glucose transporters in muscle cells.

  14. Sodium Glucose Co-transporter Type 2 (SGLT2) Inhibitors: Targeting the Kidney to Improve Glycemic Control in Diabetes Mellitus

    OpenAIRE

    Bays, Harold

    2013-01-01

    Although hyperglycemia is a key therapeutic focus in the management of patients with type 2 diabetes mellitus (T2DM), many patients experience sub-optimal glycemic control. Current glucose-lowering agents involve the targeting of various body organs. Sodium glucose co-transporter type 2 (SGLT2) inhibitors target the kidney, reduce renal glucose reabsorption, and increase urinary glucose elimination, thus lowering glucose blood levels. This review examines some of the key efficacy and safety d...

  15. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  16. Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars.

    Science.gov (United States)

    Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Harada, Hidemitsu; Takata, Hiroki; Baba, Otto; Ohshima, Hayato

    2012-03-01

    Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Transport of alpha- and beta-D-glucose by the intact human red cell

    Energy Technology Data Exchange (ETDEWEB)

    Carruthers, A.; Melchior, D.L.

    1985-07-16

    The kinetics of alpha- and beta-D-glucose mutarotation and the transport of these anomers by intact human red cells were determined at 0.6 and 36.6 degrees C. The mutarotation coefficients for alpha- and beta-D-glucose in cell-free tris(hydroxymethyl)aminomethane medium (pH 7.4) at 0.6 degrees C are (2.25 +/- 0.2) and (1.73 +/- 0.42) X 10(-3) min-1, respectively, and at 36.6 degrees C are (69 +/- 12) and (75 +/- 5) X 10(-3) min-1, respectively. These values are in good agreement with previous estimates. At 0.6 degrees C, the red cell contains no detectable mutarotase activity. Initial rates of sugar uptake were measured by using radiolabeled D-glucose and time courses of uptake by turbidimetry. The time courses of alpha- and beta-D-glucose and an equilibrium mixture of alpha- and beta-D-glucose infinite-cis entry are identical at 0.66 degrees C (n = 41) where negligible mutarotation is observed. The apparent Ki values for inhibition of radiolabeled D-glucose initial uptake by unlabeled alpha- or beta-D-glucose at 0.6 degrees C are identical (1.6 mM). The calculated Vmax parameters for uptake of the radiolabeled anomers at this temperature are also indistinguishable. The time courses of infinite-cis alpha- and beta-D-glucose uptake at 36.66 degrees C are identical (n = 40). While D-glucose mutarotation is more rapid at this temperature, the anomers of D-glucose are not transported differently by the red cell hexose transfer system.

  18. Effects of Prolonged Glucose Infusion on Insulin Signal Transduction and Glucose Transport in Rat Skeletal Muscle

    OpenAIRE

    Houdali, Basel

    2000-01-01

    Der Effekt einer in vivo Glucoseinfusion auf die Insulinwirkung im Skelettmuskel der Ratte wurde untersucht. Dazu wurden Dauerkatheter in die rechte Jugularvene implantiert und bis zum rechten Vorhof vorgeschoben. Anschließend wurden die Katheter mit einem Perfusor verbunden. Nach einer zweitägigen Erholungsphase vom Operationsstress, wurde 50%ige Glucose mit einer Infusionsrate von 2 ml/h für zwei oder fünf Tage durchgeführt. Die Kontrolltiere erhielten eine 0,45% Kochsalzinfusion für die...

  19. DAPAGLIFLOZIN: SELECTIVE SODIUM-GLUCOSE CO-TRANSPORTER-2 INHIBITOR IN TYPE 2 DIABETES

    Directory of Open Access Journals (Sweden)

    Sudhakar Pemminati

    2011-11-01

    Full Text Available Dapagliflozin is a promising new drug that targets the so far untapped renal glucose reabsorption. By inhibiting sodium-glucose co-transporter-2 (SGLT2 which is mainly localized in the S1 segment of the proximal tubule, Dapagliflozin promotes renal glucose excretion and reduces hyperglycemia in an insulin-independent manner. Dapagliflozin also produces pronounced weight loss which may be an advantage in patients on sulfonylureas and insulin. Dapagliflozin has the potential to be used as monotherapy, as well as in combination with all approved antidiabetic agents.

  20. Atypical antipsychotic drugs directly impair insulin action in adipocytes: effects on glucose transport, lipogenesis, and antilipolysis.

    Science.gov (United States)

    Vestri, Helliner S; Maianu, Lidia; Moellering, Douglas R; Garvey, W Timothy

    2007-04-01

    Treatment with second-generation antipsychotics (SGAs) has been associated with weight gain and the development of diabetes mellitus, although the mechanisms are unknown. We tested the hypothesis that SGAs exert direct cellular effects on insulin action and substrate metabolism in adipocytes. We utilized two cultured cell models including 3T3-L1 adipocytes and primary cultured rat adipocytes, and tested for effects of SGAs risperidone (RISP), clozapine (CLZ), olanzapine (OLZ), and quetiapine (QUE), together with conventional antipsychotic drugs butyrophenone (BUTY), and trifluoperazine (TFP), over a wide concentration range from 1 to 500 microM. The effects of antipsychotic drugs on basal and insulin-stimulated rates of glucose transport were studied at 3 h, 15 h, and 3 days. Both CLZ and OLZ (but not RISP) at doses as low as 5 microM were able to significantly decrease the maximal insulin-stimulated glucose transport rate by approximately 40% in 3T3-L1 cells, whereas CLZ and RISP reduced insulin-stimulated glucose transport rates in primary cultured rat adipocytes by approximately 50-70%. Conventional drugs (BUTY and TFP) did not affect glucose transport rates. Regarding intracellular glucose metabolism, both SGAs (OLZ, QUE, RISP) and conventional drugs (BUTY and TFP) increased basal and/or insulin-stimulated glucose oxidation rates, whereas rates of lipogenesis were increased by CLZ, OLZ, QUE, and BUTY. Finally, rates of lipolysis in response to isoproterenol were reduced by the SGAs (CLZ, OLZ, QUE, RISP), but not by BUTY or TFP. These experiments demonstrate that antipsychotic drugs can differentially affect insulin action and metabolism through direct cellular effects in adipocytes. However, only SGAs were able to impair the insulin-responsive glucose transport system and to impair lipolysis in adipocytes. Thus, SGAs directly induce insulin resistance and alter lipogenesis and lipolysis in favor of progressive lipid accumulation and adipocyte enlargement. These

  1. Differential regulation of glucose transport activity in yeast by specific cAMP signatures.

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Sadoine, Mayuri S C; Chermak, Diane; Frommer, Wolf B

    2013-06-15

    Successful colonization and survival in variable environments require a competitive advantage during the initial growth phase after experiencing nutrient changes. Starved yeast cells anticipate exposure to glucose by activating the Hxt5p (hexose transporter 5) glucose transporter, which provides an advantage during early phases after glucose resupply. cAMP and glucose FRET (fluorescence resonance energy transfer) sensors were used to identify three signalling pathways that co-operate in the anticipatory Hxt5p activity in glucose-starved cells: as expected the Snf1 (sucrose nonfermenting 1) AMP kinase pathway, but, surprisingly, the sugar-dependent G-protein-coupled Gpr1 (G-protein-coupled receptor 1)/cAMP/PKA (protein kinase A) pathway and the Pho85 (phosphate metabolism 85)/Plc (phospholipase C) 6/7 pathway. Gpr1/cAMP/PKA are key elements of a G-protein-coupled sugar response pathway that produces a transient cAMP peak to induce growth-related genes. A novel function of the Gpr1/cAMP/PKA pathway was identified in glucose-starved cells: during starvation the Gpr1/cAMP/PKA pathway is required to maintain Hxt5p activity in the absence of glucose-induced cAMP spiking. During starvation, cAMP levels remain low triggering expression of HXT5, whereas cAMP spiking leads to a shift to the high capacity Hxt isoforms.

  2. Flozins, inhibitors of type 2 renal sodium-glucose co-transporter – not only antihyperglycemic drugs

    OpenAIRE

    Mizerski Grzegorz; Kicinski Pawel; Jaroszynski Andrzej

    2015-01-01

    The kidneys play a crucial role in the regulation of the carbohydrate metabolism. In normal physiological conditions, the glucose that filters through the renal glomeruli is subsequently nearly totally reabsorbed in the proximal renal tubules. Two transporters are engaged in this process: sodium-glucose co-transporter type 1 (SGLT1), and sodium-glucose co-transporter type type 2 (SGLT2) - this being located in the luminal membrane of the renal tubular epithelial cells. It was found that the a...

  3. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

    OpenAIRE

    1992-01-01

    GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular dist...

  4. PACSIN3 Overexpression Increases Adipocyte Glucose Transport through GLUT1

    Science.gov (United States)

    Roach, William; Plomann, Markus

    2007-01-01

    PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes. PMID:17320047

  5. New mathematical model for fluid-glucose-albumin transport in peritoneal dialysis

    CERN Document Server

    Cherniha, Roman

    2011-01-01

    A mathematical model for fluid transport in peritoneal dialysis is constructed. The model is based on a three-component nonlinear system of two-dimensional partial differential equations for fluid, glucose and albumin transport with the relevant boundary and initial conditions. Non-constant steady-state solutions of the model are studied. The restrictions on the parameters arising in the model are established with the aim to obtain exact formulae for the non-constant steady-state solutions. As the result, the exact formulae for the fluid fluxes from blood to tissue and across the tissue were constructed together with two linear autonomous ODEs for glucose and albumin concentrations. The analytical results were checked for their applicability for the description of fluid-glucose-albumin transport during peritoneal dialysis.

  6. CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1.

    Science.gov (United States)

    Chen, Jiaying; Zhang, Can; Mi, Yang; Chen, Fuxue; Du, Dongshu

    2017-06-23

    Glioma is stemmed from the glial cells in the brain, which is accounted for about 45% of all intracranial tumors. The characteristic of glioma is invasive growth, as well as there is no obvious boundary between normal brain tissue and glioma tissue, so it is difficult to resect completely with worst prognosis. The metabolism of glioma is following the Warburg effect. Previous researches have shown that GLUT1, as a glucose transporter carrier, affected the Warburg effect, but the molecular mechanism is not very clear. CREB1 (cAMP responsive element-binding protein1) is involved in various biological processes, and relevant studies confirmed that CREB1 protein regulated the expression of GLUT1, thus mediating glucose transport in cells. Our experiments mainly reveal that the CREB1 could affect glucose transport in glioma cells by regulating the expression of GLUT1, which controlled the metabolism of glioma and affected the progression of glioma.

  7. Decreased insulin action on muscle glucose transport after eccentric contractions in rats

    DEFF Research Database (Denmark)

    Asp, S; Richter, Erik

    1996-01-01

    We have recently shown that eccentric contractions (Ecc) of rat calf muscles cause muscle damage and decreased glycogen and glucose transporter GLUT-4 protein content in the white (WG) and red gastrocnemius (RG) but not in the soleus (S) (S. Asp, S. Kristiansen, and E. A. Richter. J. Appl. Physiol....... 79: 1338-1345, 1995). To study whether these changes affect insulin action, hindlimbs were perfused at three different insulin concentrations (0, 200, and 20,000 microU/ml) 2 days after one-legged eccentric contractions of the calf muscles. Compared with control, basal glucose transport was slightly...... velocity of glycogen synthase increased similarly with increasing insulin concentrations in Ecc- and control WG and RG. We conclude that insulin action on glucose transport but not glycogen synthase activity is impaired in perfused muscle exposed to prior eccentric contractions....

  8. Influence of blood glucose on the expression of glucose transporter proteins 1 and 3 in the brain of diabetic rats

    Institute of Scientific and Technical Information of China (English)

    HOU Wei-kai; FU Chun-li; ZHANG Wen-wen; CHEN Li; XIAN Yu-xin; ZHANG Li; LAI Hong; HOU Xin-guo; XU Yu-xin; YU Ting; XU Fu-yu; SONG Jun

    2007-01-01

    Background The delivery of glucose from the blood to the brain involves its passage across the endothelial cells of the blood-brain barrier (BBB), which is mediated by the facilitative glucose transporter protein 1 (GLUT1), and then across the neural cell membranes, which is mediated by GLUT3. This study aimed to evaluate the dynamic influence of hyperglycemia on the expression of these GLUTs by measuring their expression in the brain at different blood glucose levels in a rat model of diabetes. This might help to determine the proper blood glucose threshold level in the treatment of diabetic apoplexy.Methods Diabetes mellitus was induced with streptozotocin (STZ) in 30 rats. The rats were randomly divided into 3 groups: diabetic group without blood glucose control (group DM1), diabetic rats treated with low dose insulin (group DM2),and diabetic rats treated with high dose insulin (group DM3). The mRNA and protein levels of GLUT1 and GLUT3 were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively.Results Compared with normal control rats, the GLUT1 mRNA was reduced by 46.08%, 29.80%, 19.22% (P<0.01) in DM1, DM2, and DM3 group, respectively; and the GLUT3 mRNA was reduced by 75.00%, 46.75%, and 17.89% (P<0.01)in DM1, DM2, and DM3 group, respectively. The abundance of GLUT1 and GLUT3 proteins had negative correlation with the blood glucose level (P<0.01). The density of microvessels in the brain of diabetic rats did not change significantly compared with normal rats.Conclusions Chronic hyperglycemia downregulates GLUT1 and GLUT3 expression at both mRNA and protein levels in the rat brain, which is not due to the decrease of the density of microvessels. The downregulation of GLUT1 and GLUT3 expression might be the adaptive reaction of the body to prevent excessive glucose entering the cell that may lead to cell damage.

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

    Science.gov (United States)

    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.

  10. Impairment of brain endothelial glucose transporter by methamphetamine causes blood-brain barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Murrin L Charles

    2011-03-01

    Full Text Available Abstract Background Methamphetamine (METH, an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB to date. Results In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 μM increased the expression of glucose transporter protein-1 (GLUT1 in primary human brain endothelial cell (hBEC, main component of BBB without affecting the glucose uptake. A high concentration of 200 μM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. Conclusion Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

  11. Glucose Transporter 4 (GLUT4) is Not Necessary for Overload-Induced Glucose Uptake or Hypertrophic Growth in Mouse Skeletal Muscle.

    Science.gov (United States)

    McMillin, Shawna L; Schmidt, Denise L; Kahn, Barbara B; Witczak, Carol A

    2017-03-09

    Glucose transporter 4 (GLUT4) is necessary for acute insulin- and contraction-induced skeletal muscle glucose uptake, but its role in chronic muscle loading (overload)-induced glucose uptake is unknown. Our goal was to determine if GLUT4 is required for overload-induced glucose uptake. Overload was induced in mouse plantaris muscle by unilateral synergist ablation. After 5 days, muscle weights and ex vivo [(3)H]-2-deoxy-D-glucose uptake were assessed. Overload-induced muscle glucose uptake and hypertrophic growth were not impaired in muscle-specific GLUT4 knockout mice, demonstrating that GLUT4 is not necessary for these processes. To assess which transporter(s) mediate overload-induced glucose uptake, chemical inhibitors were utilized. The facilitative GLUT inhibitor, cytochalasin B, but not the sodium-dependent glucose-co-transport inhibitor, phloridzin, prevented overload-induced uptake demonstrating that GLUT(s) mediate this effect. To assess which GLUT, hexose competition experiments were performed. Overload-induced [(3)H]-2-deoxy-D-glucose uptake was not inhibited by D-fructose, demonstrating that the fructose-transporting GLUT2, GLUT5, GLUT8, and GLUT12, do not mediate this effect. To assess additional GLUTs, immunoblots were performed. Overload increased GLUT1, GLUT3, GLUT6 and GLUT10 protein levels 2- to 5-fold. Collectively, these results demonstrate that GLUT4 is not necessary for overload-induced muscle glucose uptake or hypertrophic growth, and suggest that GLUT1, GLUT3, GLUT6 and/or GLUT10 mediate overload-induced glucose uptake.

  12. Role of Akt substrate of 160 kDa in insulin-stimulated and contraction-stimulated glucose transport

    DEFF Research Database (Denmark)

    Cartee, Gregory D; Wojtaszewski, Jørgen F P

    2007-01-01

    Insulin and exercise, the most important physiological stimuli to increase glucose transport in skeletal muscle, trigger a redistribution of GLUT4 glucose transporter proteins from the cell interior to the cell surface, thereby increasing glucose transport capacity. The most distal insulin...... signaling protein that has been linked to GLUT4 translocation, Akt substrate of 160 kDa (AS160), becomes phosphorylated in insulin-stimulated 3T3-L1 adipocytes; this is important for insulin-stimulated GLUT4 translocation and glucose transport. Insulin also induces a rapid and dose-dependent increase in AS....../contraction-stimulated glucose uptake is currently inconclusive. The distinct signaling pathways that are stimulated by insulin and exercise/contraction converge at AS160. Although AS160 phosphorylation is apparently important for insulin-stimulated GLUT4 translocation and glucose transport, it is uncertain whether elevated AS...

  13. The high-affinity immunoglobulin E receptor as pharmacological target.

    Science.gov (United States)

    Blank, Ulrich; Charles, Nicolas; Benhamou, Marc

    2016-05-05

    The high-affinity receptor for immunoglobulin E is expressed mainly on mast cells and basophils, but also on neutrophils, eosinophils, platelets, monocytes, Langerhans and dendritic cells, airway smooth muscle cells and some nerve cells. Its main function is, upon its engagement by IgE and specific antigen, to trigger a powerful defense against invading pathogens and a rapid neutralization of dangerous toxic substances introduced in the body. This powerful response could be wielded against tumors. But, when control over this receptor is lost, its unchecked activation can induce an array of diseases, some of which can lead to death. In this review we will summarize the pharmacological approaches and strategies that are currently used, or under study, to harness or wield activation of this receptor for therapeutic purposes.

  14. Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

    Science.gov (United States)

    May, J M; Qu, Z C; Beechem, J M

    1993-09-21

    The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

  15. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  16. Mammalian glucose permease GLUT1 facilitates transport of arsenic trioxide and methylarsonous acid†

    Science.gov (United States)

    Liu, Zijuan; Sanchez, Marco A.; Jiang, Xuan; Boles, Eckhard; Landfear, Scott M.; Rosen, Barry P.

    2006-01-01

    Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH)3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH)3 and CH3As(OH)2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH)3 and CH3As(OH)2. The Km of GLUT1 is to 1.2 mM for CH3As(OH)2, compared to a Km of 3 mM for glucose. Inhibition between glucose and CH3As(OH)2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH)3 and CH3As(OH)2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity. PMID:17064664

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

    Science.gov (United States)

    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.

  18. A tale of two glucose transporters: how GLUT2 re-emerged as a contender for glucose transport into the human beta cell.

    Science.gov (United States)

    van de Bunt, M; Gloyn, A L

    2012-09-01

    Finding novel causes for monogenic forms of diabetes is important as, alongside the clinical implications of such a discovery, it can identify critical proteins and pathways required for normal beta cell function in humans. It is increasingly apparent that there are significant differences between rodent and human islets. One example that has generated interest is the relative importance of the glucose transporter GLUT2 in rodent and human beta cells. The central role of GLUT2 in rodent beta cells is well established, but a number of studies have suggested that other glucose transporters, namely GLUT1 and GLUT3, may play an important role in facilitating glucose transport into human beta cells. In this issue of Diabetologia Sansbury et al (DOI: 10.1007/s00125-012-2595-0 ) report homozygous loss of function mutations in SLC2A2, which encodes GLUT2, as a rare cause of neonatal diabetes. Evidence for a beta cell defect in these subjects comes from very low birthweights, lack of endogenous insulin secretion and a requirement for insulin therapy. Neonatal diabetes is not a consistent feature of SLC2A2 mutations. It is only found in a small percentage of cases (~4%) and the diabetes largely resolves before 18 months of age. This discovery is significant as it suggests that GLUT2 plays an important role in human beta cells, but the interplay and relative roles of other transporters differ from those in rodents. This finding should encourage efforts to delineate the precise role of GLUT2 in the human beta cell at different developmental time points and is a further reminder of critical differences between human and rodent islets.

  19. Glucose uptake and transport in contracting, perfused rat muscle with different pre-contraction glycogen concentrations

    DEFF Research Database (Denmark)

    Hespel, P; Richter, Erik

    1990-01-01

    1. Glucose uptake and transport, muscle glycogen, free glucose and glucose-6-phosphate concentrations were studied in perfused resting and contracting rat skeletal muscle with different pre-contraction glycogen concentrations. Rats were pre-conditioned by a combination of swimming exercise and diet......, resulting in either low (glycogen-depleted rats), normal (control rats) or high (supercompensated rats) muscle glycogen concentrations at the time their hindlimbs were perfused. 2. Compared with control rats, pre-contraction muscle glycogen concentration was approximately 40% lower in glycogen-depleted rats......, whereas it was 40% higher in supercompensated rats. Muscle glycogen break-down correlated positively (r = 0.76; P less than 0.001) with pre-contraction muscle glycogen concentration. 3. Glucose uptake during contractions was approximately 50% higher in glycogen-depleted hindquarters than in control...

  20. Heat stress reduces intestinal barrier integrity and favors intestinal glucose transport in growing pigs.

    Science.gov (United States)

    Pearce, Sarah C; Mani, Venkatesh; Boddicker, Rebecca L; Johnson, Jay S; Weber, Thomas E; Ross, Jason W; Rhoads, Robert P; Baumgard, Lance H; Gabler, Nicholas K

    2013-01-01

    Excessive heat exposure reduces intestinal integrity and post-absorptive energetics that can inhibit wellbeing and be fatal. Therefore, our objectives were to examine how acute heat stress (HS) alters intestinal integrity and metabolism in growing pigs. Animals were exposed to either thermal neutral (TN, 21°C; 35-50% humidity; n=8) or HS conditions (35°C; 24-43% humidity; n=8) for 24 h. Compared to TN, rectal temperatures in HS pigs increased by 1.6°C and respiration rates by 2-fold (Pintestinal integrity was compromised in the HS pigs (ileum and colon TER decreased; PIntestinal permeability was accompanied by an increase in protein expression of myosin light chain kinase (PIntestinal glucose transport and blood glucose were elevated due to HS (Pintestinal integrity and increase intestinal stress and glucose transport.

  1. Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I.

    Directory of Open Access Journals (Sweden)

    Marc U Baumann

    Full Text Available 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.

  2. Glucose transporter-1 (GLUT1) deficiency syndrome: diagnosis and treatment in late childhood.

    NARCIS (Netherlands)

    Gramer, G.; Wolf, N.I.; Vater, D.; Bast, T.; Santer, R.; Kamsteeg, E.J.; Wevers, R.A.; Ebinger, F.

    2012-01-01

    BACKGROUND: Typical cases of glucose transporter-1 deficiency syndrome (GLUT1-DS) present with early-onset epilepsy. We report symptoms, diagnostic results, and effects of therapy in two patients diagnosed with GLUT1-DS at the age of 10 and 15 years, respectively. PATIENTS: Patient 1: After four cer

  3. Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle

    DEFF Research Database (Denmark)

    Wojtaszewski, Jørgen; Hansen, B F; Ursø, Birgitte

    1996-01-01

    The role of phosphatidylinositol (PI) 3-kinase for insulin- and contraction-stimulated muscle glucose transport was investigated in rat skeletal muscle perfused with a cell-free perfusate. The insulin receptor substrate-1-associated PI 3-kinase activity was increased sixfold upon insulin stimulat...

  4. Glucose transporter-1 (GLUT1) deficiency syndrome: diagnosis and treatment in late childhood.

    NARCIS (Netherlands)

    Gramer, G.; Wolf, N.I.; Vater, D.; Bast, T.; Santer, R.; Kamsteeg, E.J.; Wevers, R.A.; Ebinger, F.

    2012-01-01

    BACKGROUND: Typical cases of glucose transporter-1 deficiency syndrome (GLUT1-DS) present with early-onset epilepsy. We report symptoms, diagnostic results, and effects of therapy in two patients diagnosed with GLUT1-DS at the age of 10 and 15 years, respectively. PATIENTS: Patient 1: After four cer

  5. Glucose transporter-1 (GLUT1) deficiency syndrome: diagnosis and treatment in late childhood.

    NARCIS (Netherlands)

    Gramer, G.; Wolf, N.I.; Vater, D.; Bast, T.; Santer, R.; Kamsteeg, E.J.; Wevers, R.A.; Ebinger, F.

