Novel Use Of Fluorescent Glucose Analogues To Identify A New Class Of Triazine-based Insulin Mimetics Possessing Useful Secondary Effects
-based insulin mimetics possessing useful secondary effects D. Jung, H. Ha, X. Zheng, Y. Chang and D. R. Williams, Mol. BioSyst., 2011,7, 346 If you are not the author of this article and you wish to reproduce material from it in a third party non-RSC publication you must formally request permission using RightsLink. Go to our Instructions for using RightsLink page for details. Authors contributing to RSC publications (journal articles, books or book chapters) do not need to formally request permission to reproduce material contained in this article provided that the correct acknowledgement is given with the reproduced material. Reproduced material should be attributed as follows: Reproduced from Ref. XX with permission from the Centre National de la Recherche Scientifique (CNRS) and The Royal Society of Chemistry. Reproduced from Ref. XX with permission from the PCCP Owner Societies. Reproduced from Ref. XX with permission from the European Society for Photobiology, the European Photochemistry Association, and The Royal Society of Chemistry. For reproduction of material from all other RSC journals and books: Reproduced from Ref. XX with permission from The Royal Society of Chemistry. If the material has been adapted instead of reproduced from the original RSC publication "Reproduced from" can be substituted with "Adapted from". In all cases the Ref. XX is the XXth reference in the list of references. If you are the author of this article you do not need to formally request permission to reproduce figures, diagrams etc. contained in this article in third party publications or in a thesis or dissertation provided that the correct acknowledgement is given with the reproduced material. Reproduced material should be attributed as follows: [Original citation] - Reproduced b Continue reading >>
- A Novel Intervention Including Individualized Nutritional Recommendations Reduces Hemoglobin A1c Level, Medication Use, and Weight in Type 2 Diabetes
- A Novel Intervention Including Individualized Nutritional Recommendations Reduces Hemoglobin A1c Level, Medication Use, and Weight in Type 2 Diabetes
- Researchers identify key mechanism by which obesity causes type 2 diabetes
Facs-based Glucose Uptake Assay Of Mouse Embryonic Fibroblasts And Breast Cancer Cells Using 2-nbdg Probe
Cell culture hood (Thermo Fisher Scientific, Forma class II, A2) Cell culture incubator (Thermo Fisher Scientific, Forma series II) FACSCalibur flow cytometer (BD Biosciences) FlowJo software version 10.0.8 or newer (FlowJo) Mouse embryonic fibroblasts (MEFs) are isolated from the embryos of C57BL/6 WT mouse (13.5 days) (Dong et al., 2015). Culture the MEF cells until reaching 80-90% confluence in 10 cm Petri dishes with DMEM growth medium in a humidified cell culture incubator (37 C, 5% CO2). Note: Dont use MEFs beyond passage 3. MEFs usually become senescent at about passage 4 to 5. Remove culture medium and wash cells one time with 10 ml 1x PBS. Note: Use room temperature or pre-warmed 1x PBS from Step A3 to Step A9. Using chilled 1x PBS after Step A9. Trypsinize cells using 4 ml of 0.05% trypsin-EDTA for 3 min at 37 C. Transfer cells to 15 ml polystyrene centrifuge tubes. Harvest cells at 200 x g for 5 min by centrifugation. Wash pelleted cells one time with 5 ml 1x PBS. Count cells using a hemocytometer chamber. Incubate 1 x 106 MEF cells in a 37 C water bath for 2 h with 1 ml of PBS containing 100 M 2-NBDG. Incubate the same number of MEFs in the water bath with 1 ml PBS without 2-NBDG as a negative control. Pellet the cells at 200 x g for 5 min by centrifugation. After washing the cells with chilled 1x PBS, the cells are pelleted at 200 x g for 5 min by centrifugation. Resuspend cells in 0.5 ml of ice-cold 1x PBS with 2% FBS. Note: Always keep cells on ice after this step. Filter cells through a 35 m nylon mesh (the cell-strainer cap of the 5 ml round-bottom polystyrene tubes) to obtain a uniform single-cell suspension in a 5 ml tube. Keep the samples on ice until analysis on a flow cytometer. Perform flow cytometric analysis on a FACSCalibur flow cytometer. Acq Continue reading >>
Use Of Flow Cytometry For Quantitative Analysis Of Metabolism Of Viable But Non-culturable (vbnc) salmonella
A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 110 mM H2O2 in LuriaBertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment. Salmonella is one of the most frequent causes of food-borne diseases in Japan, and is detected not only in food but also in the natural environment, such as air-borne dust, manure, river water, soil, and so on. 1 5 ) Among Salmonella spp., Salmonella enterica serovar Enteritidis (SE) is a common cause of salmonellosis and sometime causes mas Continue reading >>
- Mutual Involvement in Families With Type 2 Diabetes Through Web-Based Health Care Solutions: Quantitative Survey Study of Family Preferences, Challenges, and Potentials
- Improving Blood Flow to the Feet
- Effect of Probiotics on Glucose and Lipid Metabolism in Type 2 Diabetes Mellitus: A Meta-Analysis of 12 Randomized Controlled Trials
What Is The Apropriate Protocol To Measure Glucose Uptake In 3t3-l1 Cells And L6 Cells Using 2-nbdg?
Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes [Show abstract] [Hide abstract] ABSTRACT: Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and -ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells. Response to Comment on: Takeda et al. Loss of ACE2 Exaggerates High-Calorie DietInduced Insulin Resistance by Reduction of GLUT4 in Mice. Diabetes 2013;62:223233 [Show abstract] [Hide abstract] ABSTRACT: We thank Dr. Chhabra and Dr. Lazartigues for their thoughtful comments on the role of ACE type 2 (ACE2) in glycemic control (1). In db/db mice or in C57Bl/6J mice infused with angiotensin II, their group has shown that ACE2 can help protect against hyperglycemia by promoting pancreatic insulin secretion (2,3). Continue reading >>
Simpler Yet Sensitive Glucose Uptake Assay
Simpler Yet Sensitive Glucose Uptake Assay Comparison of Common Methods to Monitor Glucose Uptake Glucose is the primary source of energy for many organisms, and the uptake of glucose is a critical process. Glucose is transported across the cells membrane and trapped by being phosphorylated. In mammalian cells, this is performed by a family of glucose transporters (GLUT) and a few intracellular hexose kinases. Note that measuring glucose uptake is not the same as measuring glucose consumption. Glucose uptake occurs on a rapid time scale of 10 minutes or less and specifically measures transporter activity, whereas changes in glucose concentration involve a multitude of pathways and typically take several hours. We can learn several things from measuring glucose uptake. Changes in glucose uptake can reflect overall changes in metabolism, but there are many specific processes as well. Three common cellular model systems are used for three different purposes in studying glucose uptake: 1) Cancer cells, such as HCT116 and many others, to monitor glucose transporter overexpression, identify glucose transporter inhibitors, or measure general changes in metabolism;2) Fat and muscle cells, such as 3T3-L1, 3T3-L1-MBX, L6, C2C12, to monitor GLUT4 translocation in response to insulin; and3) Immunologically relevant cells, such as T cells or macrophages, to follow the transformation of certain cell types from one stage to another. Figure 1. Panels A and B. Comparison of lum-inescent and radioactive assays. Glucose up-take by 20,000 or 50,000 HCT116 cells initiated with 1 mM 2DG and with 1 Ci 3H-2DG for 10 minutes was measured with luminescent (Panel A) or radioactive (Panel B) assays. Cytochalasin B (50 M), a known glucose transporter inhibitor, was added to determine assay backgro Continue reading >>
Impact Of Glo1 Knock Down On Glut4 Trafficking And Glucose Uptake In L6 Myoblasts
Impact of GLO1 Knock Down on GLUT4 Trafficking and Glucose Uptake in L6 Myoblasts Contributed equally to this work with: Britta Engelbrecht, Bernd Stratmann Affiliation: Ruhr-University Bochum, Diabetes Center, Heart and Diabetes Center NRW, Bad Oeynhausen, Germany Contributed equally to this work with: Britta Engelbrecht, Bernd Stratmann Affiliation: Ruhr-University Bochum, Diabetes Center, Heart and Diabetes Center NRW, Bad Oeynhausen, Germany Affiliation: Institute of Forensic Medicine, University of Bonn, Bonn, Germany Affiliation: Ruhr-University Bochum, Diabetes Center, Heart and Diabetes Center NRW, Bad Oeynhausen, Germany Affiliation: Ruhr-University Bochum, Diabetes Center, Heart and Diabetes Center NRW, Bad Oeynhausen, Germany Methylglyoxal (MG), a highly reactive -dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA kno Continue reading >>
2-nbdg Glucose Uptake Assay Kit (cell-based)
For Research Use Only! Not For Use in Humans. Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolisms and homeostasis. Cancer cells exhibit increased glucose uptake to support their high proliferation rate. 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) is a fluorescent deoxyglucose analog that can be taken up by cells through glucose transporters. However, 2-NBDG cannot be fully utilized in glycolysis because of its modification and thus accumulates inside the cells. Fluorescence generated by this fluorescent glucose analog is proportional to glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. This easy to use non-radioactive kit allows imaging and accurate measurement of glucose uptake in cultured cells in response to insulin, growth factors etc. Vous serez peut-tre galement intress par les produits suivants : Continue reading >>
