Assays of Glucose Entry, Glucose Transporter Amount, and Translocation

  • Jean-François Tanti
  • Mireille Cormont
  • Thierry Grémeaux
  • Yannick Le Marchand-Brustel
Part of the Methods in Molecular Biology™ book series (MIMB, volume 155)

Abstract

Glucose enters the cell by a carrier-mediated, facilitated diffusion mechanism, which, in most tissues, exhibits no energy or counter-ion requirements. In adipose tissues and skeletal muscle, glucose entry is acutely regulated by insulin and other hormones (1,2). Indeed, in those tissues, glucose transporter 4 (GLUT4) is the chief isoform which is, in basal conditions, retained in a specific intracellular storage compartment (3). The GLUT4-containing vesicles are translocated to the plasma membrane in response to insulin, thus allowing for the massive entry of glucose into the cells (1,2). Adipocytes also contain a small proportion of the ubiquituously expressed glucose transporter, GLUT1, which is at a similar level at the plasma membranes and inside the cell (3). Because of this basal distribution, insulin effect on GLUT1 translocation is minor.

Keywords

Toluene Fluoride Brittle Adenosine Syringe 

1 Introduction

Glucose enters the cell by a carrier-mediated, facilitated diffusion mechanism, which, in most tissues, exhibits no energy or counter-ion requirements. In adipose tissues and skeletal muscle, glucose entry is acutely regulated by insulin and other hormones (1,2). Indeed, in those tissues, glucose transporter 4 (GLUT4) is the chief isoform which is, in basal conditions, retained in a specific intracellular storage compartment (3). The GLUT4-containing vesicles are translocated to the plasma membrane in response to insulin, thus allowing for the massive entry of glucose into the cells (1,2). Adipocytes also contain a small proportion of the ubiquituously expressed glucose transporter, GLUT1, which is at a similar level at the plasma membranes and inside the cell (3). Because of this basal distribution, insulin effect on GLUT1 translocation is minor.

The isolated adipocyte is a very convenient system for measuring glucose transport in basal conditions. Further, it is both highly insulin sensitive (EC50 approx 0.1 nM insulin) and responsive (10- to 20-fold stimulation). This system can be used to look for an insulinomimetic effect of drugs (in a search for new therapeutic agents for diabetes) or to determine the ability of cells from patients to respond to insulin. More recently, the possibility of measuring GLUT4 translocation in adipocytes expressing an epitope-tagged GLUT4 transporter has been taken as a way of determining the possible molecular mechanism of insulin action.

This chapter describes the techniques allowing for the measurement of glucose uptake, using glucose itself or glucose analogs (2-deoxyglucose [DOG] and 3 O-methyl glucose [OMG]). The DOG method was first described (4, 5, 6), and is still widely used. The principle of this technique is that labeled 2-DOG is transported into the cells with high affinity, and phosphorylated, but not further metabolized. Labeled 2-DOG phosphate is trapped in the cell, and the rate of uptake can be taken as a measure of unidirectional transport. By contrast, 3 OMG is not phosphorylated, and quickly equilibrates across the cell membrane (7). The third (and easiest) method is based on the premise that glucose metabolism (glucose incorporation into lipids) provides a measurement of glucose transport at very low glucose concentration (<5 μM) (8,9). The chapter then describes the method allowing for a direct measurement of GLUT translocation, using an epitope-tagged GLUT4 as a reporter gene (10,11). Finally, because glucose transporter translocation can be directly followed by immunoblotting GLUT4 (and GLUT1) in the subcellular fractions obtained from control or treated adipocytes, the transporter immunodetection is briefly described. The sheet assay, which is used to visualize GLUT4 translocation in 3T3-L1 adipocytes, after cell sonication, is not described here, since it does not apply to normal adipocytes (12).

2 Materials

  1. 1.
    Krebs-Ringer bicarbonate HEPES (KRBH) buffer: Prepare the following 10X stock solutions (store at 4°C):
    1. a.