    2012-01-01

    BACKGROUND: Typical cases of glucose transporter-1 deficiency syndrome (GLUT1-DS) present with early-onset epilepsy. We report symptoms, diagnostic results, and effects of therapy in two patients diagnosed with GLUT1-DS at the age of 10 and 15 years, respectively. PATIENTS: Patient 1: After four

  6. Regulation of glucose transport by ROCK1 differs from that of ROCK2 and is controlled by actin polymerization.

    Science.gov (United States)

    Chun, Kwang-Hoon; Araki, Kazushi; Jee, Yuna; Lee, Dae-Ho; Oh, Byung-Chul; Huang, Hu; Park, Kyong Soo; Lee, Sam W; Zabolotny, Janice M; Kim, Young-Bum

    2012-04-01

    A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

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

    Science.gov (United States)

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

    2016-06-07

    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.

  8. Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Koh, Ho-Jin; Toyoda, Taro; Fujii, Nobuharu;

    2010-01-01

    . Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction...

  9. Peritoneal transport dynamics of glucose and icodextrin: the in vitro comparative studies.

    Science.gov (United States)

    Czyzewska, Krystyna; Szary, Beata; Grzelak, Teresa

    2005-01-01

    We performed in vitro experiments with the isolated rabbit parietal peritoneum to evaluate the importance of fluid stirring intensification and of chemical modification of mesothelium and interstitium to the peritoneal transport of glucose and icodextrin. We used a mathematical model of mass transport to calculate the diffusive permeability coefficient, P, in centimeters per second. In control conditions (intact tissue; stirring rate: 11 mL/min), the rate of glucose (2.0 g/dL) transfer remained constant, and no differences were observed for transport from the interstitial to the mesothelial (I-->M) side of the membrane or in the opposite direction (M-->I). The value of P (+/- standard error of the mean) was 2.731 +/- 0.472 x 10(-4) cm/s. In contrast, the icodextrin (7.5 g/dL) I-->M transport rate was higher than that for M-->I (P: 0.319 +/- 0.038 x 10(-4) cm/s and 0.194 +/- 0.035 x 10(-4) cm/s respectively). Dynamics of the icodextrin M-->I transfer were constant, but I-->M increased by 50% over time. The intensification of the stirring rate increased the value of P at varying rates: the increase was greater for icodextrin than for glucose, and greater for the I-->M transport direction than for the M-->I direction for both solutes. Chemical modification (by 2.5 mmol/L sodium deoxycholate) increased glucose and icodextrin I-->M transfer a mean of 41% and 81% respectively, but increased M-->I transfer by 70% and 224% respectively. The dynamics of glucose and icodextrin peritoneal transfer in vitro are different: glucose diffusion is constant, but I-->M icodextrin transfer increases over time and is greater than M-->I transfer Fluid stirring intensification and chemical injury to the peritoneum enhance diffusion of glucose and icodextrin. Glucose and icodextrin M-->I transfer but not I-->M transfer is restricted more by tissue barriers than by stagnant fluid layers.

  10. Effects of electroacupuncture on microcirculatory blood flow and glucose transporter function in the hippocampus

    Institute of Scientific and Technical Information of China (English)

    Yan Lu; Bingbing Han; Shijun Wang

    2011-01-01

    Nerve cell metabolism in post brain ischemia depends on increased microcirculation perfusion and transport function of microvascular endothelial cells. In the present study, a rat model of middle cerebral artery occlusion was established to investigate the influence of electroacupuncture(EA)on hippocampal CA1 cerebral blood flow and glucose transporter 1(GLUT1)expression in the microvascular endothelial cp.lls. Following EA at Neiguan(PC 6), the cerebral blood flow in the ischemic hippocampal CA1 region was significantly elevated, the number and microvascular integrated absorbance of the GLUTl-positive cells were significantly increased, nerve cell damage was ameliorated, and GLUT1 protein expression in the ischemic hippocampus was significantly increased. Results demonstrate that EA increased the cerebral blood flow of the hippocampal CA1 region and improved the glucose transport function, thereby attenuating neuronal injuries.

  11. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  12. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight

    OpenAIRE

    Awadhesh Kumar Singh

    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 com...

  13. Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4.

    Science.gov (United States)

    Kraegen, E W; Sowden, J A; Halstead, M B; Clark, P W; Rodnick, K J; Chisholm, D J; James, D E

    1993-01-01

    Our aim was to study glucose transporters GLUT1 and GLUT4 in relation to in vivo glucose uptake in rat cardiac and skeletal muscle. The levels of both transporters were of a similar order of magnitude in whole muscle tissue (GLUT1/GLUT4 ratio varied from 0.1 to 0.6), suggesting that both may have an important physiological role in regulating muscle glucose metabolism. GLUT4 correlated very strongly (r2 = 0.97) with maximal insulin-stimulated glucose uptake (Rg' max., estimated using the glucose clamp plus 2-deoxy[3H]glucose bolus technique) in six skeletal muscles and heart. A distinct difference in regulation of the two transporters was evident in heart: in 5 h-fasted rats, basal glucose uptake and GLUT1 levels in heart were very high and both were reduced, by 90 and 60% respectively, by 48 h fasting. However, in heart (and in red skeletal muscle), neither GLUT4 levels nor Rg' max. were reduced by 48 h fasting. GLUT1 was shown to be specifically expressed in cardiac myocytes, because intracellular vesicles enriched in GLUT4 contained significant levels of GLUT1. In conclusion, the high association of muscle GLUT4 content with insulin responsiveness in different muscles, and the preservation of both with fasting, supports a predominant role of GLUT4 in insulin-mediated glucose uptake. GLUT1 may play an important role in mediating cardiac muscle glucose uptake in the basal metabolic state. Marked changes in GLUT1 expression with alterations in the metabolic state, such as prolonged fasting, may play an important role in cardiac glucose metabolism. Images Figure 1 Figure 2 PMID:8216230

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

    Energy Technology Data Exchange (ETDEWEB)

    Tomioka, Shigemasa, E-mail: tomioka@dent.tokushima-u.ac.jp [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Kaneko, Miyuki [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Satomura, Kazuhito [First Department of Oral and Maxillofacial Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Mikyu, Tomiko; Nakajo, Nobuyoshi [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan)

    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.

  15. Expression of glucocorticoid receptor and glucose transporter-1 during placental development in the diabetic rat

    Directory of Open Access Journals (Sweden)

    Ramazan Demir

    2011-07-01

    Full Text Available In various tissues, glucocorticoids (GCs are known to downregulate glucose transport systems; however, their effects on glucose transporters (GLUTs in the placenta of a diabetic rat are unknown. Glucocorticoid hormone action within the cell is regulated by the glucocorticoid receptor (GR. Thus, this study was designed to investigate the relationship between GR and glucose transporter expression in the placenta of the diabetic rat. Our immunohistochemical results indicated that GR and glucose transporter protein 1 (GLUT 1 are expressed ubiquitously in the trophoblast and endothelial cells of the labyrinthine zone, where maternal fetal transport takes place in the rat placenta. Expression of GR in the junctional zone of the rat placenta was detected in giant cells, and in some spongiotrophoblast cells, but not in the glycogen cells. GLUT 1 was present, especially in glycogen cells during early pregnancy, and in the spongiotrophoblast cells of the junctional zone during late pregnancy. Amounts of GR and GLUT 1 protein were increased towards the end of gestation both in the control and the diabetic placenta. However, at days 17 and 19 of gestation, only the placental GR protein was significantly increased in the streptozotocin-induced diabetic rats compared to control rats. Diabetes led to a significant decrease in placental weight at gestation day 15. In contrast, at gestational days 17 and 21, the weights of the diabetic placenta were significantly increased as compared with the controls. Moreover, diabetes induced fetus intrauterine growth retardation at gestational days 13, 17 and 21. In conclusion, the localization pattern of GR and GLUT 1 proteins in the same cell types led us to believe that there might be a relationship between GR and GLUT 1 expressions at the cellular level. GLUT 1 does not play a pivotal role in diabetic pregnancies. However, placental growth abnormalities during diabetic pregnancy may be related to the amount of GR

  16. Lack of SLC2A1 (glucose transporter 1) mutations in 30 Italian patients with alternating hemiplegia of childhood.

    Science.gov (United States)

    De Grandis, Elisa; Stagnaro, Michela; Biancheri, Roberta; Giannotta, Melania; Gobbi, Giuseppe; Traverso, Monica; Veneselli, Edvige; Zara, Federico

    2013-07-01

    Alternating hemiplegia of childhood is a rare, predominantly sporadic disorder. Diagnosis is clinical, and little is known about genetics. Glucose transporter 1 deficiency syndrome shares with alternating hemiplegia of childhood paroxysmal and nonparoxysmal symptoms. The aim of the study was to investigate glucose transporter 1 mutations in 30 Italian patients. Genetic material was analyzed by DNA amplification and glucose transporter 1 region sequencing. Mutational analysis findings of the SLC2A1 gene were negative in all patients. The pattern of movement disorders was reviewed. Interictal dystonia and multiple paroxysmal events were typical of alternating hemiplegia of childhood. In conclusion, alternating hemiplegia of childhood is a heterogeneous clinical condition, and although glucose transporter 1 deficiency can represent an undiagnosed cause of this disorder, mutational analysis is not routinely recommended. Alternatively, a careful clinical analysis and the 3-O-methyl-D-glucose uptake test can allow prompt identification of a subgroup of patients with alternating hemiplegia of childhood treatable with a ketogenic diet.

  17. Development and characterization of high affinity leptins and leptin antagonists.

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-02-11

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

  18. Development and Characterization of High Affinity Leptins and Leptin Antagonists*

    Science.gov (United States)

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-01-01

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin. PMID:21119198

  19. Water transport by the Na+/glucose cotransporter under isotonic conditions

    DEFF Research Database (Denmark)

    Zeuthen, T; Meinild, A K; Klaerke, D A;

    1997-01-01

    Solute cotransport in the Na+/glucose cotransporter is directly coupled to significant water fluxes. The water fluxes are energized by the downhill fluxes of the other substrates by a mechanism within the protein itself. In the present paper we investigate the Na+/glucose cotransporter expressed...... in Xenopus oocytes. We present a method which allows short-term exposures to sugar under voltage clamp conditions. We demonstrate that water is cotransported with the solutes despite no osmotic differences between the external and intracellular solutions. There is a fixed ratio of 195:1 between the number...... of water molecules and the number of Na+ ions transported, equivalent to 390 water molecules per glucose molecule. Unstirred layer effects are ruled out on the basis of experiments on native oocytes incubated with the ionophores gramicidin D or nystatin....

  20. Ca2+ effects on glucose transport and fatty acid oxidation in L6 skeletal muscle cell cultures

    Directory of Open Access Journals (Sweden)

    Darrick Balu

    2016-03-01

    We did find a Ca2+ stimulation (using either caffeine or ionomycin of fatty acid oxidation. This was observed in the absence (but not the presence of added glucose. We conclude that Ca2+ stimulates fatty acid oxidation at a mitochondrial site, secondary to malonyl CoA inhibition (represented by the presence of glucose in our experiments. In summary, the experiments resolve a controversy on Ca2+ stimulation of glucose transport by skeletal muscle, introduce an important experimental consideration for the measurement of glucose transport, and uncover a new site of action for Ca2+ stimulation of fatty acid oxidation.

  1. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative......γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...... understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...

  2. Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model.

    Science.gov (United States)

    Xiao, Changting; Quinton, V Margaret; Cant, John P

    2004-04-01

    Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-d-[1-(3)H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37 degrees C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 +/- 5.7% and 67.4 +/- 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.

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

    Science.gov (United States)

    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.

  4. Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

    Science.gov (United States)

    Ueda, Manabu; Furuyashiki, Takashi; Yamada, Kayo; Aoki, Yukiko; Sakane, Iwao; Fukuda, Itsuko; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2010-11-01

    In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 μM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.

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

    Science.gov (United States)

    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. Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis

    Science.gov (United States)

    Liu, T; Kishton, R J; Macintyre, A N; Gerriets, V A; Xiang, H; Liu, X; Abel, E D; Rizzieri, D; Locasale, J W; Rathmell, J C

    2014-01-01

    The metabolic profiles of cancer cells have long been acknowledged to be altered and to provide new therapeutic opportunities. In particular, a wide range of both solid and liquid tumors use aerobic glycolysis to supply energy and support cell growth. This metabolic program leads to high rates of glucose consumption through glycolysis with secretion of lactate even in the presence of oxygen. Identifying the limiting events in aerobic glycolysis and the response of cancer cells to metabolic inhibition is now essential to exploit this potential metabolic dependency. Here, we examine the role of glucose uptake and the glucose transporter Glut1 in the metabolism and metabolic stress response of BCR-Abl+ B-cell acute lymphoblastic leukemia cells (B-ALL). B-ALL cells were highly glycolytic and primary human B-ALL samples were dependent on glycolysis. We show B-ALL cells express multiple glucose transporters and conditional genetic deletion of Glut1 led to a partial loss of glucose uptake. This reduced glucose transport capacity, however, was sufficient to metabolically reprogram B-ALL cells to decrease anabolic and increase catabolic flux. Cell proliferation decreased and a limited degree of apoptosis was also observed. Importantly, Glut1-deficient B-ALL cells failed to accumulate in vivo and leukemic progression was suppressed by Glut1 deletion. Similarly, pharmacologic inhibition of aerobic glycolysis with moderate doses of 2-deoxyglucose (2-DG) slowed B-ALL cell proliferation, but extensive apoptosis only occurred at high doses. Nevertheless, 2-DG induced the pro-apoptotic protein Bim and sensitized B-ALL cells to the tyrosine kinase inhibitor Dasatinib in vivo. Together, these data show that despite expression of multiple glucose transporters, B-ALL cells are reliant on Glut1 to maintain aerobic glycolysis and anabolic metabolism. Further, partial inhibition of glucose metabolism is sufficient to sensitize cancer cells to specifically targeted therapies, suggesting

  7. Comparative study of glucose transporters GLUT-2 and GLUT-5 in ostriches gastrointestinal tract

    Directory of Open Access Journals (Sweden)

    Piret Hussar

    2016-10-01

    Full Text Available The knowledge about transport of sugars in animals and birds gastrointestinal tract is very important for science as carbohydrates are the main energy source of food. Since until now there is few information about the localization of glucose transporters - integral membrane proteins that mediate the transport of glucose and related substances across the cellular membranes - in birds gastrointestinal tract, the aim of the present study was to localize glucose transporters-2 and -5 (GLUT-2 and -5 in three parts of the ostriches gastrointestinal tract – proventriculus, duodenum and ileum - comparatively in ostrich chicken in their early ontogenesis period. Material from the superficial gland zone of the proventriculus, duodenum and terminal zone of the ileum were collected from eight female ostriches (Struthio camelus var. Domesticus: two chickens after hatching, three 7 and three 30-days old ostriches. The material was fixed with 10% formalin, embedded into paraffin, slices 7 μm thick were cut followed by immunohistochemical staining with polyclonal primary antibodies Rabbit anti-GLUT-2 and Rabbit anti-GLUT-5, carried out according to the manufacturers guidelines (IHC kit, Abcam, UK. The results showed that the staining for both antibodies was weaker in all parts of the gastrointestinal tract of ostriches after hatching compared to 7 and 30 days old ostriches showing that the gastrointestinal tract of ostriches immediately after hatching is not entirely capable of transportation of carbohydrates. The results of our study may indicate the possibility of a close relationship between feeding and the ability to transport sugars in the gastrointestinal tract.

  8. Association between coenzyme Q10 and glucose transporter (GLUT1) deficiency

    OpenAIRE

    Yubero, Delia; O’Callaghan, Mar; Montero, Raquel; Ormazabal, Aida; Armstrong, Judith; Espinos, Carmina; Rodríguez, Maria A; Jou, Cristina; Castejon, Esperanza; Aracil, Maria A; Cascajo, Maria V; Gavilan, Angela; Briones, Paz; Jimenez-Mallebrera, Cecilia; Pineda, Mercedes

    2014-01-01

    Background It has been demonstrated that glucose transporter (GLUT1) deficiency in a mouse model causes a diminished cerebral lipid synthesis. This deficient lipid biosynthesis could contribute to secondary CoQ deficiency. We report here, for the first time an association between GLUT1 and coenzyme Q10 deficiency in a pediatric patient. Case presentation We report a 15 year-old girl with truncal ataxia, nystagmus, dysarthria and myoclonic epilepsy as the main clinical features. Blood lactate ...

  9. Molecular cloning of glucose transporter 1 in grouper Epinephelus coioides and effects of an acute hyperglycemia stress on its expression and glucose tolerance.

    Science.gov (United States)

    Liu, Hongyu; Dong, Xiaohui; Chi, Shuyan; Yang, Qihui; Zhang, Shuang; Chen, Liqiao; Tan, Beiping

    2017-02-01

    The glucose transporter family proteins play pivotal roles in glucose metabolism. In this study, we successfully cloned the orange spotted grouper (Epinephelus coioides) glucose transporter 1 (EcGlut1) gene (GenBank accession: JQ623903). The full-length EcGlut1 cDNA was 2126 bp with a 1476 bp ORF, a 437bp5'-UTR and 223bp3'-UTR. EcGlut1 is predicted to encode a 491 amino acid protein with a MW of 53.9 kDa, a pI of 8.66 and a Pfam domain. Bioinformatics analysis revealed that EcGlut1 was evolutionally conserved between fishes with 80-89 % amino acid identities. EcGlut1 was expressed predominantly in heart and liver and at lower levels in muscle, intestine, stomach and brain. We also investigated the effect of acute hyperglycemia stress on EcGlut1 expression. In glucose tolerance test, changes in EcGlut1 mRNA expression in response to glucose injection and glucose metabolism-related indictors were assessed at the same time. Glucose injection significantly suppressed EcGlut1 mRNA expression in liver at 12 h and in brain at 24 h postinjection (P < 0.05). EcGlut1 mRNA levels in heart were increased at 6 h (P < 0.05). Plasma glucose level increased significantly and reached its maximum at 3 h postinjection (P < 0.05). The spatiotemporal expression of EcGlut1 and glucose metabolism suggested that orange spotted grouper might rely on fat anabolism to reduce acute hyperglycemia stress and the delayed transcription of EcGlut1 gene might be one reason for glucose intolerance in E. coioides.

  10. Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus)

    Science.gov (United States)

    Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Delgado, María Jesús; Valenciano, Ana Isabel; Unniappan, Suraj

    2017-01-01

    Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish. PMID:28338019

  11. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  12. Icodextrin peritoneal transport in vitro: effect of sodium deoxycholate, glucose, and methylglyoxal.

    Science.gov (United States)

    Szary, Beata; Grzelak, Teresa; Czyzewska, Krystyna

    2007-02-01

    The aim of the in vitro studies was to examine the effect of sodium deoxycholate, glucose, and methylglyoxal on icodextrin peritoneal transfer. The rabbit peritoneum in a modified Ussing chamber was an experimental model. Transport and morphometric analyses were performed. In the first of them, the icodextrin (7.5 g/dL) diffusion from the mesothelial to the interstitial side of the membrane, expressed as a diffusive permeability coefficient (P), was evaluated in the control stage, after chemical modification of the membrane using sodium deoxycholate (104 mg/dL), after the addition of glucose (1.8 g/dL) and methylglyoxal (1 mg/dL), in the separate experimental series. In the second morphometric studies, the thickness and transverse cross-section surface area of native tissue, in 75 min of experiment and after application of sodium deoxycholate, were investigated. In the control conditions, the rate of glucose polymer passage remained constant. A mean value of P +/- SD was 0.194 +/- 0.126 (x10(-4), cm/s) during 120 min of the study. The transfer of icodextrin was enhanced by 224% after 3 min of incubation of the peritoneum with sodium deoxycholate. The introduction of glucose into the circulating medium with icodextrin caused the increase of P values for glucose polymer by 94% during 60 min. In the same conditions, the usage of methylglyoxal did not change transport parameters. Both thickness and transverse cross-section surface area of the native tissue in 75 min of the study did not differ. It was 4.87 microm and 12.50 x 10(2) microm(2) for the mesothelial layer, and 63.83 microm and 208.10 x 10(2) microm(2) for the whole peritoneal membrane. The application of sodium deoxycholate caused the decrease of mesothelium thickness by 20% but the increase of thickness and transverse cross-section surface area of the peritoneum by 37% in comparison with 75 min of experiment. In conclusion, sodium deoxycholate and glucose, but not methylglyoxal, intensify peritoneal

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

    Directory of Open Access Journals (Sweden)

    Hongbin Tu

    2015-11-01

    Full Text Available 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.

  14. Expression of GLUT1 and GLUT3 glucose transporters in endometrial and breast cancers.

    Science.gov (United States)

    Krzeslak, Anna; Wojcik-Krowiranda, Katarzyna; Forma, Ewa; Jozwiak, Paweł; Romanowicz, Hanna; Bienkiewicz, Andrzej; Brys, Magdalena

    2012-07-01

    Cancer cells have accelerated metabolism and high glucose requirements. The up-regulation of specific glucose transporters may represent a key mechanism by which malignant cells may achieve increased glucose uptake to support the high rate of glycolysis. In present study we analyzed the mRNA and protein expression of GLUT1 and GLUT3 glucose transporters by quantitative real-time polymerase chain reaction (Q-PCR) and Western blotting technique in 76 cases of endometrial carcinoma and 70 cases of breast carcinoma. SLC2A1 and SLCA2A3 mRNAs expression was found, respectively in 100% and 97.4% samples of endometrial cancers and only in 50% and 40% samples of breast cancers. In endometrial cancers GLUT1 and GLUT3 protein expression was identified in 67.1% and 30.3% of cases. Analogously, in breast cancers in 48.7% and 21% of samples, respectively. The results showed that both endometrial and breast poorly differentiated tumors (grade 2 and 3) had significantly higher GLUT1 and GLUT3 expression than well-differentiated tumors (grade 1). Statistically significant association was found between SLCA2A3 mRNA expression and estrogen and progesterone receptors status in breast cancers. GLUT1 has been reported to be involved in the uptake of glucose by endometrial and breast carcinoma cells earlier and the present study determined that GLUT3 expression is also involved. GLUT1 and GLUT3 seem to be important markers in endometrial and breast tumors differentiation.

  15. Placental glucose and amino acid transport in calorie-restricted wild-type and Glut3 null heterozygous mice.

    Science.gov (United States)

    Ganguly, Amit; Collis, Laura; Devaskar, Sherin U

    2012-08-01

    Calorie restriction (CR) decreased placenta and fetal weights in wild-type (wt) and glucose transporter (Glut) 3 heterozygous null (glut3(+/-)) mice. Because placental nutrient transport is a primary energy determinant of placentofetal growth, we examined key transport systems. Maternal CR reduced intra- and transplacental glucose and leucine transport but enhanced system A amino acid transport in wt mice. These transport perturbations were accompanied by reduced placental Glut3 and leucine amino acid transporter (LAT) family member 2, no change in Glut1 and LAT family member 1, but increased sodium coupled neutral amino acid transporter (SNAT) and SNAT2 expression. We also noted decreased total and active phosphorylated forms of mammalian target of rapamycin, which is the intracellular nutrient sensor, the downstream total P70S6 kinase, and pS6 ribosomal protein with no change in total and phosphorylated 4E-binding protein 1. To determine the role of placental Glut3 in mediating CR-induced placental transport changes, we next investigated the effect of gestational CR in glut3(+/-) mice. In glut3(+/-) mice, a key role of placental Glut3 in mediating transplacental and intraplacental glucose transport was established. In addition, reduced Glut3 results in a compensatory increase of leucine and system A transplacental transport. On the other hand, diminished Glut3-mediated intraplacental glucose transport reduced leucine transport and mammalian target of rapamycin and preserved LAT and enhancing SNAT. CR in glut3(+/-) mice further reduced transplacental glucose transport and enhanced system A amino acid transport, although the increased leucine transport was lost. In addition, increased Glut3 was seen and preserved Glut1, LAT, and SNAT. These placental changes collectively protect survival of wt and glut3(+/-) fetuses against maternal CR-imposed reduction of macromolecular nutrients.

  16. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    OpenAIRE

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide...