The Glucose Transporter Glut1 Is Selectively Essential For Cd4t Cell Activation And Effector Function - Sciencedirect
Volume 20, Issue 1 , 1 July 2014, Pages 61-72 The Glucose Transporter Glut1 Is Selectively Essential for CD4T Cell Activation and Effector Function CD4 Tcells express multiple glucose transporters, including Gluts 1, 3, 6, and 8 Glut1 has nonredundant function in activated, but not resting, CD4 Tcells CD4 Th1 and Th17 selectively require Glut1 invivo to regulate immunologic diseases Targeting Tcell glucose metabolism invivo can selectively impact effector cells CD4 Tcell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or oxidative metabolic programs invitro, requirements and mechanisms that control Tcell glucose uptake and metabolism invivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metabolism and function of thymocytes and Teff. Resting Tcells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease invivo. Treg cells, incontrast, were enriched invivo and appeared functionally unaffected and able to suppress Teff, irrespective of Glut1 expression. These data show a selective invivo requirement for Glut1 in metabolic reprogramming of CD4 Tcell activation and Teff expansion and survival. Continue reading >>
Flow Cytometric Method Of Estimation Of Glucose Uptake Using 2nbdg
Flow Cytometric Method of Estimation of Glucose Uptake Using 2NBDG George Washington University, Washington DC 2NBDG is a fluorescent analogue of glucose which represents amount of glucose uptake by cells. Measuring the amount of fluorescence by a flow cytometric method in cells stained with 2-NBDG gives an accurate estimation of the glucose uptake by the cells. In this method, cells undergo a short-time incubation in a glucose free media (to normalize the glucose uptake rate across groups) followed by a 20-30 min. incubation with 0.1-0.2mM 2NBDG. The cells are washed and fluorescence measured in the FL-1 (green fluorescence) channel. It is important to note that 2NBDG assay is an index only for the glucose uptake and should not be compared for glycolytic rate or metabolism. Culture cells O/N in 96 well plate with approximately 5X10 5 cells per well. Make duplicate wells for each group and add/ treat cells with any modulators/ inhibitors/specific culture medium. After O/N incubation, spin down cells and add 100uL of glucose free media to each well and incubate 60 mins. to overnight depending on the type of cells (for T cells 60 min. is sufficient). Timing of incubation should be long enough to show sufficient differences in the end result. Make a working solution of 2-NBDG so that the concentration is 300uM. This is achieved by a 1:100 dilution of the supplied 2-NBDG solution which has a con. of 30mM. It is easy to add 10ul of 2NBDG to 1 ml of glucose free media (300uM) concentration. Next add 100ul of the working 2NBDG to each well containing 100ul of glucose free media (total volume 200ul.) Do not add 2NBDG to one well which is the negative control/isotype. Incubate for 20-30 mins. at 37 degree C ( for T cells; time may vary for other cell type). Wash cells twice wit Continue reading >>
2-nbdg Glucose Uptake Assay Kit (cell-based) Kit-1012 - Creative Biomart
Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolisms and homeostasis. Cancer cells exhibit increased glucose uptake to support their high proliferation rate. 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) is a fluorescent deoxyglucose analog that can be taken up by cells through glucose transporters. However, 2-NBDG cannot be fully utilized in glycolysis because of its modification and thus accumulates inside the cells. Fluorescence generated by this fluorescent glucose analog is proportional to glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. This easy to use non-radioactive kit allows imaging and accurate measurement of glucose uptake in cultured cells in response to insulin, growth factors etc. Measurement of glucose uptake in response to insulin, growth factors, cytokines, mitogens and nutrients, etc. Dual-staining of glucose transporters and glucose uptake Analysis of glucose metabolism and cell signaling in various cell types Continue reading >>