      1.2 M NaCl, 40 mM KH2PO4, 10 mM MgSO4, 7.5 mM CaCl2 (see Note 1 ).

       
    2. b.

      100 mM NaHCO3.

       
    3. c.

      300 mM HEPES, pH 7.4.

      To prepare fresh KRBH buffer, mix 10 mL of each stock solution, and add water to 100 mL.

       
     
  2. 2.

    d-Glucose stock solution: Dissolve anhydrous glucose in water at a concentration of 10 mg/mL (store in aliquots at −20°C).

     
  3. 3.

    Adipocyte incubation buffer: To KRBH buffer, add glucose (200X dilution of the glucose solution) and 1% (w/v) bovine serum albumin (BSA) (see Note 2 ). Adjust the pH to 7.4.

     
  4. 4.

    Dulbecco’s modified Eagle’s medium (DMEM).

     
  5. 5.

    Laemmli buffer: 70 mM Tris-HCl, pH 7.0, 3% sodium dodecyl sulfate (SDS), 11% glycerol (13), final concentrations. Stock solutions of this buffer (2X or 4X) are usually prepared.

     
  6. 6.

    1 M HEPES, pH 7.4.

     
  7. 7.

    Potassium cyanide (KCN) stock solution: Dissolve KCN in water at a concentration of 200 mM, and store in aliquots at −20°C (see Note 3 ).

     
  8. 8.

    Insulin: Commercial insulin preparations (rapid type), used for diabetic patient care, can be used.

     
  9. 9.

    Antibodies (Abs) against Myc epitope (Santa Cruz) (see Note 4 ).

     
  10. 10.

    125I-iodinated protein A (see Note 5 ).

     
  11. 11.

    Diisononylphthalate (oil of density 0.972, which allows for the separation of fat cells from the incubation medium) (6).

     
  12. 12.

    Gentamicin solution at 10 mg/mL.

     
  13. 13.

    2059 Falcon tubes see Note 6 ).

     
  14. 14.

    20- and 6-mL polyethylene scintillation vials see Note 6 ).

     
  15. 15.

    Microtubes (400-μL, Beckman type) see Note 6 ).

     
  16. 16.

    15-mL polystyrene flat-bottomed tubes see Note 6 ).

     
  17. 17.

    Nonpyrogenic syringes without needle.

     
  18. 18.

    0.4-cm-gap electroporation cuvets.

     
  19. 19.

    Electroporator system see Note 7 ).

     
  20. 20.

    2-Deoxy-d-glucose and 3-O-methyl-d-glucose solution: Dissolve 2-deoxy-d-glucose or 3-O-methyl-d-glucose in water, at a concentration of 100 mM, and store in aliquots at −20°C.

     
  21. 21.
    Radiolabeled sugars:
    1. a.

      Radiolabeled deoxy-d-glucose, 2-[3H(G)]: 259 GBq/mmol (7 Ci/mmol), 1 mCi/mL in EtOH:water (9∶1); store at −20°C see Note 8 ).

       
    2. b.

      Radiolabeled 3-O-methyl-D-3H glucose: 2.8 TBq/mmol (75.2 Ci/mmol), 1 mCi/mL in EtOH:water (9∶1) see Note 8 ).

       
    3. c.

      Radiolabeled [3-3H]-d-glucose: 647.5 Gbq/mmol (17.5 Ci/mmol), 1 mCi/mL in EtOH:water (9∶1) see Note 8 ).

       
     
  22. 22.

    Cytochalasin B solution: Dissolve cytochalasin B in dimethyl sulfoxide (DMSO) at a concentration of 1.5 mM, and store in aliquots at −20°C see Note 9 ).

     
  23. 23.

    Toluene/butyl PBD scintillation cocktail: Dissolve 4 g butyl PBD (Serva 15075) per liter of toluene.

     
  24. 24.

    Commercial scintillation fluid accepting a small proportion of water (e.g., Ready Safe, Packard).