  17. Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model

    Directory of Open Access Journals (Sweden)

    João M N Duarte

    2009-10-01

    Full Text Available Glucose supply from blood to brain occurs through facilitative transporter proteins. A near linear relation between brain and plasma glucose has been experimentally determined and described by a reversible model of enzyme kinetics. A conformational four-state exchange model accounting for trans-acceleration and asymmetry of the carrier was included in a recently developed multi-compartmental model of glucose transport. Based on this model, we demonstrate that brain glucose (Gbrain as function of plasma glucose (Gplasma can be described by a single analytical equation namely comprising three kinetic compartments: blood, endothelial cells and brain. Transport was described by four parameters: apparent half saturation constant Kt, apparent maximum rate constant Tmax, glucose consumption rate CMRglc, and the iso-inhibition constant Kii that suggests Gbrain as inhibitor of the isomerisation of the unloaded carrier. Previous published data, where Gbrain was quantified as a function of plasma glucose by either biochemical methods or NMR spectroscopy, were used to determine the aforementioned kinetic parameters. Glucose transport was characterized by Kt ranging from 1.5 to 3.5 mM, Tmax/CMRglc from 4.6 to 5.6, and Kii from 51 to 149 mM. It was noteworthy that Kt was on the order of a few mM, as previously determined from the reversible model. The conformational four-state exchange model of glucose transport into the brain includes both efflux and transport inhibition by Gbrain, predicting that Gbrain eventually approaches a maximum concentration. However, since Kii largely exceeds Gplasma, iso-inhibition is unlikely to be of substantial importance for plasma glucose below 25 mM. As a consequence, the reversible model can account for most experimental observations under euglycaemia and moderate cases of hypo- and hyperglycaemia.

  18. Myocardial glucose transporters and glycolytic metabolism during ischemia in hyperglycemic diabetic swine.

    Science.gov (United States)

    Stanley, W C; Hall, J L; Smith, K R; Cartee, G D; Hacker, T A; Wisneski, J A

    1994-01-01

    We assessed the effects of 4 weeks of streptozocin-induced diabetes on regional myocardial glycolytic metabolism during ischemia in anesthetized open-chest domestic swine. Diabetic animals were hyperglycemic (12.0 +/- 2.1 v 6.6 +/- .5 mmol/L), and had lower fasting insulin levels (27 +/- 8 v 79 +/- 19 pmol/L). Myocardial glycolytic metabolism was studied with coronary flow controlled by an extracorporeal perfusion circuit. Left anterior descending coronary artery (LAD) flow was decreased by 50% for 45 minutes and left circumflex (CFX) flow was constant. Myocardial glucose uptake and extraction were measured with D-[6-3H]-2-deoxyglucose (DG) and myocardial blood flow was measured with microspheres. The rate of glucose conversion to lactate and lactate uptake and output were assessed with a continuous infusion of [6-14C]glucose and [U-13C]lactate into the coronary perfusion circuit. Both diabetic and nondiabetic animals had sharp decreases in subendocardial blood flow during ischemia (from 1.21 +/- .10 to 0.43 +/- .08 mL.g-1.min-1 in the nondiabetic group, and from 1.30 +/- .15 to 0.55 +/- .11 in the diabetic group). Diabetes had no significant effect on myocardial glucose uptake or glucose conversion to lactate under either well-perfused or ischemic conditions. Forty-five minutes of ischemia resulted in significant glycogen depletion in the subendocardium in both nondiabetic and diabetic animals, with no differences between the two groups. Glycolytic metabolism is not impaired in hyperglycemic diabetic swine after 1 month of the disease when compared with that in normoglycemic nondiabetic animals. The myocardial content of the insulin-regulatable glucose transporter (GLUT 4) was measured in left ventricular biopsies.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Science.gov (United States)

    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.

  20. Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle

    DEFF Research Database (Denmark)

    Ai, Hua; Ihlemann, Jacob; Hellsten, Ylva

    2002-01-01

    )- and alpha(2)-isoforms of AMPK. Expression of both isoforms varied with fiber types, and alpha(2) was highly expressed in nuclei. In conclusion, AICAR-stimulated glucose transport varies with muscle fiber type and nutritional state. AMPK is unlikely to be the sole mediator of contraction-stimulated glucose...

  1. Effect of vanadate on glucose transporter (GLUT4) intrinsic activity in skeletal muscle plasma membrane giant vesicles

    DEFF Research Database (Denmark)

    Kristiansen, S; Youn, J; Richter, Erik

    1996-01-01

    for 1.5 h with concentrations of vanadate ranging from 3 to 40 mmol l-1 at 34 degrees C before being used for determination of glucose transport. The dose-response curve showed that vanadate decreased the specific D-glucose uptake by a maximum of 70% compared with a control preparation. The vanadate...

  2. Epigenetic Regulation of Glucose Transporters in Non-Small Cell Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    O' Byrne, Kenneth J.; Baird, Anne-Marie; Kilmartin, Lisa; Leonard, Jennifer; Sacevich, Calen; Gray, Steven G., E-mail: sgray@stjames.ie [Department of Clinical Medicine, Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James' s Hospital, Dublin 8 (Ireland)

    2011-03-25

    Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy.

  3. Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking.

    Science.gov (United States)

    Wasik, Anita A; Polianskyte-Prause, Zydrune; Dong, Meng-Qiu; Shaw, Andrey S; Yates, John R; Farquhar, Marilyn G; Lehtonen, Sanna

    2012-09-01

    Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.

  4. DHHC7 Palmitoylates Glucose Transporter 4 (Glut4) and Regulates Glut4 Membrane Translocation.

    Science.gov (United States)

    Du, Keyong; Murakami, Shoko; Sun, Yingmin; Kilpatrick, Casey L; Luscher, Bernhard

    2017-02-17

    Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane plays a key role in the dynamic regulation of glucose homeostasis. We recently showed that this process is critically dependent on palmitoylation of Glut4 at Cys-223. To gain further insights into the regulation of Glut4 palmitoylation, we set out to identify the palmitoyl acyltransferase (PAT) involved. Here we report that among 23 mammalian DHHC proteins, DHHC7 is the major Glut4 PAT, based on evidence that ectopic expression of DHHC7 increased Glut4 palmitoylation, whereas DHHC7 knockdown in 3T3-L1 adipocytes and DHHC7 KO in adipose tissue and muscle decreased Glut4 palmitoylation. Moreover, inactivation of DHHC7 suppressed insulin-dependent Glut4 membrane translocation in both 3T3-L1 adipocytes and primary adipocytes. Finally, DHHC7 KO mice developed hyperglycemia and glucose intolerance, thereby confirming that DHHC7 represents the principal PAT for Glut4 and that this mechanism is essential for insulin-regulated glucose homeostasis.

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

    Science.gov (United States)

    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.

  6. Glucose transporter-1 (GLUT-1) immunoreactivity in benign, premalignant and malignant lesions of the gallbladder.

    Science.gov (United States)

    Legan, Mateja; Tevžič, Spela; Tolar, Ana; Luzar, Boštjan; Marolt, Vera Ferlan

    2011-03-01

    GLUT-1 is a transmembrane glucose transport protein that allows the facilitated transport of glucose into cells, normally expressed in tissues which depend mainly on glucose metabolism. Enhanced expression of GLUT-1 can also be found in a large spectrum of carcinomas. This study aimed to investigate GLUT-1 expression in gallbladder tissue: from normal tissue samples, hyperplasias, low-grade and high-grade dysplasias to gallbladder carcinomas. In all, 115 archived samples of gallbladder tissue from 68 patients, presented after cholecystectomy, were immunohistochemically stained for GLUT-1. According to the intensity of GLUT-1 immunoreactivity, samples were divided into negative (stained 0-10% of cells stained), positive with weak to moderate (10-50%) and positive with strong (>50%) GLUT-1 expression. The GLUT-1 immunoreactivity of the samples showed a characteristic increase from premalignant lesions to carcinomas. Normal gallbladder tissue samples did not express GLUT-1 (100%). Weak expression was shown only focally in hyperplasias, but to a greater extent with low-grade dysplasias (20%), high-grade dysplasias (40%) and carcinomas (51.8%). Normal gallbladder tissue is GLUT-1 negative. GLUT-1 expression in carcinoma tissue is significantly higher than in dysplastic lesions. Strong GLUT-1 expression indicates 100% specificity for detecting gallbladder carcinomas. Therefore, GLUT-1 is a candidate as a diagnostic as well as a tissue prognostic marker in gallbladder carcinoma patients.

  7. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

    NARCIS (Netherlands)

    Bosch, R.R.; Bazuine, M.; Wake, M.M.; Span, P.N.; Olthaar, A.J.; Schurmann, A.; Maassen, J.A.; Hermus, A.R.M.M.; Willems, P.H.G.M.; Sweep, C.G.J.

    2003-01-01

    The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rat

  8. Analysis of metabolism of 6FDG: a PET glucose transport tracer

    Energy Technology Data Exchange (ETDEWEB)

    Muzic, Raymond F., E-mail: raymond.muzic@case.edu [Department of Radiology, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106 (United States); Chandramouli, Visvanathan [Department of Radiology, Case Western Reserve University, Cleveland, OH 44106 (United States); Huang, Hsuan-Ming [Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106 (United States); Wu Chunying; Wang Yanming [Department of Radiology, Case Western Reserve University, Cleveland, OH 44106 (United States); Ismail-Beigi, Faramarz [Department of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States)

    2011-07-15

    Introduction: We are developing {sup 18}F-labeled 6-fluoro-6-deoxy-D-glucose ([{sup 18}F]6FDG) as a tracer of glucose transport. As part of this process it is important to characterize and quantify putative metabolites. In contrast to the ubiquitous positron emission tomography (PET) tracer {sup 18}F-labeled 2-fluoro-2-deoxy-D-glucose ([{sup 18}F]2FDG) which is phosphorylated and trapped intracellularly, the substitution of fluorine for a hydroxyl group at carbon-6 in [{sup 18}F]6FDG should prevent its phosphorylation. Consequently, [{sup 18}F]6FDG has the potential to trace the transport step of glucose metabolism without the confounding effects of phosphorylation and subsequent steps of metabolism. Herein the focus is to determine whether, and the degree to which, [{sup 18}F]6FDG remains unchanged following intravenous injection. Methods: Biodistribution studies were performed using 6FDG labeled with {sup 18}F or with the longer-lived radionuclides {sup 3}H and {sup 14}C. Tissues were harvested at 1, 6, and 24 h following intravenous administration and radioactivity was extracted from the tissues and analyzed using a combination of ion exchange columns, high-performance liquid chromatography, and chemical reactivity. Results: At the 1 h time-point, the vast majority of radioactivity in the liver, brain, heart, skeletal muscle, and blood was identified as 6FDG. At the 6-h and 24-h time points, there was evidence of a minor amount of radioactive material that appeared to be 6-fluoro-6-deoxy-D-sorbitol and possibly 6-fluoro-6-deoxy-D-gluconic acid. Conclusion: On the time scale typical of PET imaging studies radioactive metabolites of [{sup 18}F]6FDG are negligible.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Christian D Young

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

  12. Pharmacokinetic and pharmacodynamic profile of empagliflozin, a sodium glucose co-transporter 2 inhibitor.

    Science.gov (United States)

    Scheen, André J

    2014-03-01

    Empagliflozin is an orally active, potent and selective inhibitor of sodium glucose co-transporter 2 (SGLT2), currently in clinical development to improve glycaemic control in adults with type 2 diabetes mellitus (T2DM). SGLT2 inhibitors, including empagliflozin, are the first pharmacological class of antidiabetes agents to target the kidney in order to remove excess glucose from the body and, thus, offer new options for T2DM management. SGLT2 inhibitors exert their effects independently of insulin. Following single and multiple oral doses (0.5-800 mg), empagliflozin was rapidly absorbed and reached peak plasma concentrations after approximately 1.33-3.0 h, before showing a biphasic decline. The mean terminal half-life ranged from 5.6 to 13.1 h in single rising-dose studies, and from 10.3 to 18.8 h in multiple-dose studies. Following multiple oral doses, increases in exposure were dose-proportional and trough concentrations remained constant after day 6, indicating a steady state had been reached. Oral clearance at steady state was similar to corresponding single-dose values, suggesting linear pharmacokinetics with respect to time. No clinically relevant alterations in pharmacokinetics were observed in mild to severe hepatic impairment, or in mild to severe renal impairment and end-stage renal disease. Clinical studies did not reveal any relevant drug-drug interactions with several other drugs commonly prescribed to patients with T2DM, including warfarin. Urinary glucose excretion (UGE) rates were higher with empagliflozin versus placebo and increased with dose, but no relevant impact on 24-h urine volume was observed. Increased UGE resulted in proportional reductions in fasting plasma glucose and mean daily glucose concentrations.

  13. Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoshida, Aya; Wei, Dandan; Nomura, Wataru; Izawa, Shingo; Inoue, Yoshiharu

    2012-01-02

    Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.

  14. Topology mapping of insulin-regulated glucose transporter GLUT4 using computational biology.

    Science.gov (United States)

    Chakraborty, Chiranjib; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Agoramoorthy, Govindasamy

    2013-01-01

    The type 2 diabetes is increasing rapidly around the globe. The primary cause for this is insulin resistance due to the disruption of the insulin signal transduction mechanism. Insulin signal transduction stimulates glucose transport through the glucose transporter GLUT4, by promoting the exocytosis process. Understanding the structural topology of GLUT4 mechanism will increase our understanding of the dynamic activities about glucose transport and its regulation in the membrane environment. However, little is known about the topology of GLUT4. In this article, we have determined the amino acid composition, disulfide topology, structure conformation pattern of GLUT4. The amino acid composition portrays that leucine composition is the highest contributing to 15.5% among all other amino acids. Three cysteine residues such as Cys223, Cys361, and Cys363 were observed and the last two were associated with one disulfide bond formation. We have generated surface cavities to know the clefts/pockets on the surface of this protein that showed few irregular cavities placed mostly in the transmembrane-helical part. Besides, topology mapping of 12 transmembrane-helixes was done to predict N- and O-glycosylation sites and to show the highly glycosylated GLUT4 that includes both N- and O-glycosylation sites. Furthermore, hydrophobic segment and molecular charge distribution were analyzed. This article shows that bioinformatics tools can provide a rapid methodology to predict the topology of GLUT4. It also provides insights into the structural details and structural functioning relationships in the human GLUT4. The results can be of great help to advance future drug development research using GLUT4 as a target protein.

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

    Science.gov (United States)

    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.

  16. Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts.

    Science.gov (United States)

    Wagstaff, P; Kang, H Y; Mylott, D; Robbins, P J; White, M K

    1995-01-01

    Vertebrate cells that are transformed by oncogenes such as v-src or are stimulated by mitogens have increased rates of glucose uptake. In rodent cells, the mechanisms whereby glucose transport is up-regulated are well understood. Stimulation of glucose transport involves an elevation in mRNA encoding the GLUT1 glucose transporter that is controlled at the levels of both transcription and mRNA stability. Cloning and sequencing of chicken GLUT1 cDNA showed that it shares 95% amino acid sequence similarity to mammalian GLUT1s. Nevertheless, unlike mammalian GLUT1 mRNA, it was not induced by v-src, serum addition, or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in chicken embryo fibroblasts. Rather, the induction of glucose transport in chicken embryo fibroblasts by v-src, serum, and 12-O-tetradecanoylphorbol 13-acetate was associated with induction of GLUT3 mRNA level and GLUT3 transcription. Rat fibroblasts were also found to express both GLUT1 and GLUT3 isoforms, but v-src induced GLUT1 and not GLUT3. This suggests that animal cells require both a basal and an upregulatable glucose transporter and that these functions have been subsumed by different GLUT isoforms in avian and mammalian cells. Images PMID:8589457

  17. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  18. Overexpression of glucose transporter-1 (GLUT-1) predicts poor prognosis in epithelial ovarian cancer.

    Science.gov (United States)

    Cho, Hanbyoul; Lee, You Sun; Kim, Julie; Chung, Joon-Yong; Kim, Jae-Hoon

    2013-11-01

    Illumina microarray was used to identify differentially expressed genes in three epithelial ovarian cancer (EOC) cells. To validate the microarray data, mRNA and protein level of glucose transporter-1 (GLUT-1) was examined. GLUT-1 had an EOC/normal cells ratio of 5.51 based on microarray. Real-time PCR and immunohistochemistry demonstrated that GLUT-1 expression was significantly increased in EOC (p = .029 and p GLUT-1 overexpression (HR = 4.80, p = .027) and lymph node metastases (HR = 8.35, p = .016) conferred a significantly worse overall survival. In conclusion, GLUT-1 expression is remarkably upregulated in EOC and predicts a poor overall survival.

  19. Water transport by the Na+/glucose cotransporter under isotonic conditions

    DEFF Research Database (Denmark)

    Zeuthen, T; Meinild, A K; Klaerke, D A

    1997-01-01

    in Xenopus oocytes. We present a method which allows short-term exposures to sugar under voltage clamp conditions. We demonstrate that water is cotransported with the solutes despite no osmotic differences between the external and intracellular solutions. There is a fixed ratio of 195:1 between the number...... of water molecules and the number of Na+ ions transported, equivalent to 390 water molecules per glucose molecule. Unstirred layer effects are ruled out on the basis of experiments on native oocytes incubated with the ionophores gramicidin D or nystatin....

  20. Novel Roles for the Insulin-Regulated Glucose Transporter-4 in Hippocampally Dependent Memory.

    Science.gov (United States)

    Pearson-Leary, Jiah; McNay, Ewan C

    2016-11-23

    The insulin-regulated glucose transporter-4 (GluT4) is critical for insulin- and contractile-mediated glucose uptake in skeletal muscle. GluT4 is also expressed in some hippocampal neurons, but its functional role in the brain is unclear. Several established molecular modulators of memory processing regulate hippocampal GluT4 trafficking and hippocampal memory formation is limited by both glucose metabolism and insulin signaling. Therefore, we hypothesized that hippocampal GluT4 might be involved in memory processes. Here, we show that, in male rats, hippocampal GluT4 translocates to the plasma membrane after memory training and that acute, selective intrahippocampal inhibition of GluT4-mediated glucose transport impaired memory acquisition, but not memory retrieval. Other studies have shown that prolonged systemic GluT4 blockade causes insulin resistance. Unexpectedly, we found that prolonged hippocampal blockade of glucose transport through GluT4-upregulated markers of hippocampal insulin signaling prevented task-associated depletion of hippocampal glucose and enhanced both working and short-term memory while also impairing long-term memory. These effects were accompanied by increased expression of hippocampal AMPA GluR1 subunits and the neuronal GluT3, but decreased expression of hippocampal brain-derived neurotrophic factor, consistent with impaired ability to form long-term memories. Our findings are the first to show the cognitive impact of brain GluT4 modulation. They identify GluT4 as a key regulator of hippocampal memory processing and also suggest differential regulation of GluT4 in the hippocampus from that in peripheral tissues. The role of insulin-regulated glucose transporter-4 (GluT4) in the brain is unclear. In the current study, we demonstrate that GluT4 is a critical component of hippocampal memory processes. Memory training increased hippocampal GluT4 translocation and memory acquisition was impaired by GluT4 blockade. Unexpectedly, whereas long

  1. Effects of glucose and insulin on the H9c2 (2-1) cell proliferation may be mediated through regulating glucose transporter 4 expression

    Institute of Scientific and Technical Information of China (English)

    LIU Qian; HUANG Qing-xian; LOU Fu-chen; ZHANG Li; WANG Kun; YU Shan; XU Hua

    2013-01-01

    Background The change of glucose transporter 4 (GLUT4) expression could influence glucose uptake in the myocardial cells and then effect myocardial metabolism,which maybe one of the factor for the diabetes cardiovascular disease.This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2 (2-1) cell proliferation and its GLUT4 expression in vitro,and evaluate the correlation between myocardial cells proliferation and GLUT4 expression.This might be helpful for understanding the relationship between glucose metabolism and cardiovascular disease.Methods According to glucose concentrations in culture medium,cultured H9c2 rat myocardial cells were divided into five groups:control group (NC,glucose concentration 5.0 mmol/L),low glucose group (LG,glucose concentration 0.1 mmol/L),high glucose group 1 (HG1,glucose concentration 10 mmol/L),high glucose group 2 (HG2,glucose concentration 15 mmol/L),high glucose group 3 (HG3,glucose concentration 20 mmol/L).Then according to different insulin concentrations in culture medium,each group was further divided into two subgroups:normal insulin subgroup (INSc,insulin concentration 3.8 mU/L),high insulin subgroup (INSh,insulin concentration 7.6 mU/L).H9c2 (2-1) cells were cultured for 1,2,3 days,the proliferation of cells were assayed by cell counting Kit-8 assay,the expressions of GLUT4 mRNA and protein were detected with RT-PCR and Western Blotting technique,and the relation between myocardial cells proliferation and GLUT4 expression was evaluated.Results Compared with NC group,cell proliferation (OD value) was lower in LG,HG2,HG3 group but higher in HG1 group on the second and the third day (P <0.05).There was a negative correlation between OD value and the glucose level in HG1,HG2,HG3 groups (P <0.05).OD value in INSc subgroups was lower than that in INSh subgroups (P <0.05).GLUT4 mRNA was lower in LG,HG2,HG3 groups than that in NC group (P <0.05).Compared with NC group,GLUT4 m

  2. The Kv1.3 channel blocker Vm24 enhances muscle glucose transporter 4 mobilization but does not reduce body-weight gain in diet-induced obese male rats.

    Science.gov (United States)

    Jaimes-Hoy, Lorraine; Gurrola, Georgina B; Cisneros, Miguel; Joseph-Bravo, Patricia; Possani, Lourival D; Charli, Jean-Louis

    2017-07-15

    Voltage-gated potassium channels 1.3 (Kv1.3) can be targeted to reduce diet-induced obesity and insulin resistance in mice. Since species-specific differences in Kv1.3 expression and pharmacology have been observed, we tested the effect of Vm24, a high-affinity specific blocker of Kv1.3 channels from Vaejovis mexicanus smithi, on body weight (BW), glucose tolerance and insulin resistance in diet-induced obese rats. Young adult male Wistar rats were switched to a high-fat/high-fructose (HFF) diet. Eighteen days later animals were divided in two groups: vehicle and Vm24 group. Subcutaneous injections were applied every other day until sacrifice 2months later. An additional cohort was maintained on standard chow. The HFF diet promoted obesity. Treatment with Vm24 did not alter various metabolic parameters such as food intake, BW gain, visceral white adipose tissue mass, adipocyte diameter, serum glucose, leptin and thyroid hormone concentrations, brown adipose tissue mass or uncoupling protein-1 expression, and insulin tolerance. Vm24 did reduce basal and glucose-stimulated serum insulin concentrations, serum C-peptide concentration, increased QUICKI, and tended to lower HOMA-IR. Vm24 treatment did not change the activation of insulin receptor substrate-1, but enhanced protein-kinase B activation and membrane glucose-transporter 4 (GLUT4) protein levels in skeletal muscle. In conclusion, in male rats, long-term blockade of Kv1.3 channels with Vm24 does not reduce weight gain and visceral adiposity induced by HFF diet; instead, it reduces serum insulin concentration, and enhances GLUT4 mobilization in skeletal muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Elevated skeletal muscle glucose transporter levels in exercise-trained middle-aged men.

    Science.gov (United States)

    Houmard, J A; Egan, P C; Neufer, P D; Friedman, J E; Wheeler, W S; Israel, R G; Dohm, G L

    1991-10-01

    Exercise training has been proposed to improve whole body insulin sensitivity through a postreceptor adaptation in skeletal muscle. This study examined if levels of the insulin-responsive muscle glucose transporter protein (GLUT-4) were associated with improved insulin sensitivity in trained vs. sedentary middle-aged individuals. Muscle GLUT-4 levels and oral glucose tolerance test (OGTT) responses were obtained in age-matched trained and sedentary men (n = 11). Plasma insulin levels during the OGTT were significantly lower (P less than 0.01) in the trained men, whereas no differences were seen in plasma glucose responses. GLUT-4 protein content was approximately twofold higher in the trained men (2.41 +/- 0.17 vs. 1.36 +/- 0.11 micrograms standard, P less than 0.001). OGTT responses and GLUT-4 levels were not altered 15-18 h after a standard exercise bout in six representative sedentary subjects. These data suggest that GLUT-4 levels are increased in conjunction with insulin sensitivity in chronically exercise-trained middle-aged men. This finding suggests a possible mechanism for the improved insulin sensitivity observed with exercise training in humans.