Prime Pubmed | 2-nbdg As A Fluorescent Indicator For Direct Glucose Uptake Measuremen
Type your tag names separated by a space and hit enter 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research. Continue reading >>
2-nbdg Glucose Uptake Assay Kit (cell-based)
2-NBDG Glucose Uptake Assay Kit (Cell-Based) Details for Product No. ABIN2693663, Supplier: Log in to see Log in to see the original catalogue number and supplier of this product. Detection (D), Functional Studies (Func), Flow Cytometry (FACS), Quantification (Q) Submit your validation data for this product and get a full refund. Shipping to: United States ( Change Change ) Shipping to: United States ( Change Change ) Submit your validation data for this product and get a full refund. Fluorescence generated by this fluorescent glucose analog is proportional to glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. This easy to use non-radioactive kit allows imaging and accurate measurement of glucose uptake in cultured cells in response to insulin, growth factors etc. Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolisms and homeostasis. Cancer cells exhibit increased glucose uptake to support their high proliferation rate. 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) is a fluorescent deoxyglucose analog that can be taken up by cells through glucose transporters. However, 2-NBDG cannot be fully utilized in glycolysis because of its modification and thus accumulates inside the cells. Fluorescence generated by this fluorescent glucose analog is proportional to glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. This easy to use non-radioactive kit allows imaging and accurat Continue reading >>
2-nbdg Glucose Uptake Assay Kit (cell-based)
For Research Use Only! Not For Use in Humans. Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolisms and homeostasis. Cancer cells exhibit increased glucose uptake to support their high proliferation rate. 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) is a fluorescent deoxyglucose analog that can be taken up by cells through glucose transporters. However, 2-NBDG cannot be fully utilized in glycolysis because of its modification and thus accumulates inside the cells. Fluorescence generated by this fluorescent glucose analog is proportional to glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. This easy to use non-radioactive kit allows imaging and accurate measurement of glucose uptake in cultured cells in response to insulin, growth factors etc. Sie knnten auch an folgenden Produkten interessiert sein: Continue reading >>
Glucose Uptake Assay Kit (cell-based) (ab204702)
Glucose Uptake Assay Kit (Cell-based) (ab204702) Glucose Uptake Assay Kit (Cell-based)(ab204702) is an easy-to-use, non-radioactive kit allows imaging and accurate measurement of glucose uptake in cultured cells in response to insulin and growth factors. The assay is based ona fluorescent glucose analog, which just like glucose can be taken up by cells through glucose transporters. However, this glucose analog cannot be fully utilized in glycolysis because of its modification and thus accumulates inside the cells. Fluorescence generated by this fluorescent glucose analog is proportional to the glucose uptake by the cells and can be used to measure glucose uptake using fluorescent microscopy and flow cytometry. To validate the assay, the kit includes phloretin, a natural phenol that inhibits glucose uptake. Glucose is a ubiquitous energy source in most organisms and plays a pivotal role in cellular metabolism and homeostasis. Cancer cells exhibit increased glucose uptake to support their high proliferation rate. Suitable for: FM , Flow Cyt , Functional Studies more details Continue reading >>
Glucose Transporter 1-mediated Glucose Uptake Is Limiting For B-cell Acute Lymphoblastic Leukemia Anabolic Metabolism And Resistance To Apoptosis
Original Article | Open Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis Cell Death & Disease volume 5, page e1470 (2014) 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 t Continue reading >>