     
  25. 25.

    Phosphate-buffered saline (PBS).

     
  26. 26.

    Tris-buffered saline (TBS)-Tween: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20.

     
  27. 27.

    Fat-skimmed milk.

     
  28. 28.

    Rabbit anti-GLUT4 and anti-GLUT1 Abs: Abs directed against the C-terminus of GLUT4 and GLUT1, respectively (East Acres Biological, Southbridge, MA).

     
  29. 29.

    Horseradish peroxydase (HRP) conjugated Donkey antirabbit (Amersham).

     
  30. 30.

    Electrochemiluminescence (ECL) detection kit (Amersham).

     
  31. 31.

    Vinyl-covered exposure cassettes.

     
  32. 32.

    Sensitive X-ray film (Sigma, Amersham).

     
  33. 33.

    Nitrocellulose or polyvinylidene fluoride (PVDF) membranes (Millipore).

     

3 Methods

3.1 Measurements of Glucose Uptake by Freshly Isolated Adipocytes

3.1.1 DOG Uptake

  1. 1.

    Isolate adipocytes from epididymal fat pads of male Wistar rats (170–200 g) by collagenase digestion see Note 10 ).

     
  2. 2.

    Wash the isolated adipocytes 2× by flotation in a 50-mL syringe with 30 mL KRBH buffer (which should be prewarmed at 37°C see Note 11 ).

     
  3. 3.

    Resuspend adipocytes as a 30% (v/v) cell suspension see Note 12 ) in incubation buffer without glucose.

     
  4. 4.

    Prepare a series of 15-mL flat-bottomed tubes with 100 μL assay buffer without glucose containing various insulin dilutions (0–100 nM).

     
  5. 5.

    Pipet in triplicates 100 μL adipocyte suspension, and put it in a water bath at 37°C see Note 13 ). Incubate the cells for 10–20 min with the hormone.

     
  6. 6.

    Add in each tube, with a 15-s interval, 50 μL incubation buffer without glucose containing 0.5 mM deoxy-d-glucose and 0.5 μCi of 3H deoxy-d-glucose, and incubate for 2 min at 37°C, with gentle shaking see Note 14 ).

     
  7. 7.

    Stop the transport by adding, with a 15-s interval, 10 μL 1.5 mM cytochalasin B.

     
  8. 8.

    Pipet 240 μL adipocyte suspension in a 400-μL microtube (Beckman type) already containing 100 μL diisononylphthalate.

     
  9. 9.

    Centrifuge 1 min at 6000g in a microcentrifuge at room temperature (RT) to separate the adipocytes from the medium see Note 15 ).

     
  10. 10.

    Cut the tubes through the dinonylphthalate layer, and put the top part of the tube with the fat cake in 6-mL polyethylene scintillation vials.

     
  11. 11

    Add 4 mL liquid scintillation (Ready Safe) in the vials, and vortex well.

     
  12. 12

    Count the radioactivity associated with the cells in a α-counter, using the tritium program.

     
  13. 13.

    Substract the nonspecific transport from all the values see Note 16 ).

     

3.1.2 3-OMG Uptake

  1. 1.

    Proceed as described in Subheading 3.1.1. , steps 1–5.

     
  2. 2.

    Add 50 μL incubation buffer, without glucose, containing 0.5 mM 3-O-methyl-d-glucose, 1 μCi 3-O-methyl-d-3H glucose.

     
  3. 3.

    Incubate for 30 s for basal condition or 5 s for insulin condition see Note 17 ) at 37°C, with gentle shaking.

     
  4. 4.

    Stop the transport with 10 μL 1.5 mM cytochalasin B.

     
  5. 5.

    Proceed as described in Subheading 3.1.1. , steps 8–13.

     

3.1.3 Measurement of Glucose Incorporation into Lipids at Low Glucose Concentration

  1. 1.

    Proceed as described in Subheading 3.1.1. , steps 1 and 2.

     
  2. 2.