  4. Effect of puerarin on the P13K pathway for glucose transportation and insulin signal transduction in adipocytes

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ying; ZHOU You; YIN Hui-jun; ZHANG Ying

    2009-01-01

    To explore the effect of puerarin on insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and protein expression of protein kinase B(PKB) in the P13K pathway of the glucose consumption, transportation and insulin signal transduction in 3T3-L1 adipocytes with insulin resistance. The insulin resistance 3T3-L1 adiocytes model was established by free fatty acid induction. The model cells were managed with puerarin in different concentrations. Glucose consumption was detected with glucose oxidase method, glucose transportation rate was determined by 2-deoxy-3 H glucose ingesting method, and the IR, IRS-1 and PKB expression were determined by Western blot. Glucose consumption and transportation were significantly decreased in the model adipocytes, but increased after treated with puerarin (P < 0. 01 ). Moreover, the level of tyrosine phosphorylation of IR subunit βwas higher in the puerarin treated groups, and that of IRS-1 was higher in the group treated with low dose puerarin than that in the model group. The 3T3-L1 adipocytes of insulin resistance model could be induced by free fatty acid successfully, puerarin could promote the glucose utilization in them to alleviate the insulin resistance, which may be related with the action in advancing the tyrosine phosphorylation of IR and IRS-1.

  5. Zinc transporter 5 and zinc transporter 7 induced by high glucose protects peritoneal mesothelial cells from undergoing apoptosis.

    Science.gov (United States)

    Zhang, Xiuli; Liang, Dan; Guo, Baolei; Deng, Wenyan; Chi, Zhi-Hong; Cai, Yuan; Wang, Lining; Ma, Jianfei

    2013-04-01

    Zinc is an essential micronutrient and cytoprotectant involved in many types of apoptosis. The zinc transporter family SLC30A (ZnTs) is an important factor in the regulation of zinc homeostasis; however, its function in apoptosis in peritoneal mesothelial cells (PMCs) remains unknown. This study explores the regulation of zinc transporters and how they play a role in cell survival, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations, and the molecular mechanism involved. The messenger RNA (mRNA) transcripts were quantitatively measured by real-time polymerase chain reaction for all known nine zinc transport exporters (SLC30A1-8,10), as well as in primary RPMCs and the cells cultured under nonstimulated and HG-stimulated conditions. While many zinc transporters were constitutively expressed, ZnT5 mRNA and ZnT7 mRNA were strongly induced by HG. Overexpression of ZnT5 and ZnT7 respectively resulted in a decrease in the expression of caspace 3, caspace 8, BAX, and AIF and coincided with cell survival in the presence of HG. Inhibition of ZnT5 and ZnT7 expression using considerable siRNA-mediated knockdown of RPMCs was examined and, afterwards, the impact on cell apoptosis was investigated. Increased levels of apoptosis were observed after knockdown of ZnT5 and ZnT7. Furthermore, overexpression of ZnT5 and ZnT7 is accompanied by activation of PI3K/Akt pathway and inhibiting HG-induced apoptosis. This study suggests that the zinc transporting system in RPMCs is influenced by exposure to HG, particularly ZnT5 and ZnT7. This may account for the inhibition of HG-induced RPMC apoptosis and peritoneum injury, likely through targeting PI3K/Akt pathway-mediated cell survival.

  6. Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

    Science.gov (United States)

    Bogan, Jonathan S; Rubin, Bradley R; Yu, Chenfei; Löffler, Michael G; Orme, Charisse M; Belman, Jonathan P; McNally, Leah J; Hao, Mingming; Cresswell, James A

    2012-07-06

    To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

  7. Immunohistochemical Evaluation of Glucose Transporter Type 1 in Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Pereira, Karuza Maria Alves; Feitosa, Sthefane Gomes; Lima, Ana Thayssa Tomaz; Luna, Ealber Carvalho Macedo; Cavalcante, Roberta Barroso; de Lima, Kenio Costa; Chaves, Filipe Nobre; Costa, Fábio Wildson Gurgel

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity and some of these have been documented in association or preceded by oral epithelial dysplasia (OED). Aggressive cancers with fast growth have demonstrated overexpression of some glucose transporters (GLUTs). Thus, the aim of this study was to analyze the immunohistochemical expression of the glucose transporter, GLUT-1, in OEDs and OSCCs, seeking to better elucidate the biological behavior of neoplasias. Fifteen cases were selected this research of both lesions. Five areas were analyzed from each case by counting the percentage of positive cells at 400x magnification. Immunoreactivity of GLUT-1 was observed in 100% of the samples ranging from 54.2% to 86.2% for the OSCC and 73.9% to 97.4% for the OED. Statistical test revealed that there was greater overexpression of GLUT-1 in OED than the OSCC (p=0.01). It is believed the high expression of GLUT-1 may reflect the involvement of GLUT-1 in early stages of oral carcinogenesis.

  8. Paraoxonase 2 Facilitates Pancreatic Cancer Growth and Metastasis by Stimulating GLUT1-Mediated Glucose Transport.

    Science.gov (United States)

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Sun, Lisha; Gandotra, Neeru; Ho, Thuy; Cai, Guoping; Cline, Gary; Kumar, Priti; Cowles, Robert A; Wajapeyee, Narendra

    2017-08-17

    Metabolic deregulation is a hallmark of human cancers, and the glycolytic and glutamine metabolism pathways were shown to be deregulated in pancreatic ductal adenocarcinoma (PDAC). To identify new metabolic regulators of PDAC tumor growth and metastasis, we systematically knocked down metabolic genes that were overexpressed in human PDAC tumor samples using short hairpin RNAs. We found that p53 transcriptionally represses paraoxonase 2 (PON2), which regulates GLUT1-mediated glucose transport via stomatin. The loss of PON2 initiates the cellular starvation response and activates AMP-activated protein kinase (AMPK). In turn, AMPK activates FOXO3A and its transcriptional target, PUMA, which induces anoikis to suppress PDAC tumor growth and metastasis. Pharmacological or genetic activation of AMPK, similar to PON2 inhibition, blocks PDAC tumor growth. Collectively, our results identify PON2 as a new modulator of glucose transport that regulates a pharmacologically tractable pathway necessary for PDAC tumor growth and metastasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Modified Atkins diet therapy for a case with glucose transporter type 1 deficiency syndrome.

    Science.gov (United States)

    Ito, Susumu; Oguni, Hirokazu; Ito, Yasushi; Ishigaki, Keiko; Ohinata, Junko; Osawa, Makiko

    2008-03-01

    Glucose transporter type 1 deficiency syndrome (GLUT-1 DS), giving rise to impaired glucose transport across the blood-brain barrier, is characterized by infantile seizures, complex motor disorders, global developmental delay, acquired microcephaly, and hypoglycorrhachia. GLUT-1 DS can be treated effectively with a ketogenic diet because it can provide an alternative fuel for brain metabolism; however, the excessive restriction of food intake involved frequently makes it difficult for patients to initiate or continue the diet. Recently, the modified Atkins diet, which is much less restrictive in terms of the total calorie and protein intake than the classical ketogenic diet, has been shown to be effective and well tolerated in children with intractable epilepsy. We successfully introduced the modified Atkins diet to a 7-year-old boy with GLUT-1 DS, whose caregivers refused ketogenic diet treatment because of strong concerns over restricting the diet. The modified Atkins diet should be considered for patients with GLUT-1 DS as an alternative to the traditional ketogenic diet.

  10. Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

    Directory of Open Access Journals (Sweden)

    Jennine M Dawicki-McKenna

    Full Text Available In adipocytes, vesicles containing glucose transporter-4 (GLUT4 redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin, preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

  11. A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

    Science.gov (United States)

    Lee, Eunice E; Ma, Jing; Sacharidou, Anastasia; Mi, Wentao; Salato, Valerie K; Nguyen, Nam; Jiang, Youxing; Pascual, Juan M; North, Paula E; Shaul, Philip W; Mettlen, Marcel; Wang, Richard C

    2015-06-04

    Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

  12. Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

    Directory of Open Access Journals (Sweden)

    Amanda E Siglin

    Full Text Available Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC and the dynactin p150(Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

  13. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    2013-01-01

    Full Text Available Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB with high affinity (KD values from 2.55 to 36.27 nM. RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p. administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  14. Molecular mechanisms beyond glucose transport in diabetes-related male infertility.

    Science.gov (United States)

    Alves, M G; Martins, A D; Rato, L; Moreira, P I; Socorro, S; Oliveira, P F

    2013-05-01

    Diabetes mellitus (DM) is one of the greatest public health threats in modern societies. Although during a few years it was suggested that DM had no significant effect in male reproductive function, this view has been challenged in recent years. The increasing incidence of DM worldwide will inevitably result in a higher prevalence of this pathology in men of reproductive age and subfertility or infertility associated with DM is expected to dramatically rise in upcoming years. From a clinical perspective, the evaluation of semen parameters, as well as spermatozoa deoxyribonucleic acid (DNA) integrity, are often studied due to their direct implications in natural and assisted conception. Nevertheless, recent studies based on the molecular mechanisms beyond glucose transport in testicular cells provide new insights in DM-induced alterations in male reproductive health. Testicular cells have their own glucose sensing machinery that react to hormonal fluctuations and have several mechanisms to counteract hyper- and hypoglycemic events. Moreover, the metabolic cooperation between testicular cells is crucial for normal spermatogenesis. Sertoli cells (SCs), which are the main components of blood-testis barrier, are not only responsible for the physical support of germ cells but also for lactate production that is then metabolized by the developing germ cells. Any alteration in this tied metabolic cooperation may have a dramatic consequence in male fertility potential. Therefore, we present an overview of the clinical significance of DM in the male reproductive health with emphasis on the molecular mechanisms beyond glucose fluctuation and transport in testicular cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier.

    Science.gov (United States)

    Duarte, João M N; Gruetter, Rolf

    2012-05-01

    Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.

  16. Experimental lead poisoning and intestinal transport of glucose, amino acids, and sodium.

    Science.gov (United States)

    Wapnir, R A; Exeni, R A; McVicar, M; Lipshitz, F

    1977-03-01

    Juvenile rats fed a diet containing 1% lead acetate for 7 weeks, in addition to an impaired growth rate and renal function derangements, suffered malabsorption of glucose and certain amino acids, as assessed by an in vivo perfusion technique. The reduction in glucose absorption ranged between 10% and 31% when the carbohydrate was pumped in concentrations of 2-80 mM. This alteration was compatible with a noncompetitive type of transport inhibition. The intestinal absorption of glycine, lysine, and phenylalanine were, respectively, decreased 22, 18, and 15% when these amino acids were present at 1 mM levels. Sodium transport was severely reduced (57.6 +/- 17.9 (SEM) vs. 124.2 +/- 17.4 muEq/min-cm) and intestinal mucosa (Na+-K+)-ATPase was concomitantly lower in the lead-intoxicated rats (186.4 +/- 19.0 vs 268.4 +/- 29.8 nmol P/min-mg protein). However, this enzyme was not altered in liver and kidney. Furthermore, intestinal mucosa fructose-1,6-diphosphatase, succinic dehydrogenase, pyruvate kinase, and tryptophan hydroxylase were not different in experimental and control animals. These studies substantiate the presence of functional and biochemical abnormalities in the intestinal mucosa of young rats when fed substantial amounts of a soluble lead salt. It is, therefore, reasonable to accept the possibility that physiologic damage occurs in tissues directly subjected to high and persistent levels of a toxic agents, as it occurs in other organs, underscoring the parallelism between transport mechanisms at the renal and intestinal levels.

  17. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

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    Arieh Riskin

    2015-10-01

    Full Text Available 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.

  18. The effects of sodium-glucose co-transporter 2 inhibitors in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Storgaard, Heidi; Gluud, Lise Lotte; Christensen, Mikkel

    2014-01-01

    to the knowledge regarding the beneficial and harmful effects of SGLT-2i in patients with type 2 diabetes. We plan to publish the study irrespective of the results. RESULTS: The study will be disseminated by peer-review publication and conference presentation. TRIAL REGISTRATION NUMBER: PROSPERO CRD42014008960......INTRODUCTION: Sodium-glucose co-transporter 2 inhibitors (SGLT-2i) increase urinary glucose excretion through a reduced renal glucose reabsorption. We plan to perform a systematic review of SGLT-2i for treatment of type 2 diabetes. METHODS AND ANALYSIS: A systematic review with meta......-analyses of randomised clinical trials on SGLT-2i versus placebo, other oral glucose lowering drugs or insulin for patients with type 2 diabetes will be performed. The primary end point will be the glycated haemoglobin. Secondary end points will include changes in body weight, body mass index, fasting plasma glucose...

  19. In Vitro Infection of Trypanosoma cruzi Causes Decrease in Glucose Transporter Protein-1 (GLUT1 Expression in Explants of Human Placental Villi Cultured under Normal and High Glucose Concentrations

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    Luciana Mezzano

    2012-01-01

    Full Text Available Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1 is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations.

  20. Insulin-sensitive regulation of glucose transport and GLUT4 translocation in skeletal muscle of GLUT1 transgenic mice.

    Science.gov (United States)

    Etgen, G J; Zavadoski, W J; Holman, G D; Gibbs, E M

    1999-01-01

    Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased approximately 3-8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased approximately 4-14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity approximately 2-3-fold in isolated muscles and to a much greater extent ( approximately 7-20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to approximately 50-75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 approximately 2-fold in isolated EDL and approximately 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a

  1. Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells

    OpenAIRE

    Pyla, Rajkumar; Poulose, Ninu; Jun, John Y.; Segar, Lakshman

    2013-01-01

    Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1–5) and novel GLUT isoforms (GLUT6–14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms h...

  2. Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca2+ release

    Directory of Open Access Journals (Sweden)

    Thomas E. Jensen

    2014-10-01

    Full Text Available Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm.

  3. The biological activity of Coccinia indica on glucose transporter 1 (GLUT1 promoter

    Directory of Open Access Journals (Sweden)

    Juntipa Purintrapiban

    2009-08-01

    Full Text Available Plant derivatives with purported hypoglycemic properties have been used in traditional medicine around the world. Coccinia indica (ivy gourd is used in traditional medicine to treat diabetics in many countries. C. indica is able to cause a reduction in blood glucose level and has shown hypoglycemic activity in vitro and in vivo. However, the mechanism of this effect remains unknown. In this study, we generated the pGL3-glucose transporter 1 (GLUT1 promoter to elucidate the molecular mechanism of the regulation of GLUT1 gene expression in response to a water extract of C. indica stem (CIextract. A fragment of 2.1 kb of rat GLUT1 promoter, located at -2,106 to +134, was linked to firefly luciferase. The regulating transcription was analyzed in transient expression assay after transfection and exposure of L6 myocytes with the GLUT1 promoter system and CI extract, respectively. Under normal condition (5 mM glucose, promoter activity induced by 0.15 mg CI extract was markedly increased by 5.71 fold from the basal value. CI extract was more effective than 2 mM metformin. Surprisingly, promoter activity in hyperglycemic condition (15 mM glucose induced by 0.50 mg CI was increased by 1.63 fold from the basal value. In addition, CI extract increased the 2-deoxyglucose (2-DG uptake in L6myocytes in a dose-dependent manner in both conditions, 5 mM and 15 mM glucose. GLUT1 protein was determined by Western blot analysis and the level also increased in a dose-dependent fashion. Interestingly, the activity of the -273 to +134 of GLUT1 promoter was increased by 2.12 fold from the basal value. This site is the transcription initiation site containing GC box and TATA box. These observations suggest that the hypoglycemic action of C. indica may regulate through the activation of GLUT1 promoter resulting in an increase of the GLUT1 protein expression.

  4.  The role of glucose transporter 1 (GLUT1 in the diagnosis and therapy of tumors

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    Paweł Jóźwiak

    2012-03-01

    Full Text Available  Malignant cells are known to enhance glucose metabolism, to increase glucose uptake and to inhibit the process of oxidative phosphorylation. Accelerated glycolysis is one of the biochemical characteristics of cancer cells that allow them to compensate the inefficient extraction of energy from glucose in order to continue their uncontrolled growth and proliferation. Upregulation of glucose transport across the plasma membrane is mediated by a family of facilitated glucose transporter proteins named GLUT. Overexpression of GLUTs, especially the hypoxia-responsive GLUT1, has been frequently observed in various human carcinomas. Many studies have reported a correlation between GLUT1 expression level and the grade of tumor aggressiveness, which suggests that GLUT1 expression may be of prognostic significance. Therefore, GLUT1 is a key rate-limiting factor in the transport and glucose metabolism in cancer cells. This paper presents the current state of knowledge on GLUT1 regulation as well as its utility in the diagnosis and therapy of cancers.

  5. Immunohistochemical expression of the glucose transporters Glut-1 and Glut-3 in human malignant melanomas and benign melanocytic lesions

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    Parente Paola

    2008-09-01

    Full Text Available Abstract Background Reported data indicate that cancer cells have increased rates of glucose metabolism, as determined by 18FDG-PET imaging in patients with malignancies. The results of many studies have demonstrated that the expression of glucose transporters, especially Glut-1, is increased in a variety of malignancies. This study was undertaken to assess the differential expression of Glut-1 and Glut-3 by benign and malignant melanocytic lesions. Methods Immunohistochemical staining for Glut-1 and Glut-3 was performed on paraffin-embedded tissue sections prepared from melanocytic nevi (12 cases, Spitz nevi (12 cases and primary cutaneous malignant melanomas (20 cases. Results We observed immunoreactivity for Glut-1 in all melanocytic nevi, 9 of the 12 Spitz nevi and in 9 of the 20 malignant melanomas, whereas Glut-3 was expressed in all the melanocytic lesions, both benign and malignant. Conclusion These findings indicate that the glucose transporters Glut-1 and Glut-3 play a role in the glucose metabolism of melanocytic cells. Glut-1 was present in the majority of benign nevi, whereas its expression was downregulated in 55% of malignant melanomas. Our results suggest that glucose transporter Glut-1 expression can significantly discriminate between human malignant melanoma and benign melanocytic nevi, and support the idea that additional mechanisms other than Glut-1 may contribute to glucose uptake in melanomas.

  6. The Influence of Initial Peritoneal Transport Characteristics, Inflammation, and High Glucose Exposure on Prognosis for Peritoneal Membrane Function

    Science.gov (United States)

    Fernández-Reyes, M. José; Bajo, M. Auxiliadora; Del Peso, Gloria; Ossorio, Marta; Díaz, Raquel; Carretero, Beatriz; Selgas, Rafael

    2012-01-01

    ♦ Background: Fast transport status, acquired with time on peritoneal dialysis (PD), is a pathology induced by peritoneal exposure to bioincompatible solutions. Fast transport has important clinical consequences and should be prevented. ♦ Objective: We analyzed the repercussions of initial peritoneal transport characteristics on the prognosis for peritoneal membrane function, and also whether the influence of peritonitis and high exposure to glucose are different according to the initial peritoneal transport characteristics or the moment when such events occur. ♦ Methods: The study included 275 peritoneal dialysis patients with at least 2 peritoneal function studies (at baseline and 1 year). Peritoneal kinetic studies were performed at baseline and annually. Those studies consist of a 4-hour dwell with glucose (1.5% during 1981 - 1990, and 2.27% during 1991 - 2002) to calculate the peritoneal mass transfer coefficients of urea and creatinine (milliliters per minute) using a previously described mathematical model. ♦ Results: Membrane prognosis and technique survival were independent of baseline transport characteristics. Fast transport and ultrafiltration (UF) failure are reversible conditions, provided that peritonitis and high glucose exposure are avoided during the early dialysis period. The first year on PD is a main determining factor for the membrane’s future, and the mass transfer coefficient of creatinine at year 1 is the best functional predictor of future PD history. After 5 years on dialysis, permeability frequently increases, and UF decreases. Icodextrin is associated with peritoneal protection. ♦ Conclusions: Peritoneal membrane prognosis is independent of baseline transport characteristics. Intrinsic fast transport and low UF are reversible conditions when peritonitis and high glucose exposure are avoided during the early dialysis period. Icodextrin helps in glucose avoidance and is associated with peritoneal protection. PMID:22473036

  7. The sentrin-conjugating enzyme mUbc9 interacts with GLUT4 and GLUT1 glucose transporters and regulates transporter levels in skeletal muscle cells

    Science.gov (United States)

    Giorgino, Francesco; de Robertis, Ottilia; Laviola, Luigi; Montrone, Carmela; Perrini, Sebastio; McCowen, Karen C.; Smith, Robert J.

    2000-01-01

    Glucose transport in insulin-regulated tissues is mediated by the GLUT4 and GLUT1 transporters. Using the yeast two-hybrid system, we have cloned the sentrin-conjugating enzyme mUbc9 as a protein that interacts with the GLUT4 COOH-terminal intracellular domain. The mUbc9 enzyme was found to bind directly to GLUT4 and GLUT1 through an 11-aa sequence common to the two transporters and to modify both transporters covalently by conjugation with the mUbc9 substrate, sentrin. Overexpression of mUbc9 in L6 skeletal muscle cells decreased GLUT1 transporter abundance 65%, resulting in decreased basal glucose transport. By contrast, mUbc9 overexpression increased GLUT4 abundance 8-fold, leading to enhanced transport stimulation by insulin. A dominant-negative mUbc9 mutant lacking catalytic activity had effects opposite to those of wild-type mUbc9. The regulation of GLUT4 and GLUT1 was specific, as evidenced by an absence of mUbc9 interaction with or regulation of the GLUT3 transporter isoform in L6 skeletal muscle cells. The mUbc9 sentrin-conjugating enzyme represents a novel regulator of GLUT1 and GLUT4 protein levels with potential importance as a determinant of basal and insulin-stimulated glucose uptake in normal and pathophysiological states. PMID:10655495

  8. Intestinal D-glucose transport and membrane fluidity along crypt-villus axis of streptozocin-induced diabetic rats.

    Science.gov (United States)

    Dudeja, P K; Wali, R K; Klitzke, A; Brasitus, T A

    1990-10-01

    Diabetes was induced in male Lewis rats by a single injection of streptozocin (50 mg/kg body wt ip). After 10-14 days, diabetic and age- and sex-matched control animals were killed, and their proximal small intestines were removed. Villus-tip, mid-villus, and lower-villus enterocytes were harvested from each group with a method that combined divalent cation chelation with mild mechanical dissociation. These fractions were used as starting material to prepare brush-border membrane vesicles. Preparations from each of these fractions were then analyzed and compared with respect to their Na(+)-gradient-dependent and Na(+)-independent D-glucose transport, lipid fluidity, and lipid composition. The results of these experiments demonstrated that 1) maximum rates of Na(+)-gradient-dependent D-glucose transport (Vmax) were greatest in membrane vesicles prepared from mature cells (villus tip and mid villus) of control rats; 2) the glucose concentration producing half-maximal rates of transport (Km), however, was significantly lower in lower-villus membrane vesicles of control rats, suggesting that a distinct glucose transporter existed in the membranes of these relatively immature enterocytes; 3) Na(+)-gradient-dependent, but not Na(+)-independent, D-glucose uptake was greater in diabetic membrane vesicles prepared from mid-villus and lower-villus fractions but not in vesicles prepared from villus-tip cells; and 4) no obvious relationship between alterations in membrane lipid fluidity and enhanced uptake of Na(+)-gradient-dependent D-glucose by these transporter(s) could be established in this experimental model of acute diabetes mellitus.

  9. Phylogenetic analysis and tissue distribution of elasmobranch glucose transporters and their response to feeding

    Directory of Open Access Journals (Sweden)

    Courtney A. Deck

    2016-03-01

    Full Text Available 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.

  10. Conditioning causes an increase in glucose transporter-4 levels in mononuclear cells in sled dogs.

    Science.gov (United States)

    Schnurr, Theresia M; Reynolds, Arleigh J; Gustafson, Sally J; Duffy, Lawrence K; Dunlap, Kriya L

    2014-10-01

    This study was designed to investigate the effects of physical conditioning on the expression of the insulin sensitive glucose transporter-4 protein (GLUT4) on mononuclear cells and HOMA-IR levels in dogs and compared to results reported in human skeletal muscle and the skeletal muscle of rodent models. Blood was sampled from conditioned dogs (n = 8) and sedentary dogs (n = 8). The conditioned dogs were exercised four months prior the experiment and were following a uniform training protocol, whereas the sedentary dogs were not. GLUT4 expression in mononuclear cells and plasma insulin levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA). Blood glucose levels were determined using blood plasma. HOMA-IR was calculated using plasma insulin and blood glucose levels using the linear approximation formula. Our results indicate that the state of conditioning had a significant effect on the GLUT4 expression at the surface of mononuclear cells. HOMA-IR was also affected by conditioning in dogs. GLUT4 levels in mononuclear cells of sled dogs were inversely correlated with the homeostasis model assessment of insulin sensitivity. This study demonstrates that conditioning increases GLUT4 levels in mononuclear cells of sled dogs as it has been previously reported in skeletal muscle. Our results support the potential of white blood cells as a proxy tissue for studying insulin signaling and may lead to development of a minimally invasive and direct marker of insulin resistance. This may be the first report of GLUT4 in mononuclear cells in response to exercise and measured with ELISA.

  11. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

    Science.gov (United States)

    Ryndak, Michelle B; Wang, Shuishu; Smith, Issar; Rodriguez, G Marcela

    2010-02-01

    Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

  12. Exercise-induced increase in glucose transport, GLUT-4, and VAMP-2 in plasma membrane from human muscle

    DEFF Research Database (Denmark)

    Kristiansen, S; Hargreaves, Mark; Richter, Erik

    1996-01-01

    contractions may induce trafficking of GLUT-4-containing vesicles via a mechanism similar to neurotransmitter release. Our results demonstrate for the first time exercise-induced translocation of GLUT-4 and VAMP-2 to the plasma membrane of human muscle and increased sarcolemmal glucose transport.......A major effect of muscle contractions is an increase in sarcolemmal glucose transport. We have used a recently developed technique to produce sarcolemmal giant vesicles from human muscle biopsy samples obtained before and after exercise. Six men exercised for 10 min at 50% maximal O2 uptake (Vo2max...

  13. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight

    Directory of Open Access Journals (Sweden)

    Awadhesh Kumar Singh

    2015-01-01

    Full Text Available 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.

  14. A potential role for glucose transporters in the evolution of human brain size.

    Science.gov (United States)

    Fedrigo, Olivier; Pfefferle, Adam D; Babbitt, Courtney C; Haygood, Ralph; Wall, Christine E; Wray, Gregory A

    2011-01-01

    Differences in cognitive abilities and the relatively large brain are among the most striking differences between humans and their closest primate relatives. The energy trade-off hypothesis predicts that a major shift in energy allocation among tissues occurred during human origins in order to support the remarkable expansion of a metabolically expensive brain. However, the molecular basis of this adaptive scenario is unknown. Two glucose transporters (SLC2A1 and SLC2A4) are promising candidates and present intriguing mutations in humans, resulting, respectively, in microcephaly and disruptions in whole-body glucose homeostasis. We compared SLC2A1 and SLC2A4 expression between humans, chimpanzees and macaques, and found compensatory and biologically significant expression changes on the human lineage within cerebral cortex and skeletal muscle, consistent with mediating an energy trade-off. We also show that these two genes are likely to have undergone adaptation and participated in the development and maintenance of a larger brain in the human lineage by modulating brain and skeletal muscle energy allocation. We found that these two genes show human-specific signatures of positive selection on known regulatory elements within their 5'-untranslated region, suggesting an adaptation of their regulation during human origins. This study represents the first case where adaptive, functional and genetic lines of evidence implicate specific genes in the evolution of human brain size. Copyright © 2011 S. Karger AG, Basel.

  15. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight

    Science.gov (United States)

    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

  16. Small Molecule PET Tracers for Transporter Imaging.

    Science.gov (United States)

    Kilbourn, Michael R

    2017-09-01

    As the field of PET has expanded and an ever-increasing number and variety of compounds have been radiolabeled as potential in vivo tracers of biochemistry, transporters have become important primary targets or facilitators of radiotracer uptake and distribution. A transporter can be the primary target through the development of a specific high-affinity radioligand: examples are the multiple high-affinity radioligands for the neuronal membrane neurotransmitter or vesicular transporters, used to image nerve terminals in the brain. The goal of a radiotracer might be to study the function of a transporter through the use of a radiolabeled substrate, such as the application of 3-O-[(11)C]methyl]glucose to measure rates of glucose transport through the blood-brain barrier. In many cases, transporters are required for radiotracer distributions, but the targeted biochemistries might be unrelated: an example is the use of 2-deoxy-2-[(18)F]FDG for imaging glucose metabolism, where initial passage of the radiotracer through cell membranes requires the action of specific glucose transporters. Finally, there are transporters such as p-glycoprotein that function to extrude small molecules from tissues, and can effectively work against successful uptake of radiotracers. The diversity of structures and functions of transporters, their importance in human health and disease, and their role in therapeutic drug disposition suggest that in vivo imaging of transporter location and function will continue to be a point of emphasis in PET radiopharmaceutical development. In this review, the variety of transporters and their importance for in vivo PET radiotracer development and application are discussed. Transporters have thus joined the other major protein targets such as G-protein coupled receptors, ligand-gated ion channels, enzymes, and aggregated proteins as of high interest for understanding human health and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Glucose transporter 1 (GLUT1) expression is associated with intestinal type of gastric carcinoma.

    Science.gov (United States)

    Kim, W. S.; Kim, Y. Y.; Jang, S. J.; Kimm, K.; Jung, M. H.

    2000-01-01

    Increased expression of glucose transporter1 (GLUT1) has been reported in many human cancers. We hypothesized that the degree of GLUT1 might provide a useful biological information in gastric adenocarcinoma. RT-PCR and immunostaining were used to analyze GLUT1 expression in gastric cancer. RT-PCR showed GLUT1 expression was not largely detected in normal gastric tissue but was detected in cancerous gastric tissue of counterpart. By immunohistochemistry, GLUT1 protein was absent in normal gastric epithelium and intestinal metaplasia. 11 of 65 patients with gastric adenocarcinoma had specific GLUT1 immunostaining in a plasma membrane pattern with varied intensities. GLUT1 protein did not show any significant correlation with tumor stage and nodal metastasis (p>0.05 by Mann-Whitney test). However, the positive immunostaining for GLUT1 is associated with intestinal differentiation (p=0.003). Our results suggest that GLUT1 protein is associated with intestinal type of gastric cancer. PMID:10983690

  18. Unusual phenotype of glucose transport protein type 1 deficiency syndrome: A case report and literature review

    Directory of Open Access Journals (Sweden)

    Annio Posar

    2014-01-01

    Full Text Available 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.

  19. In vivo measurements of brain glucose transport using the reversible Michaelis-Menten model and simultaneous measurements of cerebral blood flow changes during hypoglycemia.

    Science.gov (United States)

    Choi, I Y; Lee, S P; Kim, S G; Gruetter, R

    2001-06-01

    Glucose is the major substrate that sustains normal brain function. When the brain glucose concentration approaches zero, glucose transport across the blood-brain barrier becomes rate limiting for metabolism during, for example, increased metabolic activity and hypoglycemia. Steady-state brain glucose concentrations in alpha-chloralose anesthetized rats were measured noninvasively as a function of plasma glucose. The relation between brain and plasma glucose was linear at 4.5 to 30 mmol/L plasma glucose, which is consistent with the reversible Michaelis-Menten model. When the model was fitted to the brain glucose measurements, the apparent Michaelis-Menten constant, Kt, was 3.3 +/- 1.0 mmol/L, and the ratio of the maximal transport rate relative to CMRglc, Tmax/CMRglc, was 2.7 +/- 0.1. This Kt is comparable to the authors' previous human data, suggesting that glucose transport kinetics in humans and rats are similar. Cerebral blood flow (CBF) was simultaneously assessed and constant above 2 mmol/L plasma glucose at 73 +/- 6 mL 100 g(-1) min(-1). Extrapolation of the reversible Michaelis-Menten model to hypoglycemia correctly predicted the plasma glucose concentration (2.1 +/- 0.6 mmol/L) at which brain glucose concentrations approached zero. At this point, CBF increased sharply by 57% +/- 22%, suggesting that brain glucose concentration is the signal that triggers defense mechanisms aimed at improving glucose delivery to the brain during hypoglycemia.

  20. Development of a glucose binding protein biosensor

    Science.gov (United States)

    Dweik, M.; Milanick, M.; Grant, S.

    2007-09-01

    Glucose binding protein (GBP) is a monomeric periplasmic protein. It is synthesized in the cytoplasm of Escherichia coli which functions as a receptor for transport D-glucose. GBP binds glucose with high affinity. The binding mechanism is based on a hinge motion due to the protein conformational change. This change was utilized as an optical sensing mechanism by applying Fluorescence Resonance Energy Transfer (FRET). The wild-type GBP lacks cysteine in its structure, but by introducing a single cysteine at a specific site by site-directed mutagenesis, this ensured single-label attachment at specific sites with a fluorescent probe. The other sites were amino sites, which were labeled with second fluorophore. The near IR FRET pair, Alexa Fluor 680 (AF680) and Alexa Fluor 750(AF750), was utilized. The AF680 targeted the amine sites, which was the donor fluorophore, while the AF750 labeled the single cysteine site, which was the acceptor fluorophore. The sensing system strategy was based on the fluorescence changes of the probe as the protein undergoes a structural change upon binding. This biosensor had the ability to detect down to 10 uM concentrations of glucose. Next the probes were uploaded into red blood cells via hypo osmotic dialysis. The sensor responded to glucose while encapsulated with the red cells. These results showed the feasibility of an intracellular glucose biosensor.

  1. Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

    Science.gov (United States)

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V; Bevan, Michael W

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

  2. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Milne, J.L.; Coukell, M.B. (York Univ., North York, Ontario (Canada))

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  3. Muscle Mitochondrial ATP Synthesis and Glucose Transport/Phosphorylation in Type 2 Diabetes

    OpenAIRE

    Julia Szendroedi; Schmid, Albrecht I; Marek Chmelik; Christian Toth; Attila Brehm; Martin Krssak; Peter Nowotny; Michael Wolzt; Werner Waldhausl; Michael Roden

    2007-01-01

    Editors' Summary Background. Diabetes mellitus is an increasingly common chronic disease characterized by high blood sugar (glucose) levels. In normal individuals, blood sugar levels are maintained by the hormone insulin. Insulin is released by the pancreas when blood glucose levels rise after eating (glucose is produced by the digestion of food) and “instructs” insulin-responsive muscle and fat cells to take up glucose from the bloodstream. The cells then use glucose as a fuel or convert it ...

  4. Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate

    Directory of Open Access Journals (Sweden)

    Timothy eHackmann

    2015-06-01

    Full Text Available From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio spp., we have re-evaluated the contribution of electron transport phosphorylation 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 electron transport phosphorylation. 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 (NADred during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH+ or ΔμNa+ from oxidation of reduced Fd and reduction of oxidized NAD (NADox. Further, 47 genomes possessed all 6 subunits of the Ech, which generates ΔμH+ from oxidation of reduced Fd (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 3 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.

  5. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    Science.gov (United States)

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  6. Uptake and phloem transport of glucose-fipronil conjugate in Ricinus communis involve a carrier-mediated mechanism.

    Science.gov (United States)

    Wu, Han-Xiang; Yang, Wen; Zhang, Zhi-Xiang; Huang, Ting; Yao, Guang-Kai; Xu, Han-Hong

    2012-06-20

    Some compounds containing glucose are absorbed via the monosaccharide transporters of the plasma membrane. A glucose-fipronil conjugate, N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-d-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF), has been synthesized in our previous work. GTF exhibits moderate phloem mobility in Ricinus communis. In the current paper, we demonstrate that the uptake of GTF by Ricinus seedling cotyledon discs is partly mediated by an active carrier system (K(m)1 = 0.17 mM; V(max)1 = 2.2 nmol cm(-2) h(-1)). Four compounds [d-glucose, sucrose, phloridzin, and carbonyl cyanide m-chlorophenylhydrazone (CCCP)] were examined for their effect on GTF uptake. Phloridzin as well as CCCP markedly inhibit GTF uptake, and d-glucose weakly competes with it. The phloem transport of GTF in Ricinus seedlings is found to involve an active carrier-mediated mechanism that effectively contributes to the GTF phloem loading. The results prove that adding a glucose core is a reasonable and feasible approach to confer phloem mobility to fipronil by utilizing plant monosaccharide transporters.

  7. Zinc finger protein 407 (ZFP407) regulates insulin-stimulated glucose uptake and glucose transporter 4 (Glut4) mRNA.

    Science.gov (United States)

    Buchner, David A; Charrier, Alyssa; Srinivasan, Ethan; Wang, Li; Paulsen, Michelle T; Ljungman, Mats; Bridges, Dave; Saltiel, Alan R

    2015-03-06

    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.

  8. Sodium-glucose co-transporter 2 (SGLT2 inhibitors: a growing class of anti-diabetic agents

    Directory of Open Access Journals (Sweden)

    Eva M Vivian

    2014-12-01

    Full Text Available 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.

  9. Fat gain with physical detraining is correlated with increased glucose transport and oxidation in periepididymal white adipose tissue in rats

    Energy Technology Data Exchange (ETDEWEB)

    Sertié, R.A.L.; Andreotti, S. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Proença, A.R.G. [Laboratório de Biotecnologia, Faculdade de Ciências Aplicadas, Universidade Estadual de Campinas, Limeira, SP (Brazil); Campaña, A.B.; Lima, F.B. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2015-05-26

    As it is a common observation that obesity tends to occur after discontinuation of exercise, we investigated how white adipocytes isolated from the periepididymal fat of animals with interrupted physical training transport and oxidize glucose, and whether these adaptations support the weight regain seen after 4 weeks of physical detraining. Male Wistar rats (45 days old, weighing 200 g) were divided into two groups (n=10): group D (detrained), trained for 8 weeks and detrained for 4 weeks; and group S (sedentary). The physical exercise was carried out on a treadmill for 60 min/day, 5 days/week for 8 weeks, at 50-60% of the maximum running capacity. After the training protocol, adipocytes isolated from the periepididymal adipose tissue were submitted to glucose uptake and oxidation tests. Adipocytes from detrained animals increased their glucose uptake capacity by 18.5% compared with those from sedentary animals (P<0.05). The same cells also showed a greater glucose oxidation capacity in response to insulin stimulation (34.55%) compared with those from the S group (P<0.05). We hypothesize that, owing to the more intense glucose entrance into adipose cells from detrained rats, more substrate became available for triacylglycerol synthesis. Furthermore, this increased glucose oxidation rate allowed an increase in energy supply for triacylglycerol synthesis. Thus, physical detraining might play a role as a possible obesogenic factor for increasing glucose uptake and oxidation by adipocytes.

  10. Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.

    Science.gov (United States)

    el-Kebbi, I M; Roser, S; Pollet, R J; Cushman, S W; Wilson, C M

    1994-01-01

    We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA. PMID:8037688

  11. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites.

    Science.gov (United States)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina; Vogensen, Stine B; Lehel, Szabolcs; Thiesen, Louise; Bundgaard, Christoffer; Clausen, Rasmus P; Knudsen, Gitte M; Herth, Matthias M; Wellendorph, Petrine; Frølund, Bente

    2017-01-18

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA.

  12. Sertraline and its metabolite desmethylsertraline, but not bupropion or its three major metabolites, have high affinity for P-glycoprotein.

    Science.gov (United States)

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-02-01

    The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood-brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertraline, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic P(i) in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis-Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The V(max)/K(m) values of sertraline (1.6 min(-1) x 10(-3)) and desmethylsertraline (1.4 min(-1) x 10(-3)) were comparable with that of verapamil (1.7 min(-1) x 10(-3)). Bupropion and its three metabolites showed very weak affinity for P-gp, with V(max)/K(m) values lower than 0.01 min(-1) x 10(-3). The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug-drug interactions among antidepressants.

  13. Exercise-induced increase in glucose transport, GLUT-4, and VAMP-2 in plasma membrane from human muscle

    DEFF Research Database (Denmark)

    Kristiansen, S; Hargreaves, Mark; Richter, Erik

    1996-01-01

    ) and then to fatigue at 100% Vo2max (5.7 +/- 0.2 min). Vesicle glucose transport at 5 mM increased from 3.3 +/- 0.6 pmol.microgram-1.min-1 at rest to 6.6 +/- 1.0 pmol.microgram-1.min-1 at fatigue (mean +/- SE, n = 6, P increase in glucose transport was associated with a 1.6-fold increase in vesicle GLUT......A major effect of muscle contractions is an increase in sarcolemmal glucose transport. We have used a recently developed technique to produce sarcolemmal giant vesicles from human muscle biopsy samples obtained before and after exercise. Six men exercised for 10 min at 50% maximal O2 uptake (Vo2max......-4 protein content. Glucose transport normalized to GLUT-4 protein content also increased with exercise, suggesting increased intrinsic activity of GLUT-4. Furthermore, exercise resulted in a 1.4-fold increase in sarcolemmal vesicle-associated membrane protein (VAMP-2) content, suggesting that muscle...

  14. In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

    Science.gov (United States)

    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

    2015-06-05

    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.

  15. 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.

    Science.gov (United States)

    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.

  16. 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

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2014-01-01

    Full Text Available This study was to investigate the antidiabetic and antihyperlipidemic effects of (E-3-[3, 4-dihydroxyphenyl-1-(piperidin-1-ylprop-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.

  17. Effects of acute hyperinsulinemia on insulin signal transduction and glucose transporters in ovine fetal skeletal muscle.

    Science.gov (United States)

    Anderson, Marianne S; Thamotharan, M; Kao, Doris; Devaskar, Sherin U; Qiao, Liping; Friedman, Jacob E; Hay, William W

    2005-02-01

    To test the effects of acute fetal hyperinsulinemia on the pattern and time course of insulin signaling in ovine fetal skeletal muscle, we measured selected signal transduction proteins in the mitogenic, protein synthetic, and metabolic pathways in the skeletal muscle of normally growing fetal sheep in utero. In experiment 1, 4-h hyperinsulinemic-euglycemic clamps were conducted in anesthetized twin fetuses to produce selective fetal hyperinsulinemia-euglycemia in one twin and euinsulinemia-euglycemia in the other. Serial skeletal muscle biopsies were taken from each fetus during the clamp and assayed by Western blot for selected insulin signal transduction proteins. Tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1, and the p85 subunit of phosphatidylinositol 3-kinase doubled at 30 min and gradually returned to control values by 240 min. Phosphorylation of extracellular signal-regulated kinase 1,2 was increased fivefold through 120 min of insulin infusion and decreased to control concentration by 240 min. Protein kinase B phosphorylation doubled at 30 min and remained elevated throughout the study. Phosphorylation of p70 S6K increased fourfold at 30, 60, and 120 min. In the second experiment, a separate group of nonanesthetized singleton fetuses was clamped to intermediate and high hyperinsulinemic-euglycemic conditions for 1 h. GLUT4 increased fourfold in the plasma membrane at 1 h, and hindlimb glucose uptake increased significantly at the higher insulin concentration. These data demonstrate that an acute increase in fetal plasma insulin concentration stimulates a unique pattern of insulin signal transduction proteins in intact skeletal muscle, thereby increasing pathways for mRNA translation, glucose transport, and cell growth.

  18. Ginsenosides, ingredients of the root of Panax ginseng, are not substrates but inhibitors of sodium-glucose transporter 1.

    Science.gov (United States)

    Gao, Shengli; Kushida, Hirotaka; Makino, Toshiaki

    2017-01-01

    Recent pharmacokinetic studies have revealed that ginsenosides, the major ingredients of ginseng (the roots of Panax ginseng), are present in the plasma collected from subjects receiving ginseng, and speculated that ginsenosides might be actively transported via glucose transporters. We evaluated whether ginsenosides Rb1 and Rg1, and their metabolites from enteric bacteria act as substrates of sodium-glucose cotransporter (SGLT) 1, the major glucose transporter expressed on the apical side of intestinal epithelial cells. First, we evaluated the competing effects of ginseng extract and ginsenosides on the uptake of [(14)C]methyl-glucose, a substrate of SGLT1, by SGLT1-overexpressing HEK293 cells. A boiling water extract of ginseng inhibited SGLT1 in a concentration-dependent manner with an IC50 value of 0.85 mg/ml. By activity-guided fractionation, we determined that the fraction containing ginsenosides displayed an inhibitory effect on SGLT1. Of the ginsenosides evaluated, protopanaxatriol-type ginsenosides were not found to inhibit SGLT1, whereas protopanaxadiol-type ginsenosides, including ginsenosides Rd, Rg3, Rh2, F2 and compound K, exhibited significant inhibitory effects on SGLT1, with ginsenoside F2 having the highest activity with an IC50 value of 23.0 µM. Next, we measured the uptake of ginsenoside F2 and compound K into Caco-2 cells, a cell line frequently used to evaluate the intestinal absorption of drugs. The uptake of ginsenoside F2 and compound K into Caco-2 cells was not competitively inhibited by glucose. Furthermore, the uptake of ginsenoside F2 and compound K into SGLT1-overexpressing HEK293 cells was not significantly higher than into mock cells. Ginsenoside F2 and compound K did not appear to be substrates of SGLT1, although these compounds could inhibit SGLT1. Ginsenosides might be absorbed by passive diffusion through the intestinal membrane or actively transported via unknown transporters other than SGLT1.

  19. A new class of fluorescent boronic acids that have extraordinarily high affinities for diols in aqueous solution at physiological pH.

    Science.gov (United States)

    Cheng, Yunfeng; Ni, Nanting; Yang, Wenqian; Wang, Binghe

    2010-12-03

    The boronic acid group is an important recognition moiety for sensor design. Herein, we report a series of isoquinolinylboronic acids that have extraordinarily high affinities for diol-containing compounds at physiological pH. In addition, 5- and 8-isoquinolinylboronic acids also showed fairly high binding affinities towards D-glucose (K(a)=42 and 46 M(-1), respectively). For the first time, weak but encouraging binding of cis-cyclohexanediol was found for these boronic acids. Such binding was coupled with significant fluorescence changes. Furthermore, 4- and 6-isoquinolinylboronic acids also showed the ability to complex methyl α-D-glucopyranose (K(a)=3 and 2 M(-1), respectively).

  20. Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

    Directory of Open Access Journals (Sweden)

    Mirick Gary

    2009-04-01

    Full Text Available Abstract Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL (DvLPBaPPP2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a

  1. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  2. Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter.

    Science.gov (United States)

    Inukai, K; Asano, T; Katagiri, H; Anai, M; Funaki, M; Ishihara, H; Tsukuda, K; Kikuchi, M; Yazaki, Y; Oka, Y

    1994-01-01

    A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site. Images Figure 1 Figure 2 PMID:8092986

  3. Insulin Resistance in Nondiabetic Peritoneal Dialysis Patients: Associations with Body Composition, Peritoneal Transport, and Peritoneal Glucose Absorption.

    Science.gov (United States)

    Bernardo, Ana Paula; Oliveira, Jose C; Santos, Olivia; Carvalho, Maria J; Cabrita, Antonio; Rodrigues, Anabela

    2015-12-07

    Insulin resistance has been associated with cardiovascular disease in peritoneal dialysis patients. Few studies have addressed the impact of fast transport status or dialysis prescription on insulin resistance. The aim of this study was to test whether insulin resistance is associated with obesity parameters, peritoneal transport rate, and glucose absorption. Insulin resistance was evaluated with homeostasis model assessment method (HOMA-IR), additionally corrected by adiponectin (HOMA-AD). Enrolled patients were prevalent nondiabetics attending at Santo António Hospital Peritoneal Dialysis Unit, who were free of hospitalization or infectious events in the previous 3 months (51 patients aged 50.4 ± 15.9 years, 59% women). Leptin, adiponectin, insulin-like growth factor-binding protein 1 (IGFBP-1), and daily glucose absorption were also measured. Lean tissue index, fat tissue index (FTI), and relative fat mass (rel.FM) were assessed using multifrequency bioimpedance. Patients were categorized according to dialysate to plasma creatinine ratio at 4 hours, 3.86% peritoneal equilibration test, and obesity parameters. Obesity was present in 49% of patients according to rel.FM. HOMA-IR correlated better with FTI than with body mass index. Significant correlations were found in obese, but not in nonobese patients, between HOMA-IR and leptin, leptin/adiponectin ratio (LAR), and IGFBP-1. HOMA-IR correlated with HOMA-AD, but did not correlate with glucose absorption or transport rate. There were no significant differences in insulin resistance indices, glucose absorption, and body composition parameters between fast and nonfast transporters. A total of 18 patients (35.3%) who had insulin resistance presented with higher LAR and rel.FM (7.3 [12.3, interquartile range] versus 0.7 [1.4, interquartile range], Pinsulin resistance. FTI and LAR were independent correlates of HOMA-IR in multivariate analysis adjusted for glucose absorption and small-solute transport (r=0

  4. High-affinity olfactory receptor for the death-associated odor cadaverine

    OpenAIRE

    2013-01-01

    Cadaverine and putrescine, two diamines emanating from decaying flesh, are strongly repulsive odors to humans but serve as innate attractive or social cues in other species. Here we show that zebrafish, a vertebrate model system, exhibit powerful and innate avoidance behavior to both diamines, and identify a high-affinity olfactory receptor for cadaverine.

  5. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; Ruiter, de M.V.; Cornelissen, J.J.L.M.; Jonkheijm, P.

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  6. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue;

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects...

  7. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective radiol

  8. N-Oxide analogs of WAY-100635 : new high affinity 5-HT1A receptor antagonists

    NARCIS (Netherlands)

    Marchais-Oberwinkler, S; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, HV

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective radiol

  9. Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

    NARCIS (Netherlands)

    Sankaran, S.; de Ruiter, Mark Vincent; Cornelissen, Jeroen Johannes Lambertus Maria; Jonkheijm, Pascal

    2015-01-01

    Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly

  10. High affinity, bioavailable 3-amino-1,4-benzodiazepine-based gamma-secretase inhibitors.

    Science.gov (United States)

    Owens, Andrew P; Nadin, Alan; Talbot, Adam C; Clarke, Earl E; Harrison, Timothy; Lewis, Huw D; Reilly, Michael; Wrigley, Jonathan D J; Castro, José L

    2003-11-17

    In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.

  11. The dual aptamer approach: rational design of a high-affinity FAD aptamer.

    Science.gov (United States)

    Merkle, T; Holder, I T; Hartig, J S

    2016-01-14

    A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP- and FMN-aptamers. Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.

  12. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K...

  13. Effect of training and detraining on skeletal muscle glucose transporter (GLUT4) content in rats.

    Science.gov (United States)

    Neufer, P D; Shinebarger, M H; Dohm, G L

    1992-09-01

    The aim of the present study was to examine the effects of treadmill exercise training and detraining on the skeletal muscle fiber type specific expression of the insulin-regulated glucose transporter protein (GLUT4) in rats. GLUT4 protein content was determined by Western and dot-blot analysis, using a polyclonal antibody raised against the carboxy-terminal peptide. Rats were sacrificed 24 h after the last training session. There were no significant changes in muscle GLUT4 after 1 day or 1 week of training. Six weeks of training increased GLUT4 protein content 1.4- to 1.7-fold (p < 0.05) over controls in the soleus and red vastus lateralis, whereas no significant change was evident in the white vastus lateralis muscle. GLUT4 protein content in both soleus and red vastus lateralis muscle returned to near control values after 7 days of detraining. Similar to GLUT4, citrate synthase activity showed no change after 1 day or 1 week of training, increased 1.8-fold over controls after 6 weeks of training, but returned to control values after 7 days detraining. These findings demonstrate that muscle GLUT4 protein is increased in rats with as little as 6 weeks of treadmill exercise training but that the adaptation is lost within 1 week of detraining. It is suggested that expression of the GLUT4 protein is coordinated with the well-documented adaptations in oxidative enzyme activity with endurance training and detraining.

  14. Apigenin inhibits the proliferation of adenoid cystic carcinoma via suppression of glucose transporter-1

    Science.gov (United States)

    FANG, JIN; BAO, YANG-YANG; ZHOU, SHUI-HONG; FAN, JUN

    2015-01-01

    Apigenin is a natural phyto-oestrogen flavonoid, which exerts various biological effects, including anti-oxidative, anti-inflammatory and anticancer activities. In addition, apigenin has recently been reported to target hypoxic markers; however, there are currently no studies regarding the association between apigenin and glucose transporter-1 (GLUT-1) in adenoid cystic carcinoma (ACC). The present study investigated whether apigenin inhibits the proliferation of ACC cells or suppresses the expression of GLUT-1 in ACC cells. The results of the present study demonstrated that apigenin inhibits ACC-2 cell growth in a dose- and time-dependent manner. Treatment with apigenin also induced apoptosis and G2/M-phase arrest in a dose- and time-dependent manner. Corresponding with the above results, the expression levels of GLUT-1 were significantly decreased following treatment in a dose- and time-dependent manner. These results suggest that the inhibition of ACC-2 cell growth by apigenin may be due to the decreased expression of GLUT-1. PMID:26300442

  15. Impairment of tight junctions and glucose transport in endothelial cells of human cerebral cavernous malformations.

    Science.gov (United States)

    Schneider, Hannah; Errede, Mariella; Ulrich, Nils H; Virgintino, Daniela; Frei, Karl; Bertalanffy, Helmut

    2011-06-01

    Cerebral cavernous malformations (CCMs) often cause hemorrhages that can result in severe clinical manifestations, including hemiparesis and seizures. The underlying mechanisms of the aggressive behavior of CCMs are undetermined to date, but alterations of vascular matrix components may be involved. We compared the localization of the tight junction proteins (TJPs) in 12 CCM specimens and the expression of glucose transporter 1 (GLUT-1), which is sensitive to alterations in TJP levels, in 5 CCM specimens with those in 5 control temporal lobectomy specimens without CCM by immunofluorescence microscopy. The TJPs occludin, claudin-5, and zonula occludens ZO-1 were downregulated at intercellular contact sites and partly redistributed within the surrounding tissue in the CCM samples; there was also a marked reduction of GLUT-1 immunoreactivity compared with that in control specimens. Corresponding analysis using quantitative real-time reverse transcription polymerase chain reaction on 8 CCM and 8 control specimens revealed significant downregulation of mRNA expression of occludin, claudin-5, ZO-1, and GLUT-1. The altered expression and localization of the TJPs at interendothelial contact sites accompanied by a reduction of GLUT-1 expression in dilated CCM microvessels likely affect vascular matrix stability and may contribute to hemorrhages of CCMs.

  16. Relation Between Fluorodeoxyglucose Uptake and Glucose Transporter 1 Expression in Gastric signet Ring Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Bong Hoi; Song, Hee Sung; An, Young Sil; Han, Sang Uk; Kim, Jang Hee; Yoon, Joon kee [Ajou Univ. School of medicine, Suwon (Korea, Republic of)

    2011-03-15

    Gastic signet ring cell carcinoma (GSRC) is known to have low fluorodeoxyglucose (FDG) uptake. The aim of the study was to investigate the relation between FDG uptake and glucose transporter (GLUT) 1 expression and clinicopathologic parameters in cases of GSRC. Forty patients (28 men, mean age 54{+-}12 years) with histologically confirmed GSRC who underwent pre operative [{sup 18}F]FDG PET/CT were enrolled. Maximum standardized uptake values (SUVmax) were compared with clinicopathologic parameters and GLUT 1 expression. Cases were divided based on GLUT 1 expression in tumor tissues into a membranous group (n=17) and a cytoplasmic group (n=23). Mean SUVmax was significantly higher in the membranous group than in the cytoplasmic group (6.06{+-}2.79 vs, 3.67{+-}1.54, P=0.03). Gastric wall invasion, depth of invasion, extent of LN metastasis, overall stage, and tumor size were found to be related to SUVmax. On the other hand, age, sex, and the presence of distant metastasis were not related to SUVmax. Multiveriate analysis revealed that membranous GLCT 1 expression and the extent of LN metastasis independently predicted high FDG uptake. This study demonstrates that high FDG uptake is mediated by membranous GLUT 1 expression in GSRC.

  17. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells

    Science.gov (United States)

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B.; Bischoff, Joyce

    2014-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin and VEGFR2, they converted to a mesenchymal phenotype after three weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to re-differentiate into endothelial cells, or into pericyte/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in infantile hemangioma. PMID:25187207

  18. Genetic changes during a laboratory adaptive evolution process that allowed fast growth in glucose to an Escherichia coli strain lacking the major glucose transport system

    Directory of Open Access Journals (Sweden)

    Aguilar César

    2012-08-01

    Full Text Available Abstract Background Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS, which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400% increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways. Results Whole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna and in other putative regulatory loci (yjjU, rssA and ypdA. In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented. Conclusions The deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased

  19. Role of the water extract from Coccinia indica stem on the stimulation of glucose transport in L8 myotubes

    Directory of Open Access Journals (Sweden)

    Chaweewan Jansakul

    2006-11-01

    Full Text Available Hypoglycemic effect of Coccinia indica used for treatment of diabetes in traditional remedies has known to relate with increased transport of glucose into peripheral tissues. However, the cellular mechanisms for this effect remain unclear. This present study reports that the water extract (WE of C. indica stem exhibited a dose-dependent induction of 2-deoxyglucose (2-DG uptake in rat L8 myotubes. Maximal uptake was observed with approximately 3-fold increase in 2-DG transport in 16 h treatment compared with the control. Effect of WE was stronger than that of 1 mM metformin. The effects of insulin and WE were additive. WE-induced glucose uptake was significantly inhibited by cycloheximide and partially reversed by SB203580. GLUT1 protein was markedly increased in response to WE. Conversely, WE had no effect on GLUT4 protein level. Redistribution of GLUT4 to the plasma membrane was demonstrated. Triterpenoids and carbohydrates were detected in WE. In conclusion, new GLUT1 protein synthesis is necessary for WEstimulated glucose transport while p38-MAPK-dependent activation of transporter intrinsic activity partly contributes to WE action. These results may explain and support the use of C. indica for the prevention and treatment of diabetes.

  20. Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, S.A.; Buxton, J.M.; Czech, M.P. (Univ. of Massachusetts Medical Center, Worcester (United States))

    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.

  1. Novel and High Affinity 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues as Atypical Dopamine Transporter Inhibitors

    DEFF Research Database (Denmark)

    Cao, Jianjing; Slack, Rachel D.; Bakare, Oluyomi M.

    2016-01-01

    pharmacological profiles in animal models of cocaine and methamphetamine abuse. Herein, we report a series of modafinil analogues that have an atypical DAT inhibitor profile. We extended SAR by chemically manipulating the oxidation states of the sulfoxide and the amide functional groups, halogenating the phenyl...

  2. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death

    DEFF Research Database (Denmark)

    Nørholm, Morten Helge Hauberg; Nour-Eldin, Hussam H; Brodersen, Peter;

    2006-01-01

    GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP......13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants....

  3. A new Michaelis-Menten-based kinetic model for transport and phosphorylation of glucose and its analogs in skeletal muscle.

    Science.gov (United States)

    Huang, Hsuan-Ming; Ismail-Beigi, Faramarz; Muzic, Raymond F

    2011-08-01

    A new model is introduced that individually resolves the delivery, transport, and phosphorylation steps of metabolism of glucose and its analogs in skeletal muscle by interpreting dynamic positron emission tomography (PET) data. The model uniquely utilizes information obtained from the competition between glucose and its radiolabeled analogs. Importantly, the model avoids use of a lumped constant which may depend on physiological state. Four basic physiologic quantities constitute our model parameters, including the fraction of total tissue space occupied by interstitial space (f(IS)), a flow-extraction product and interstitial (IS(g)) and intracellular (IC(g)) glucose concentrations. Using the values of these parameters, cellular influx (CI) and efflux (CE) of glucose, glucose phosphorylation rate (PR), and maximal transport (V(G)) and phosphorylation capacities (V(H)) can all be determined. Herein, the theoretical derivation of our model is addressed and characterizes its properties via simulation. Specifically, the model performance is evaluated by simulation of basal and euglycemic hyperinsulinemic (EH) conditions. In fitting the model-generated, synthetic data (including noise), mean estimates of all but IC(g) of the parameter values are within 5% of their values for both conditions. In addition, mean errors of CI, PR, and V(G) are less than 5% whereas those of VH and CE are not. It is concluded that under the conditions tested, the novel model can provide accurate parameter estimates and physiological quantities, except IC(g) and two quantities that are dependent on IC(g), namely CE and VH. However, the ability to estimate IC(g) seems to improve with increases in intracellular glucose concentrations as evidenced by comparing IC(g) estimates under basal vs EH conditions.

  4. Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

    Directory of Open Access Journals (Sweden)

    Zahra Maria

    Full Text Available 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.

  5. 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.

    Science.gov (United States)

    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.

  6. High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Jiahong Xue; Zuyi Yuan; Yue Wu; Yan Zhao; Zhaofei Wan

    2008-01-01

    Objective:ATP-binding cassette transporters(ABC) A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods:Incubation of human VSMCs with D-ghicose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-kB inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF-kB inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

  7. Benefits and Harms of Sodium-Glucose Co-Transporter 2 Inhibitors in Patients with Type 2 Diabetes

    DEFF Research Database (Denmark)

    Storgaard, Heidi; Gluud, Lise L; Bennett, Cathy

    2016-01-01

    OBJECTIVE: Sodium-glucose co-transporter 2 inhibitors (SGLT2-i) are a novel drug class for the treatment of diabetes. We aimed at describing the maximal benefits and risks associated with SGLT2-i for patients with type 2 diabetes. DESIGN: Systematic review and meta-analysis. DATA SOURCES AND STUDY...... increased risk in non-serious adverse events. The analyses may overestimate the intervention benefit due bias....

  8. Expression Patterns of Glucose Transporter-1 Gene and Thyroid Specific Genes in Human Papillary Thyroid Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sungeun; Chung, Junekey; Min Haesook and others

    2014-06-15

    The expression of glucose transporter-1 (Glut-1) gene and those of major thyroid-specific genes were examined in papillary carcinoma tissues, and the expressions of these genes were compared with cancer differentiation grades. Twenty-four human papillary carcinoma tissues were included in this study. The expressions of Glut-1- and thyroid-specific genes [sodium/iodide symporter (NIS), thyroid peroxidase, thyroglobulin, TSH receptor and pendrin] were analyzed by RT-PCR. Expression levels were expressed as ratios versus the expression of beta-actin. Pathologic differentiation of papillary carcinoma was classified into a relatively well-differentiated group (n=13) and relatively less differentiated group (n=11). Glut-1 gene expression was significantly higher in the less differentiated group (0.66±0.04) than in the well-differentiated group (0.59±0.07). The expression levels of the NIS, PD and TG genes were significantly higher in the well-differentiated group (NIS: 0.67±0.20, PD: 0.65±0.21, TG: 0.74±0.16) than in the less differentiated group (NIS: 0.36±0.05, PD: 0.49±0.08, TG: 0.60±0.11), respectively. A significant negative correlation was found between Glut-1 and NIS expression, and positive correlations were found between NIS and TG, and between NIS and PD. The NIS, PD and TG genes were highly expressed in well-differentiated thyroid carcinomas, whereas the Glut-1 gene was highly expressed in less differentiated thyroid carcinomas. These findings provide a molecular rationale for the management of papillary carcinoma, especially in the selection of FDG PET or radioiodine whole-body scan and I-131-based therapy.

  9. Expression of glucose transporter 4 in endometrium of polycystic ovary syndrome

    Institute of Scientific and Technical Information of China (English)

    Wang Wei; Li Xiao-dong; Hao Gui-min; Xu Su-xin; Cui Na; Cao Jin-feng

    2008-01-01

    Objective:The aim of this study was to detect the expression of glucose transporter 4(GLUT4),insulin(INS)and insulin receptor(INS-R)genes and proteins in endometrium of patients with polycystic ovarian syndrome(P-COS)and control group and investigate the relationship between GLUT4 and insulin resistance(IR).Methods:The patients with PCOS were divided into two groups:hypertestosteronemia(h-T)group and hyperin-sulinemia(h-I)group.The expression of GLUT4,INS and INS-R genes and proteins in endometrium of PCOS groups and control group was detected by immunohistochemistry and reverse transcription-polymerase chain reaction.Results:There was the expression of GLUT4,INS and INS-R proteins and genes in endometrium of PCOS groups and control group.Compared with the control group,PCOS groups were significantly lower in the expres-sion of GLUT4 protein and gene as well as INS-R protein and gene but much stronger in the expression of INS pro-tein and gene.The GLUT4 gene expression in h-I group was lower than that of h-T group but the expression of INS protein in h-I group was siginificantly higher than that of h-T group.Conclusion:The expression of GLUT4,INS and INS-R in endometrium of PCOS and control groups is con-firmed.Hyperinsulinemia and hyperandrogen can cause the expression of GLUT4 to decrease.The decreased ex-pression of GLUT4 in PCOS may be related with the IR of endometrium.

  10. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages.

    Science.gov (United States)

    Cao, Heping; Cao, Fangping; Roussel, Anne-Marie; Anderson, Richard A

    2013-12-01

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.

  11. Maltose and maltodextrin utilization by Listeria monocytogenes depend on an inducible ABC transporter which is repressed by glucose.

    Directory of Open Access Journals (Sweden)

    Shubha Gopal

    Full Text Available 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.

  12. A three-year-old boy with glucose transporter type 1 deficiency syndrome presenting with episodic ataxia.

    Science.gov (United States)

    Ohshiro-Sasaki, Akiko; Shimbo, Hiroko; Takano, Kyoko; Wada, Takahito; Osaka, Hitoshi

    2014-01-01

    Glucose transporter type 1 deficiency syndrome is a metabolic encephalopathy that results from impaired glucose transport into the brain as the result of a mutation of the SLC2A1 gene. It has been recognized recently that these patients can present with a much broader clinical spectrum than previously thought. We describe a 3-year-old boy presenting with episodic ataxia. Our patient exhibited periodic abnormal eye movements, including opsoclonus, since he was 4 months of age. At 2 years of age, he experienced acute cerebellar ataxia after a vaccination. Since then, he has had periodic attacks of ataxic gait, repeated vomiting, and abnormal eye movement. He was diagnosed as having episodic ataxia type 2 because the administration of acetazolamide seemed effective. By 3 years and 10 months of age, he exhibited mild mental retardation and mild trunk ataxia. The attacks were more likely to occur when he was hungry. Molecular analysis revealed that the SLC2A1 gene had a de novo mutation of heterozygous seven nucleotide insertion within exon 7, resulting in a frameshift. He has recently begun a modified Atkins diet; the frequency of attacks has been reduced, and his psychomotor and language skills have begun to develop. Glucose transporter type 1 deficiency syndrome should be considered in the differential diagnosis in children with episodic ataxia, even if acetazolamide is effective. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  14. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  15. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    Science.gov (United States)

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.

  16. A High-Affinity Metal-Binding Peptide From Escherichia Coli Hypb

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.C.Chan; Cao, L.; Dias, A.V.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.

  17. Decreased [{sup 18}F]fluoro-2-deoxy-D-glucose incorporation and increased glucose transport are associated with resistance to 5FU in MCF7 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Tim A.D. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom)], E-mail: t.smith@biomed.abdn.ac.uk; Sharma, Rituka I. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Wang, Weiguang G. [Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); School of Applied Sciences, University of Wolverhampton, City Campus-South, Wolverhampton WV1 1SB (United Kingdom); Welch, Andy E.; Schweiger, Lutz F. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Collie-Duguid, Elaina S.R. [Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom)

    2007-11-15

    Introduction: Tumor refractoriness to chemotherapy is frequently due to the acquisition of resistance. Resistant cells selected by exposure to chemotherapy agents may exhibit differences in [{sup 18}F]fluoro-2-deoxy-D-glucose (FDG) incorporation, as compared with sensitive cells. Methods: FDG incorporation, hexokinase (HK) activity, glucose transport and ATP content were determined in clones of 5-fluorouracil (5FU)-resistant MCF7 cells, established by long-term exposure to increasing 5FU concentrations, and in parental MCF7 cells. Results: FDG incorporation was decreased in MCF7 cells resistant to 5FU; HK activity was similar in the resistant and sensitive cells, while glucose transport was increased, as compared with sensitive cells. Treatment of cells with the glucose efflux inhibitor phloretin increased FDG incorporation to similar levels in the resistant and sensitive cells. Analysis of microarray data demonstrated the expression of GLUT1, 8 and 10 transporters in MCF7 cells. GLUT8 and 10 expression was decreased in the resistant cells, while GLUT1 was only increased in cells resistant to the lowest 5FU concentration. Conclusion: FDG incorporation in 5FU-resistant MCF7 cells is decreased, as compared with sensitive cells. Our findings also suggest that this may be due to high rates of membrane glucose transport in the resistant cells resulting in enhanced efflux of FDG. We believe that this is the first demonstration that facilitative glucose transporters can actually decrease the incorporation of FDG.

  18. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

    Science.gov (United States)

    Maute, Roy L; Gordon, Sydney R; Mayer, Aaron T; McCracken, Melissa N; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M; Manglik, Aashish; Kruse, Andrew C; Gambhir, Sanjiv S; Weissman, Irving L; Ring, Aaron M

    2015-11-24

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

  19. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  20. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  1. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

  2. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  3. [3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

    Science.gov (United States)

    Hoo, K; Legutko, B; Rizkalla, G; Deverill, M; Hawes, C R; Ellis, G J; Stensbol, T B; Krogsgaard-Larsen, P; Skolnick, P; Bleakman, D

    1999-12-01

    The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

  4. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  5. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    Science.gov (United States)

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  6. Flozins, inhibitors of type 2 renal sodium-glucose co-transporter – not only antihyperglycemic drugs

    Directory of Open Access Journals (Sweden)

    Mizerski Grzegorz

    2015-09-01

    Full Text Available The kidneys play a crucial role in the regulation of the carbohydrate metabolism. In normal physiological conditions, the glucose that filters through the renal glomeruli is subsequently nearly totally reabsorbed in the proximal renal tubules. Two transporters are engaged in this process: sodium-glucose co-transporter type 1 (SGLT1, and sodium-glucose co-transporter type type 2 (SGLT2 - this being located in the luminal membrane of the renal tubular epithelial cells. It was found that the administration of dapagliflozin, a selective SGLT2 inhibitor, in patients with type 2 diabetes, is associated with the reduction of HbA1c concentration by 0.45-1.11%. Additional benefits from the treatment with dapagliflozin are the reduction of arterial blood pressure and a permanent reduction of body weight. This outcome is related to the effect of osmotic diuresis and to the considerable loss of the glucose load by way of urine excretion. Dapagliflozin may be successfully applied in type 2 diabetes monotherapy, as well as in combined therapy (including insulin, where it is equally effective as other oral anti-diabetic drugs. Of note: serious adverse effects of dapagliflozin administration are rarely observed. What is more, episodes of severe hypoglycaemia related with the treatment occur only sporadically, most often in the course of diabetes polytherapy. The most frequent effects of the SGLT2 inhibitors are inseparably associated with the mechanism of their action (the glucuretic effect, and cover urogenital infections with a mild clinical course. At present, clinical trials are being continued of the administration of several subsequent drugs from this group, the most advanced of these being the use of canagliflozin and empagliflozin.

  7. Skeletal muscle sodium glucose co-transporters in older adults with type 2 diabetes undergoing resistance training

    Directory of Open Access Journals (Sweden)

    Francisco Castaneda, Jennifer E. Layne, Carmen Castaneda

    2006-01-01

    Full Text Available We examined the expression of the sodium-dependent glucose co-transporter system (hSGLT3 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 hSGLT3 and GLUT4 mRNA transcript levels were determined by real time RT-PCR. hSGLT3 transcripts increased by a factor of ten following resistance training compared to control subjects (0.10, P=0.03. There were no differences in GLUT4 mRNA expression levels between groups. Protein expression levels of these transporters were confirmed by immunohistochemistry and Western blotting. hSGLT3 after resistance exercise was found not to be co-localized with the nicotinic acetylcholine receptor. The change in hSGLT3 transcript levels in the vastus lateralis muscle was positively correlated with glucose uptake, as measured by the change in muscle glycogen stores (r=0.53, P=0.02; and with exercise intensity, as measured by the change in muscle strength (r=0.73, P=0.001. Group assignment was be the only independent predictor of hSGLT3 transcript levels, explaining 68% of its variability (P=0.01. Our data show that hSGLT3, but not GLTU4, expression was enhanced in skeletal muscle after 16 weeks of resistance training. This finding suggests that hSGLT3, an insulin-independent glucose transporter, is activated with exercise and it may play a significant role in glycemic control with muscle contraction. The hSGLT3 exact mechanism is not well understood and requires further investigation. However its functional significance regarding a reduction of glucose toxicity and improvement of insulin resistance is the subject of ongoing research.

  8. Empagliflozin: a new sodium-glucose co-transporter 2 (SGLT2 inhibitor for the treatment of type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Joshua J Neumiller

    2014-06-01

    Full Text Available Type 2 diabetes is increasing in prevalence worldwide, and hyperglycemia is often poorly controlled despite a number of therapeutic options. Unlike previously available agents, sodium-glucose co-transporter 2 (SGLT2 inhibitors offer an insulin-independent mechanism for improving blood glucose levels, since they promote urinary glucose excretion (UGE by inhibiting glucose reabsorption in the kidney. In addition to glucose control, SGLT2 inhibitors are associated with weight loss and blood pressure reductions, and do not increase the risk of hypoglycemia. Empagliflozin is a selective inhibitor of SGLT2, providing dose-dependent UGE increases in healthy volunteers, with up to 90 g of glucose excreted per day. It can be administered orally, and studies of people with renal or hepatic impairment indicated empagliflozin needed no dose adjustment based on pharmacokinetics. In Phase II trials in patients with type 2 diabetes, empagliflozin provided improvements in glycosylated hemoglobin (HbA1c and other measures of glycemic control when given as monotherapy or add-on to metformin, as well as reductions in weight and systolic blood pressure. As add-on to basal insulin, empagliflozin not only improved HbA1c levels but also reduced insulin doses. Across studies, empagliflozin was generally well tolerated with a similar rate of hypoglycemia to placebo; however, patients had a slightly increased frequency of genital infections, but not urinary tract infections, versus placebo. Phase III studies have also reported a good safety profile along with significant improvements in HbA1c, weight and blood pressure, with no increased risk of hypoglycemia versus placebo. Based on available data, it appears that empagliflozin may be a useful option in a range of patients; however, clinical decisions will be better informed by the results of ongoing studies, in particular, a large cardiovascular outcome study (EMPA-REG OUTCOME™.

  9. Canagliflozin: a sodium glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes mellitus.

    Science.gov (United States)

    Rosenthal, Norm; Meininger, Gary; Ways, Kirk; Polidori, David; Desai, Mehul; Qiu, Rong; Alba, Maria; Vercruysse, Frank; Balis, Dainius; Shaw, Wayne; Edwards, Robert; Bull, Scott; Di Prospero, Nicholas; Sha, Sue; Rothenberg, Paul; Canovatchel, William; Demarest, Keith

    2015-11-01

    The sodium glucose co-transporter 2 (SGLT2) inhibitor canagliflozin is a novel treatment option for adults with type 2 diabetes mellitus (T2DM). In patients with hyperglycemia, SGLT2 inhibition lowers plasma glucose levels by reducing the renal threshold for glucose (RTG ) and increasing urinary glucose excretion (UGE). Increased UGE is also associated with a mild osmotic diuresis and net caloric loss, which can lead to reductions in body weight and blood pressure (BP). After promising results from preclinical and phase I/II studies, the efficacy and safety of canagliflozin was evaluated in a comprehensive phase III development program in over 10,000 patients with T2DM on various background therapies. Canagliflozin improved glycemic control and provided reductions in body weight and BP versus placebo and active comparators in studies of up to 2 years' duration. Canagliflozin was generally well tolerated, with higher incidences of adverse events (AEs) related to the mechanism of action, including genital mycotic infections and AEs related to osmotic diuresis. Results from the preclinical and clinical studies led canagliflozin to be the first-in-class SGLT2 inhibitor approved in the United States, and support canagliflozin as a safe and effective therapeutic option across a broad range of patients with T2DM. © 2015 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

  10. Adaptive evolution in the glucose transporter 4 gene Slc2a4 in Old World fruit bats (family: Pteropodidae).

    Science.gov (United States)

    Shen, Bin; Han, Xiuqun; Zhang, Junpeng; Rossiter, Stephen J; Zhang, Shuyi

    2012-01-01

    Frugivorous and nectarivorous bats are able to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose. The glucose transporter 4 protein (GLUT4) encoded by the Slc2a4 gene plays a critical role in transmembrane skeletal muscle glucose uptake and thus glucose homeostasis. To test whether the Slc2a4 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Slc2a4 gene in a number of bat species, including four Old World fruit bats (Pteropodidae) and three New World fruit bats (Phyllostomidae). Our molecular evolutionary analyses revealed evidence that Slc2a4 has undergone a change in selection pressure in Old World fruit bats with 11 amino acid substitutions detected on the ancestral branch, whereas, no positive selection was detected in the New World fruit bats. We noted that in the former group, amino acid replacements were biased towards either Serine or Isoleucine, and, of the 11 changes, six were specific to Old World fruit bats (A133S, A164S, V377F, V386I, V441I and G459S). Our study presents preliminary evidence that the Slc2a4 gene has undergone adaptive changes in Old World fruit bats in relation to their ability to meet the demands of a high sugar diet.

  11. Adaptive evolution in the glucose transporter 4 gene Slc2a4 in Old World fruit bats (family: Pteropodidae.

    Directory of Open Access Journals (Sweden)

    Bin Shen

    Full Text Available Frugivorous and nectarivorous bats are able to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose. The glucose transporter 4 protein (GLUT4 encoded by the Slc2a4 gene plays a critical role in transmembrane skeletal muscle glucose uptake and thus glucose homeostasis. To test whether the Slc2a4 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Slc2a4 gene in a number of bat species, including four Old World fruit bats (Pteropodidae and three New World fruit bats (Phyllostomidae. Our molecular evolutionary analyses revealed evidence that Slc2a4 has undergone a change in selection pressure in Old World fruit bats with 11 amino acid substitutions detected on the ancestral branch, whereas, no positive selection was detected in the New World fruit bats. We noted that in the former group, amino acid replacements were biased towards either Serine or Isoleucine, and, of the 11 changes, six were specific to Old World fruit bats (A133S, A164S, V377F, V386I, V441I and G459S. Our study presents preliminary evidence that the Slc2a4 gene has undergone adaptive changes in Old World fruit bats in relation to their ability to meet the demands of a high sugar diet.

  12. Sodium-glucose co-transporter-2 inhibitors as add-on therapy to insulin: rationale and evidences.

    Science.gov (United States)

    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.

  13. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Institute of Scientific and Technical Information of China (English)

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral

  14. Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose.

    Science.gov (United States)

    Schürmann, A; Keller, K; Monden, I; Brown, F M; Wandel, S; Shanahan, M F; Joost, H G

    1993-01-01

    The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin. Images Figure 1 Figure 2 PMID:8452538

  15. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat

    2010-12-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  16. Adolescents with clinical type 1 diabetes display reduced red blood cell glucose transporter isoform 1 (GLUT1).

    Science.gov (United States)

    Garg, Meena; Thamotharan, Manikkavasagar; Becker, Dorothy J; Devaskar, Sherin U

    2014-11-01

    Type 1 diabetic (T1D) adolescent children on insulin therapy suffer episodes of both hyper- and hypoglycemic episodes. Glucose transporter isoform GLUT1 expressed in blood-brain barrier (BBB) and red blood cells (RBC) compensates for perturbed circulating glucose toward protecting the supply to brain and RBCs. We hypothesized that RBC-GLUT1 concentration, as a surrogate for BBB-GLUT1, is altered in T1D children. To test this hypothesis, we measured RBC-GLUT1 by enzyme-linked immunosorbent assay (ELISA) in T1D children (n = 72; mean age 15.3 ± 0.2 yr) and control children (CON; n = 11; mean age 15.6 ± 0.9 yr) after 12 h of euglycemia and during a hyperinsulinemic-hypoglycemic clamp with a nadir blood glucose of ~3.3 mmol/L for 90 min (clamp I) or ~3 mmol/L for 45 min (clamp II). Reduced baseline RBC-GLUT1 was observed in T1D (2.4 ± 0.17 ng/ng membrane protein); vs. CON (4.2 ± 0.61 ng/ng protein) (p increasing during acute hypoglycemia over the durations examined, may demonstrate a vulnerability of impaired RBC glucose transport (serving as a surrogate for BBB), especially in those with the worst control. We speculate that this may contribute to the perturbed cognition seen in T1D adolescents.

  17. Insulin sensitivity and inhibition by forskolin, dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofacial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58±0.01 fold and the GLUT4 content 1.96±0.11 fold, as well as the 2-deoxyglucose uptake 1.53±0.09 and 1.86±0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitivity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin responsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobilization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.

  18. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    Science.gov (United States)

    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.

  19. Glucose transporter distribution in the vessels of the central nervous system of the axolotl Ambystoma mexicanum (Urodela: Ambystomatidae).

    Science.gov (United States)

    Lazzari, Maurizio; Bettini, Simone; Ciani, Franco; Franceschini, Valeria

    2008-10-01

    The GLUT-1 isoform of the glucose transporter is commonly considered a reliable molecular marker of blood-brain barrier endothelia in the neural vasculature organized in a three-dimensional network of single vessels. The central nervous system of the axolotl Ambystoma mexicanum is characterized by a vascular architecture that contains both single and paired vessels. The presence and distribution of the GLUT-1 transporter are studied in this urodele using both immunoperoxidase histochemistry and immunogold technique. Light microscopy reveals immunopositivity in both parenchymal and meningeal vessels. The transverse-sectioned pairs of vessels do not show the same size. Furthermore, in the same pair, the two elements often differ in diameter. The main regions of the central nervous system show a different percentage of the paired structures. Only immunogold cytochemistry reveals different staining intensity in the two adjoined elements of a vascular pair. Colloidal gold particles show an asymmetric distribution in the endothelia of both single and paired vessels. These particles are more numerous on the abluminal surface than on the luminal one. The particle density is calculated in both vascular types. The different values could indicate functional differences between single and paired vessels and between the two adjoined elements of a pair, regarding glucose transport.

  20. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI)

    OpenAIRE

    1992-01-01

    It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast c...

  1. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  2. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    Science.gov (United States)

    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.

  3. Dissociation of glucose tracer uptake and glucose transporter distribution in the regionally ischaemic isolated rat heart: application of a new autoradiographic technique

    Energy Technology Data Exchange (ETDEWEB)

    Southworth, Richard; Medina, Rodolfo A.; Garlick, Pamela B. [Department of Radiological Sciences, Guy' s, King' s and St Thomas' School of Medicine, Guy' s Campus, London, SE1 9RT (United Kingdom); Dearling, Jason L.J.; Flynn, Aiden A.; Pedley, Barbara R. [Cancer Research UK Targeting and Imaging Group, Academic Department of Oncology, University College London, Royal Free Campus, London, NW3 2PF (United Kingdom)

    2002-10-01

    Fluorine-18 fluoro-2-deoxyglucose ({sup 18}FDG) and carbon-14 2-deoxyglucose ({sup 14}C-2-DG) are both widely used tracers of myocardial glucose uptake and phosphorylation. We have recently shown, using positron emission tomography (PET) and nuclear magnetic resonance, that ischaemia-reperfusion (I-R) causes differential changes in their uptake. We describe here the novel application of an autoradiographic technique allowing the investigation of this phenomenon at high resolution, using tracer concentrations of both analogues in the dual-perfused isolated rat heart. We also investigate the importance of glucose transporter (GLUT 1 and GLUT 4) distribution in governing the observed phosphorylated analogue accumulation. Hearts (n=5) were perfused with Krebs buffer for 40 min, made regionally zero-flow ischaemic for 40 min and reperfused for 60 min with Krebs containing tracer {sup 18}FDG (200 MBq) and tracer {sup 14}C-2-DG (0.37 MBq). Hearts were then frozen and five sections (10 {mu}m) were cut per heart, fixed and exposed on phosphor storage plates for 18 h (for {sup 18}FDG) and then for a further 9 days (for {sup 14}C-2-DG). Quantitative digital images of tracer accumulation were obtained using a phosphor plate reader. The protocol was repeated in a second group of hearts and GLUT 1 and GLUT 4 distribution analysed. Post-ischaemic accumulation of {sup 18}FDG-6-P was inhibited by 38.2%{+-}1.7% and {sup 14}C-DG-6-P by 19.0%{+-}2.2%, compared with control (P<0.05). After placing seven ''lines of interrogation'' across each heart section and analysing the phosphorylated tracer accumulation along them, a transmural gradient of both tracers was observed; this was highest at the endocardium and lowest at the epicardium. GLUT 4 translocated to the sarcolemma in the ischaemic/reperfused region (from 24%{+-}3% to 59%{+-}5%), while there was no cellular redistribution of GLUT 1. We conclude that since decreased phosphorylated tracer accumulation occurs

  4. Perfusion-independent effect of bradykinin and fosinoprilate on glucose transport in Langendorff rat hearts

    NARCIS (Netherlands)

    Rett, K; Maerker, E; Renn, W; vanGilst, W; Haering, HU

    1997-01-01

    Angiotensin-converting enzyme (ACE) inhibitor-stimulated glucose metabolism and perfusion in muscle tissue seem to be, at least in part, mediated by kinins. However, the relative contribution of direct metabolic or secondary hemodynamically induced effects is unclear, It was the aim of this study to

  5. The glucose transporter GLUT1 is required for ErbB2-induced mammary tumorigenesis.

    Science.gov (United States)

    Wellberg, Elizabeth A; Johnson, Stevi; Finlay-Schultz, Jessica; Lewis, Andrew S; Terrell, Kristina L; Sartorius, Carol A; Abel, E Dale; Muller, William J; Anderson, Steven M

    2016-12-20

    Altered tumor cell metabolism is an emerging hallmark of cancer; however, the precise role for glucose in tumor initiation is not known. GLUT1 (SLC2A1) is expressed in breast cancer cells and is likely responsible for avid glucose uptake observed in established tumors. We have shown that GLUT1 was necessary for xenograft tumor formation from primary mammary cells transformed with the polyomavirus middle-T antigen but that it was not necessary for growth after tumors had formed in vivo, suggesting a differential requirement for glucose depending on the stage of tumorigenesis. To determine whether GLUT1 is required early during mammary tumorigenesis, we crossed MMTV-NIC mice, which express activated HER2/NEU/ERBB2 and Cre recombinase, to Slc2a1 (Flox/Flox) (GLUT1(Flox/Flox)) mice to generate NIC-GLUT1(+/+), NIC-GLUT1(Flox/+), and NIC-GLUT1(Flox/Flox) mice. In addition, we evaluated effects of glucose restriction or GLUT1 inhibition on transformation in MCF10A-ERBB2 breast epithelial cells in three-dimensional culture. Finally, we utilized global gene expression profiling data of primary human breast tumors to determine the relationship between SLC2A1 and stage of tumorigenesis. All of the NIC-GLUT1(+/+) mice developed tumors in less than 200 days. In contrast, only 1 NIC-GLUT1(Flox/Flox) mouse and 1 NIC-GLUT1(Flox/+) mouse developed mammary tumors, even after 18 months. Mammary gland development was not disrupted in NIC mice lacking GLUT1; however, epithelial content of mature glands was reduced compared to NIC-GLUT1(Flox/+) mice. In MCF10A-ERBB2 cells, glucose restriction or GLUT1 inhibition blocked transformation induced by activated ERBB2 through reduced cell proliferation. In human breast cancers, SLC2A1 was higher in ductal carcinoma in situ compared to the normal breast, but lower in invasive versus in situ lesions, suggesting the requirement for GLUT1 decreases as tumors progress. This study demonstrates a strict requirement for GLUT1 in the early stages of

  6. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    Science.gov (United States)

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  7. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  8. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  9. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  10. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  11. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    Science.gov (United States)

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  12. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  13. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  14. Place of sodium-glucose co-transporter type 2 inhibitors for treatment of type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    Nasser; Mikhail

    2014-01-01

    Inhibitors of sodium-glucose co-transporter type 2(SGLT2), such as canagliflozin and dapagliflozin, are recently approved for treatment of type 2 diabetes. These agents lower blood glucose mainly by increasing urinary glucose excretion. Compared with placebo, SGLT2 inhibitors reduce hemoglobin A1c(Hb A1c) levels by an average of 0.5%-0.8% when used as monotherapy or add-on therapy. Advantages of this drug class include modest weight loss of approximately 2 kg, low risk of hypoglycemia, and decrease blood pressure of approximately 4 mm Hg systolic and 2 mm Hg diastolic. These characteristics make these agents potential add-on therapy in patients with Hb A1 c levels close to 7%-8.0%, particularly if these patients are obese, hypertensive, and/or prone for hypoglycemia. Meanwhile, these drugs are limited by high frequency of genital mycotic infections. Less common adverse effects include urinary tract infections, hypotension, dizziness, and worsening renal function. SGLT2 inhibitors should be used with caution in the elderly because of increased adverse effects, and should not be used in chronic kidney disease due to decreased or lack of efficacy and nephrotoxicity. Overall, SGLT2 inhibitors are useful addition for treatment of select groups of patients with type 2 diabetes,but their efficacy and safety need to be established in long-term clinical trials.

  15. Increased glucose transport in ras-transformed fibroblasts: a possible role for N-glycosylation of GLUT1.

    Science.gov (United States)

    Onetti, R; Baulida, J; Bassols, A

    1997-05-05

    2-Deoxyglucose uptake was enhanced in ts371 KiMuSV-NRK cells when growing at the permissive temperature to allow the expression of a transforming p21 ras protein. This change is due to a decrease in the K(m) by approximately 2.5-fold without affecting the V(max) of the transporter. The amount of the GLUT1 glucose transporter dit not increase as deduced from immunoblot experiments on total membranes. Nevertheless, ras-transformed GLUT1 displays a higher molecular mass due to an increased N-glycosylation of the protein. Experiments made in tunicamycin-treated cells indicates that a higher glycosylation is responsible for the increase in 2-deoxyglucose uptake in ras-transformed cells.

  16. Xylem-Transported Glucose as an Additional Carbon Source for Leaf Isoprene Formation in Quercus Robur L.

    Science.gov (United States)

    Graus, M.; Kreuzwieser, J.; Schnitzler, J.; Wisthaler, A.; Hansel, A.; Rennenberg, H.

    2003-04-01

    Isoprene is emitted from mature, photosynthesizing leaves of many plant species, particularly of trees. Current interest in understanding the biochemical and physiological mechanisms controlling isoprene formation is caused by the important role isoprene plays in atmospheric chemistry. Isoprene reacts with hydroxyl radicals (OH) thereby generating oxidizing agents such as ozone and organic peroxides. Ozone causes significant deterioration in air quality and can pose threats to human health therefore its control is a major goal in Europe and the United States. In recent years, much progress has been made in elucidating the pathways of isoprene biosynthesis. Nevertheless the regulatory mechanisms controlling isoprene emission are not completely understood. Light and temperature appear to be the main factors controlling short-term variations in isoprene emission. Exposure of plants to C-13 labeled carbon dioxide showed instantaneous assimilated carbon is the primary carbon source for isoprene formation. However, variations in diurnal and seasonal isoprene fluxes, which cannot be explained by temperature, light, and leaf development led to the suggestion that alternative carbon sources may exist contributing to isoprene emissions. The aim of the present study was to test whether xylem-transported carbohydrates act as additional sources for isoprene biosynthesis. For this purpose, [U-C-13] alpha-D-glucose was fed to photosynthesizing leaves via the xylem of Quercus robur L. seedlings and the incorporation of glucose derived C-13 into emitted isoprene was monitored in real time using Proton-Transfer-Reaction Mass Spectrometry (PTR-MS). A rapid incorporation of C-13 from xylem-fed glucose into single (mass 70) and double (mass 71) C-13 labeled isoprene molecules was observed after a lag phase of approximately 5 to 10 minutes. This incorporation was temperature dependent and was highest (up to 13% C-13 of total carbon emitted as isoprene) at the temperature optimum of

  17. [Effect of natural or synthetic detergents on the transport of D-glucose in the membranes of vesicles of the brush border of the intestine of the rabbit].

    Science.gov (United States)

    Favilli, F; Iantomasi, T; Stio, M; Treves, C; Vanni, P; Vincenzini, M T

    1988-01-01

    We describe here the effects of natural and synthetic detergents on the D-glucose transport into brush-border membranes of vesicles of rabbit's intestine. Two synthetic detergents: Triton X-100 and dodecyltrimethylammonium bromide have been found very strong inhibitors (more than 50 p. 100 of inhibition of maximal D-glucose uptake). Kinetic studies showed that these detergents behaved as mixed type inhibitors. The Na+-dependent transport of amino acids (aspartic acid, lysine, phenylalanine) is only poorly affected by dodecyltrimethylammonium bromide, while Triton X-100 inhibits unspecifically all the transport studied.

  18. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    OpenAIRE

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H. F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affin...

  19. Glucose transporter GLUT1 expression and clinical outcome in solid tumors: a systematic review and meta-analysis.

    Science.gov (United States)

    Wang, Ji; Ye, Chenyang; Chen, Cong; Xiong, Hanchu; Xie, Binbin; Zhou, Jichun; Chen, Yongxia; Zheng, Shu; Wang, Linbo

    2017-03-07

    Glucose transporter 1 (GLUT1), the uniporter protein encoded by the SLC2A1 gene, is a key rate-limiting factor in the transport of glucose in cancer cells, and frequently expressed in a significant proportion of human cancers. Numerous studies have reported paradoxical evidence of the relationship between GLUT1 expression and prognosis in solid human tumors. To address this discrepancy, we conducted a thorough search of Pubmed and Web of Science for studies evaluating the expression of GLUT1 and overall survival (OS) and disease-free survival (DFS) in patients with solid cancer from 1993 to April 2016. Data from published researches were extracted and computed into odds ratio (OR). A total of 26 studies including 2948 patients met our search criteria and were evaluated. Overexpression of GLUT1 was found to significantly correlate with poor 3-year OS (OR: 2.86; 95% CI, 1.90-4.32, P < 0.00001) and 5-year OS (OR: 2.52; 95% CI, 1.75-3.61, P < 0.00001) of solid tumors. Similar results were observed when analysis of DFS was performed. Subgroup analysis revealed that elevated GLUT1 expression was associated with worse prognosis of oral squamous cell carcinoma and breast cancer. Taken together, overexpression of GLUT1 is correlated with poor survival in most solid tumors, suggesting that the expression status of GLUT1 is a vital prognostic indicator and promising therapeutic target in solid tumors.

  20. A study of the role of glucose transporter 1 (Glut1) in white spot syndrome virus (WSSV) infection.

    Science.gov (United States)

    Huang, Huai-Ting; Chan, Hoi-Ling; Shih, Tsai-Yen; Chen, Li-Li

    2015-10-01

    White spot syndrome virus (WSSV) is a large enveloped DNA virus, and it causes a serious disease that has led to severe mortalities of cultured shrimps in many countries. To determine the mechanism of virus entry into the cell and to establish an antiviral strategy, the cell receptor for virus entry and receptor binding protein should be identified. A shrimp cell surface protein, glucose transporter1 (Glut1), was found to interact with WSSV in previous study. In this study, this Glut1 was confirmed to have the ability of transporting glucose, and this gene can also be found in other shrimp species. The interaction between Glut1 and some other WSSV envelope proteins in the infectome structure was verified by far western blot and His pull down assay. In vitro and in vivo neutralization using recombinant partial Glut1 revealed that the large extracellular portion of Glut1 could delay WSSV infection. Also, shrimps which were knocked-down Glut1 gene by treated with dsRNA before WSSV challenge showed decreased mortality. These results indeed provide a direction to develop efficient antiviral strategies or therapeutic methods by using Glut1.

  1. GDM-Induced Macrosomia Is Reversed by Cav-1 via AMPK-Mediated Fatty Acid Transport and GLUT1-Mediated Glucose Transport in Placenta

    Science.gov (United States)

    Wang, Di; Yang, Ruirui; Sang, Hui; Han, Linlin; Zhu, Yuexia; Lu, Yanyan; Tan, Yeke; Shang, Zhanping

    2017-01-01

    Objective To investigate if the role of Cav-1 in GDM-induced macrosomia is through regulating AMPK signaling pathway in placenta. Methods We used diagnostic criteria of gestational diabetes mellitus (GDM) and macrosomia to separate and compare placental protein and mRNA levels from GDM with macrosomia group (GDMM), GDM with normal birth weight group (GDMN) and normal glucose tolerance (NGT) with normal birth weight group (CON). Western blotting was performed to examine differentially expressed proteins of caveolin-1 (Cav-1) and Adenosine monophosphate-activated protein kinase (AMPK) signaling pathway related proteins, including phosphorylated-AMPKα(Thr172), AMPKα, phosphorylated-Acetyl-CoA carboxylase(Ser79) (p-ACC(Ser79)), ACC and glucose transporter 1 (GLUT1) in placenta between the three groups. The mRNA levels of Cav-1, AMPKα, ACC and GLUT1 in placenta were measured by real time-PCR. Results In the GDMM placenta group, both protein and mRNA levels of Cav-1 were down-regulated, while GLUT1 was up-regulated; the phosphorylation and mRNA levels of ACC and AMPKα were decreased, but total ACC protein levels were increased compared to both the GDMN (pGLUT1 protein levels. Besides, in GDMM group placental mRNA levels, NBW had a positive correlation with GLUT1 (pGLUT1 (pGLUT1. Conclusion GDM-induced macrosomias have more severe inhibition of Cav-1 expression in placenta. Cav-1 is associated with placental glucose and fatty acid transport via the induction of AMPK signaling pathway and the reduction of GLUT1 signaling pathway to reverse GDM-induced macrosomia. PMID:28125642

  2. Unaltered lactate and glucose transporter levels in the MPTP mouse model of Parkinson's disease

    DEFF Research Database (Denmark)

    Puchades, Maja; Sogn, Carl Johan; Maehlen, Jan

    2013-01-01

    BACKGROUND: Metabolic impairment contributes to development of Parkinson's disease (PD). Mitochondrial dysfunction is involved in degeneration of nigral dopamine neurons. Also, in PD there are alterations in glucose metabolism in nigro-striatal pathways, and increased cerebral lactate levels have...... of MCT1, MCT2 and GLUT1 is not changed following dopaminergic neurodegeneration. This is in contrast to findings in other neurodegenerative disease, such as mesial temporal lobe epilepsy, where there are large alterations in MCT levels....

  3. Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking

    OpenAIRE

    Wasik, A. A.; Polianskyte-Prause, Z.; Dong, M.-Q.; Shaw, A S; Yates, J R; Farquhar, M. G.; Lehtonen, S

    2012-01-01

    Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitate...

  4. Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit

    DEFF Research Database (Denmark)

    Zeuthen, T; Meinild, A K; Loo, D D;

    2001-01-01

    water transport was divided about equally between cotransport, osmosis across the SGLT1 and osmosis across the native oocyte membrane. 6. Coexpression of AQP1 with the SGLT1 increased the water permeability more than 10-fold and steady state isotonic transport was achieved after less than 2 s of sugar...

  5. Effects of muscle activity and fiber composition on glucose transport and GLUT-4.

    Science.gov (United States)

    Megeney, L A; Neufer, P D; Dohm, G L; Tan, M H; Blewett, C A; Elder, G C; Bonen, A

    1993-04-01

    We examined glucose uptake and GLUT-4 in rat muscles [soleus (Sol), plantaris (PL), extensor digitorum longus (EDL), tibialis anterior, and the red and white gastrocnemius (WG)]. In the normally innervated perfused rat hindlimb muscles the proportion of oxidative fibers was highly correlated with the muscle's insulin-stimulated 3-O-methyl-D-glucose (3-MG) uptake (R2 = 0.78) and GLUT-4 content (r = 0.94). Insulin-stimulated 3-MG uptake and GLUT-4 were also highly correlated (R2 = 0.996). In 3-day denervated muscles, insulin-stimulated 3-MG uptake was reduced in all six muscles (-41 to -14.6%, P 0.05). A very high correlation was observed between the decrements in GLUT-4 (%) and the decrements in 3-MG uptake (%; r = 0.99). The relatively greater loss in muscle activity (%) due to denervation in the Sol compared with the PL coincided with the reductions (%) in GLUT-4 and 3-MG uptake. These studies demonstrate that glucose uptake and GLUT-4 are regulated by insulin-independent means, namely the oxidative capacity of the muscle and the normal activity level of the muscle.

  6. Sodium-Glucose Linked Transporter-2 Inhibitors in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    L. Zanoli

    2015-01-01

    Full Text Available 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.

  7. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. (Univ. of Maryland, Baltimore (USA))

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  8. Dietary Lipid and Carbohydrate Interactions: Implications on Lipid and Glucose Absorption, Transport in Gilthead Sea Bream (Sparus aurata) Juveniles.

    Science.gov (United States)

    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.

  9. Broiler chickens (Ross strain) lack insulin-responsive glucose transporter GLUT4 and have GLUT8 cDNA.

    Science.gov (United States)

    Seki, Yoshinori; Sato, Kan; Kono, Tatsuyoshi; Abe, Hiroyuki; Akiba, Yukio

    2003-08-01

    Identification of insulin-responsive glucose transporter proteins, GLUT4 and GLUT8, was attempted in chickens that characteristically are hyperglycemic and insulin resistant. Northern blot analysis using rat GLUT4 cDNA probe and RT-PCR using primers designed against the conserved regions in mammalian GLUT4 cDNA were not successful in identifying GLUT4 homologue(s) in various chicken tissues. Furthermore, GLUT4 homologues could not be detected in chicken tissues by genomic Southern blot analyses using a rat GLUT4 cDNA probe. These data, therefore, suggest that the GLUT4 homologous gene is deficient in chicken tissues. However, GLUT8, another insulin-responsive glucose transporter in the blastocyst, was identified with the aid of RACE (rapid amplification of cDNA ends) reactions in the chicken testis. Chicken GLUT8 was composed of 1449 bp with a coding region for a 482 amino acid protein. The deduced amino acid sequence was 58.8, 56.3, and 56.8% identical with human, rat, and mouse GLUT8, respectively. By RT-PCR, GLUT8 mRNA expressions were detected in chicken brain, kidney, adrenal, spleen, lung, testis, and pancreas; and barely detectable in skeletal muscle, liver, adipose tissue, and heart. Here we firstly report that GLUT8 was identified in chickens, while GLUT4, a major insulin-responsive transporter in mammals, is deficient in these animals. We propose the hypothesis that the hyperglycemia and insulin resistance observable in chickens is associated with their possible deficiency of GLUT4.

  10. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis.

    Science.gov (United States)

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-08-31

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E(+)) and acquire a high-affinity conformation with an 'open' headpiece (H(+)). The canonical switchblade model of integrin activation proposes that the E(+) conformation precedes H(+), and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E(-)H(+) conformation. E(-)H(+) β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.

  11. Selection of high affine peptide ligands for detection of Clostridium Tyrobutyricum spores.

    Science.gov (United States)

    Lavilla, María; De Luis, Ruth; Pérez, María Dolores; Calvo, Miguel; Sánchez, Lourdes

    2009-11-01

    Clostridium tyrobutyricum is the main agent responsible for "late blowing" in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN); however, it is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.

  12. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.

    Science.gov (United States)

    Altschuler, Sarah E; Lewis, Karen A; Wuttke, Deborah S

    2013-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  13. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    Directory of Open Access Journals (Sweden)

    Sarah E. Altschuler

    2013-09-01

    Full Text Available The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  14. Experimental conditions can obscure the second high-affinity site in LeuT.

    Science.gov (United States)

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  15. Cytisine derivatives as high affinity nAChR ligands: synthesis and comparative molecular field analysis.

    Science.gov (United States)

    Nicolotti, O; Canu Boido, C; Sparatore, F; Carotti, A

    2002-06-01

    A number of new N-substituted cytisine derivatives were prepared and tested, along with similar compounds already described by us and others, as high affinity neuronal acetylcholine receptor ligands. Structure-affinity relationships were discussed in the light of our recently proposed pharmacophore model for nicotinic receptor agonists. The most significant physicochemical interactions modulating the receptor-ligand binding were detected at the three dimensional (3D) level by means of comparative molecular field analysis (CoMFA). The best predictive PLS model was a single-field steric model showing good statistical figures: n = 17, Q2 = 0.717, s(ev) = 0.566, r2 = 0.942, s = 0.275.

  16. A linker peptide with high affinity towards silica-containing materials.

    Science.gov (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  17. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    B binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven...... flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology...... model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting....

  18. A complex water network contributes to high-affinity binding in an antibody–antigen interface

    Directory of Open Access Journals (Sweden)

    S.F. Marino

    2016-03-01

    Full Text Available This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment, J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA. The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: “Potent anti-tumor response by targeting B cell maturation antigen (BCMA in a mouse model of multiple myeloma”, Mol. Oncol. 9 (7 (2015 pp. 1348–58.

  19. Expression of a Hybrid Human Superoxide Dismutase with a High Affinity for Heparin

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A designed heparin-affinity of human Cu, Zn-SOD is described. The natural leader peptide of P.leiognathi Cu, Zn-SOD and a heparin-binding peptide containing a stretch of 7 Arg were fused to the N-terminal and the C-terminal of human Cu, Zn-SOD respectively. The resulted hybrid enzyme had not only a normal SOD activity but also a high affinity for heparin eluted on the heparin-Sepharose column at 0.4 mol/L NaCl. Some properties, such as the optimum pH, the thermostability and the half-life in the circulation of rats, were also analyzed.

  20. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    DEFF Research Database (Denmark)

    Rao, C N; Castronovo, V; Schmitt, M C;

    1989-01-01

    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...... al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse......, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues...

  1. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    Science.gov (United States)

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  2. Hepatic OATP Transporter and Thyroid Hormone Receptor Interplay Determines Cholesterol and Glucose Homeostasis

    OpenAIRE

    Meyer zu Schwabedissen, Henriette E; Ware, Joseph A; Finkelstein, David; Chaudhry, Amarjit S.; Lemay, Sara; Leon-Ponte, Matilde; Strom, Stephen C.; Zaher, Hani; Schwarz, Ute I; Freeman, David J.; Schuetz, Erin G; Tirona, Rommel G; Kim, Richard B

    2011-01-01

    The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B-subfamily, we previously demonstrated that genetic ablation causes reduced hepatic clearance capacity for substrates. In this report we focused on the physiological function of the hepatic OATP1B transporters.

  3. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  4. The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors.

    Science.gov (United States)

    Nezami, Azin; Freire, Ernesto

    2002-12-04

    The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.

  5. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  6. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  7. Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging.

    Science.gov (United States)

    Dhar, Alok; Feiss, Michael

    2005-03-18

    DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.

  8. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    Science.gov (United States)

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  9. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

    DEFF Research Database (Denmark)

    Barres, Romain; Grémeaux, Thierry; Gual, Philippe

    2006-01-01

    a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma m......RNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport...... and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin...

  10. Transmembrane Segment 6 of the Glut1 Glucose Transporter Is an Outer Helix and Contains Amino Acid Side Chains Essential for Transport Activity*

    Science.gov (United States)

    Mueckler, Mike; Makepeace, Carol

    2008-01-01

    Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu204 and Pro205 abolished transport activity, whereas substitutions at Ile192, Pro196, Gln200, and Gly201 resulted in inhibition of activity that ranged from ∼35 to ∼80%. Cysteine substitutions at Leu188, Ser191, and Leu199 moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu188 conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters. PMID:18245775

  11. Combination of crystalloid (glucose) and colloid (icodextrin) osmotic agents markedly enhances peritoneal fluid and solute transport during the long PD dwell.

    Science.gov (United States)

    Freida, Philippe; Galach, Magda; Divino Filho, Jose C; Werynski, Andrzej; Lindholm, Bengt

    2007-01-01

    Fluid and sodium removal is often inadequate in peritoneal dialysis patients with high peritoneal solute transport rate, especially when residual renal function is declining. We studied the effects of using simultaneous crystalloid (glucose) and colloid (icodextrin) osmotic agents on the peritoneal transport of fluid, sodium, and other solutes during 15-hour single-dwell exchanges using 3.86% glucose, 7.5% icodextrin, and a combination fluid with 2.61% glucose and 6.8% icodextrin in 7 prevalent peritoneal dialysis patients with fast peritoneal solute transport rate. The combination fluid enhanced net ultrafiltration (mean 990 mL) and sodium removal (mean 158 mmol) compared with 7.5% icodextrin (mean net ultrafiltration 462 mL, mean net sodium removal 49 mmol). In contrast, the 3.86% glucose-based solution yielded negligible ultrafiltration (mean -85 mL) and sodium removal (mean 16 mmol). The combination solution resulted in significantly improved urea (+41%) and creatinine (+26%) clearances compared with 7.5% icodextrin. A solution containing both crystalloid (glucose 2.61%) and colloid (icodextrin 6.8%) osmotic agents enhanced fluid removal by twofold and sodium removal by threefold compared with 7.5% icodextrin solution during a dwell of 15 hours, indicating that such a combination solution could represent a new treatment option for anuric peritoneal dialysis patients with high peritoneal solute transport rate.

  12. Safety of Sodium-Glucose Co-Transporter 2 Inhibitors during Ramadan Fasting: Evidence, Perceptions and Guidelines

    Directory of Open Access Journals (Sweden)

    Salem A. Beshyah

    2016-06-01

    Full Text Available Sodium-glucose co-transporter 2 (SGLT2 inhibitors are a new glucose-lowering therapy for T2DM with documented benefits on blood glucose, hypertension, weight reduction and long term cardiovascular benefit. They have an inherent osmotic diuretic effect and lead to some volume loss and possible dehydration. There is some concern about the safety of using SGLT2 inhibitors in Muslim type 2 diabetes mellitus (T2DM patients during the fast during Ramadan. Currently, there is a dearth of research data to help guide physicians and reassure patients.  One study confirmed good glycemic control with less risk of hypoglycemia and no marked volume depletion. Data in the elderly and in combination with diuretics are reassuring of their safe to use in Ramadan in general. SGLT2 inhibitor-related diabetic ketoacidosis has not been reported during Ramadan and is unlikely to be relevant. Survey of physicians revealed that the majority felt that SGLT2 inhibitors are generally safe in T2DM patients during Ramadan fasting but should be discontinued in certain high risk patients. Some professional groups with interest in diabetes and Ramadan fasting included SGLT2 inhibitors in their guidelines on management of diabetes during Ramadan. They acknowledged the lack of trial data, recommended caution in high risk groups, advised regular monitoring and emphasized pre-Ramadan patients’ education. In conclusion, currently, knowledge, data and experience with SGLT2 inhibitors in Ramadan are limited. Nonetheless, stable patients with normal kidney function and low risk of dehydration may safely use the SGLT2 inhibitors therapy. Higher risk patients should be observed carefully and managed on individual basis.

  13. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  14. Studies of genetic variability of the glucose transporter 2 promoter in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Møller, A M; Jensen, N M; Pildal, J

    2001-01-01

    This study was performed to test the hypothesis that genetic variation in the promoter of the glucose transporter 2 (GLUT2) might predispose to prediabetic phenotypes or type 2 diabetes. A total of 1611 bp comprising the minimal promoter region of the GLUT2 gene were examined by combined single-strand...... conformational polymorphism and heteroduplex analysis followed by direct sequencing of identified variants on genomic DNA from 96 randomly recruited Danish type 2 diabetic patients. We identified 4 nucleotide variants, -447g-->a, -149c-->a, -122t-->c, and -44g-->a. None of the variants were positioned in known.......0% (24.4-33.6), respectively] as in 241 age-matched glucose-tolerant subjects [13.1% (9.8-16.4), 11.2% (8.3-14.1), and 33.4% (28.8-38.0), respectively]. The -447g-->a mutation was only identified in a single diabetic patient and did not show cosegregation with diabetes in the family of the proband...

  15. Brain microvasculature defects and Glut1 deficiency syndrome averted by early repletion of the glucose transporter-1 protein.

    Science.gov (United States)

    Tang, Maoxue; Gao, Guangping; Rueda, Carlos B; Yu, Hang; Thibodeaux, David N; Awano, Tomoyuki; Engelstad, Kristin M; Sanchez-Quintero, Maria-Jose; Yang, Hong; Li, Fanghua; Li, Huapeng; Su, Qin; Shetler, Kara E; Jones, Lynne; Seo, Ryan; McConathy, Jonathan; Hillman, Elizabeth M; Noebels, Jeffrey L; De Vivo, Darryl C; Monani, Umrao R

    2017-01-20

    Haploinsufficiency of the SLC2A1 gene and paucity of its translated product, the glucose transporter-1 (Glut1) protein, disrupt brain function and cause the neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). There is little to suggest how reduced Glut1 causes cognitive dysfunction and no optimal treatment for Glut1 DS. We used model mice to demonstrate that low Glut1 protein arrests cerebral angiogenesis, resulting in a profound diminution of the brain microvasculature without compromising the blood-brain barrier. Studies to define the temporal requirements for Glut1 reveal that pre-symptomatic, AAV9-mediated repletion of the protein averts brain microvasculature defects and prevents disease, whereas augmenting the protein late, during adulthood, is devoid of benefit. Still, treatment following symptom onset can be effective; Glut1 repletion in early-symptomatic mutants that have experienced sustained periods of low brain glucose nevertheless restores the cerebral microvasculature and ameliorates disease. Timely Glut1 repletion may thus constitute an effective treatment for Glut1 DS.

  16. Multiple signalling pathways redundantly control glucose transporter GLUT4 gene transcription in skeletal muscle

    DEFF Research Database (Denmark)

    Murgia, Marta; Elbenhardt Jensen, Thomas; Cusinato, Marzia

    2009-01-01

    Increased GLUT4 expression in skeletal muscle is an important benefit of regular exercise, resulting in improved insulin sensitivity and glucose tolerance. The Ca2+/calmodulin-dependent-kinase II (CaMKII), calcineurin and AMPK pathways have been implicated in GLUT4 gene regulation based...... on pharmacological evidence. Here, we have used a more specific genetic approach to establish the relative role of the three pathways in fast and slow muscles. Plasmids coding for protein inhibitors of CaMKII or calcineurin were co-transfected in vivo with a GLUT4 enhancer-reporter construct either in normal mice...... or in mice expressing a dominant negative AMPK mutant. GLUT4 reporter activity was not inhibited in the slow soleus muscle by blocking either CaMKII or calcineurin alone, but was inhibited by blocking both pathways. GLUT4 reporter activity was likewise unchanged in the soleus of dnAMPK mice...

  17. Inhibition of glucose-transporter 1 (GLUT-1) expression reversed Warburg effect in gastric cancer cell MKN45.

    Science.gov (United States)

    Zhang, Tian-Biao; Zhao, Ying; Tong, Zhao-Xue; Guan, Yi-Fu

    2015-01-01

    Glucose transporter-1 (GLUT-1) plays critical roles in cancer development and progression. Warburg effect (aerobic glycolysis) contributes greatly to tumorigenesis and could be targeted for tumor therapy. However, published data on the relationship between GLUT-1 and Warburg effect are scarce. In this study, gastric cancer cell, MKN45, was transfected with GLUT-1 shRNA using Lipofectamine 2000. Oxygen consumption, LDH activity, lactate production and cytoplasmic pyruvate were detected after MKN45 cells with GLUT-1 knockdown. In the last, hexokinase 1 (HK1), HK2, and pyruvate kinase M2 (PKM2) expression were detected by using western blot. In this study, we showed that inhibition of GLUT-1 expression reversed Warburg effect in MKN45 cells, and induced apoptosis.

  18. Effect of in vivo injection of cholera and pertussis toxin on glucose transport in rat skeletal muscle

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Han, X; Petersen, L N

    1997-01-01

    hyperinsulinimia, increased plasma free fatty acid levels, increased adenosine 3',5'-cyclic monophosphate (cAMP) concentrations in muscle, but no difference in plasma catecholamines. Twenty-five hours after CTX treatment, GLUT-4 protein in both soleus and red gastrocnemius muscles was decreased, whereas no change...... in GLUT-1 protein content was found. In contrast, GLUT-4 mRNA was unchanged, but transcripts for GLUT-1 were increased > or = 150% in all three muscles from CTX-treated rats. The findings suggest that CTX via increased cAMP impairs basal as well as insulin- and contraction-stimulated muscle glucose...... transport, at least in part from a decrease in intramuscular GLUT-4 protein....

  19. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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…

  2. Transcriptional regulation of the gene for glucose transporter GLUT4 in skeletal muscle. Effects of diabetes and fasting.

    Science.gov (United States)

    Neufer, P D; Carey, J O; Dohm, G L

    1993-07-05

    GLUT4 glucose transporter protein and mRNA levels in rat skeletal muscle are decreased with streptozotocin (STZ)-induced diabetes and increased by fasting, indicating that GLUT4 expression may be regulated at the pretranslational level. The purpose of the present study was to determine whether GLUT4 is subject to transcriptional regulation in skeletal muscle under the altered metabolic conditions of diabetes and fasting. Nuclei were isolated from red and white portions of the quadriceps and gastrocnemius/plantaris muscles of control, 7-day STZ-diabetic, and 3-day fasted rats. STZ-induced diabetes resulted in a 35% reduction in GLUT4 transcription in red skeletal muscle and thus accounted for a major portion of the corresponding 50% reduction in GLUT4 mRNA observed in red skeletal muscle. STZ-induced diabetes had no significant effect on GLUT4 transcription or mRNA in white skeletal muscle. Fasting, however, significantly increased both GLUT4 transcription (2.2-fold) and mRNA (2.9-fold) in white skeletal muscle with no change detected for either parameter in red skeletal muscle. The nearly 2-fold higher steady-state GLUT4 mRNA in red versus white skeletal muscle of control rats was not associated with any difference in basal transcription. These findings demonstrate that expression of the GLUT4 glucose transporter protein in skeletal muscle is subject to regulation in vivo at the level of transcription of the GLUT4 gene. In addition, GLUT4 transcription is regulated in a fiber type-specific manner in response to the metabolic challenges elicited by STZ-induced diabetes and fasting.

  3. Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia.

    Directory of Open Access Journals (Sweden)

    Tolunay Beker Aydemir

    Full Text Available ZIP14 (slc39A14 is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia of acute inflammation. ZIP14 can transport Zn(2+ and non-transferrin-bound Fe(2+ in vitro. Using a Zip14(-/- mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/- mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/- mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/- phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are

  4. Cardiovascular mortality and morbidity in patients with type 2 diabetes following initiation of sodium-glucose co-transporter-2 inhibitors versus other glucose-lowering drugs (CVD-REAL Nordic)

    DEFF Research Database (Denmark)

    Birkeland, Kåre I; Jørgensen, Marit E; Carstensen, Bendix

    2017-01-01

    BACKGROUND: In patients with type 2 diabetes and a high cardiovascular risk profile, the sodium-glucose co-transporter-2 (SGLT2) inhibitors empagliflozin and canagliflozin have been shown to lower cardiovascular morbidity and mortality. Using real-world data from clinical practice, we aimed......% (36 362 of 91 320) were women and prevalence of cardiovascular disease was 25% (22 686 of 91 320). 94% of the total SGLT2 inhibitor exposure time was for use of dapagliflozin, with 5% for empagliflozin, and 1% for canagliflozin. Compared with other glucose-lowering drugs, use of SGLT2 inhibitors...

  5. Transport and utilization of hexoses and pentoses in the halotolerant yeast Debaryomyces hansenii.

    Science.gov (United States)

    Nobre, A; Lucas, C; Leão, C

    1999-08-01

    Debaryomyces hansenii is a yeast species that is known for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources. Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars. Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars. Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other. Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system. Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose. The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide m-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl. When the V(max) values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.

  6. Bone effects of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Blevins, Thomas C; Farooki, Azeez

    2017-01-01

    Canagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor approved for the treatment of type 2 diabetes mellitus (T2DM), lowers blood glucose by inhibiting renal glucose reabsorption and increasing urinary glucose excretion. It has been reported that SGLT2 inhibitors may have potential adverse effects on bone, including increased fracture risk and decreased bone mineral density (BMD). Across clinical studies, canagliflozin was not associated with meaningful changes in serum or urine calcium, vitamin D, or parathyroid hormone. Minimal increases in serum phosphate and magnesium that were within normal limits were seen with canagliflozin versus placebo. Canagliflozin was associated with increases in serum collagen type 1 beta-carboxy telopeptide (beta-CTX), a bone resorption marker, and osteocalcin, a bone formation marker. Decreases in total hip BMD were seen with canagliflozin 100 and 300 mg versus placebo after 2 years (-1.7%, -2.1%, -0.8%; differences of -0.9% and -1.2%), but not at other skeletal sites (normal age-related bone loss, ~0.5-1.0%/year). Changes in beta-CTX and total hip BMD were significantly associated with weight loss, which is known to increase bone resorption markers and decrease BMD. Canagliflozin was associated with a higher fracture incidence in an interim analysis of the CANagliflozin cardioVascular Assessment Study (CANVAS) in patients with a history or high risk of cardiovascular disease (incidence per 100 patient-years of 1.6, 1.6, and 1.1 with canagliflozin 100 and 300 mg and placebo), but not in other clinical studies of patients with T2DM. Fractures tended to occur as early as 12 weeks after initiating treatment and were primarily located in the distal parts of the upper and lower extremities. The reason for increased fracture risk with canagliflozin treatment is unknown, but is likely not related to a direct effect of canagliflozin on bone-related biomarkers. Data from ongoing canagliflozin studies, including CANVAS, will

  7. Immunocytochemical analysis of glucose transporter protein-1 (GLUT-1) in typical, brain invasive, atypical and anaplastic meningioma.

    Science.gov (United States)

    van de Nes, Johannes A P; Griewank, Klaus G; Schmid, Kurt-Werner; Grabellus, Florian

    2015-02-01

    Glucose transporter-1 (GLUT-1) is one of the major isoforms of the family of glucose transporter proteins that facilitates the import of glucose in human cells to fuel anaerobic metabolism. The present study was meant to determine the extent of the anaerobic/hypoxic state of the intratumoral microenvironment by staining for GLUT-1 in intracranial non-embolized typical (WHO grade I; n = 40), brain invasive and atypical (each WHO grade II; n = 38) a