    Resuspend adipocytes as a 10–15% (v/v) cell suspension see Note 12 ) in assay buffer with 0.3 μM glucose (1∶183 dilution of the stock glucose solution).

     
  3. 3.

    Prepare a series of 20-mL polyethylene scintillation vials at 37°C, containing 50 μL of the various insulin concentrations (in assay buffer with 0.3 μM glucose) and 50 μL assay buffer with 0.3 μM glucose, 0.2 μCi [3-3H]-d-glucose.

     
  4. 4.

    Pipet every 10 s, in triplicates for each insulin concentration, 900 μL adipocyte suspension into each vial, and incubate for 1 h at 37°C in a water bath, with gentle shaking see Note 13 ).

     
  5. 5.

    Stop the reaction by adding 10 mL toluene/butyl PBD scintillation cocktail, vortex well and wait 2–12 h before counting in a β-counter, using the tritium program see Note 18 ).

     
  6. 6.

    Subtract counts obtained from blank samples to all the values see Note 19 ).

     
  7. 7.

    Do not forget to count an aliquot of the radioactive glucose solution (using a commercial scintillation fluid), which will allow expression of the results in absolute amounts of glucose taken up by the cells.

     

3.2 Determination of Glucose Transporter Translocation, Using an Epitope-Tagged GLUT4 in Transiently Transfected Rat Adipocytes

3.2.1 Transfection of Rat Adipocytes

  1. 1.

    Prepared adipocytes from epididymal fat pads of male Wistar rats (170–200 g) by collagenase digestion see Note 10 ).

     
  2. 2.

    Wash isolated adipocytes twice in a 50-mL syringe with 30 mL prewarmed (37°C) incubation buffer without BSA and once with 30 mL prewarmed DMEM see Note 11 ). Resuspend adipocytes as a 50% (v/v) cell suspension in DMEM.

     
  3. 3.

    Add 400 μL of the cell suspension see Notes 13 and 20 ) in a 0.4-cm-gap electroporation cuvet, along with the plasmid cDNAs (0.5 μg of pCIS2 GLUT4-myc see Note 21 ) and 9.5 μg of empty pCIS2), and perform electroporation with a double electric shock (800 V, 25 μF/200 V, 1050 μF) by using an Easyject electroporator system see Note 7 ).

     
  4. 4.

    Immediately following the electric shock, put the cell suspension (400 μL) in a 2059 Falcon tube containing 1.5 mL DMEM, 25 mM HEPES, pH 7.4, 5% (w/v) BSA, 100 μg/mL gentamicin see Note 22 ), and incubate for 16 h see Note 23 ) in a cell incubator at 37°C under an atmosphere of 5% CO2/95% air.

     

3.2.2 Determination of Amount of Epitope-Tagged GLUT4 at the Cell Surface

  1. 1.

    Pool the tubes containing the transfected adipocytes (three tubes/conditions), and wash adipocytes 3× in a 5-mL syringe with 4 mL incubation buffer (prewarmed at 37°C).

     
  2. 2.

    Resuspend adipocytes in incubation buffer at a 10% (v/v) suspension see Note 12 ) and transfer adipocytes (3 mL) in 20-mL scintillation vials see Note 13 ).

     
  3. 3.

    Incubate the cell suspension for 20–30 min at 37°C in a shaking water bath (50 cycles/min) in the absence or presence of insulin (100 nM final concentration).

     
  4. 4.

    Add KCN (2 mM, final concentration), and wait for 5 min see Note 24 ).

     
  5. 5.

    Transfer the vials containing the adipocyte suspension to a water bath at 25°C (all the following steps are performed at 25ΰC), and incubate with 1 μg/mL Abs to the myc epitope for 1 h, with gentle shaking.

     
  6. 6.

    Wash the cells 3× in a 5-mL syringe with 4 mL incubation buffer.

     
  7. 7.

    Resuspend the adipocytes with 450 μL incubation buffer (the total volume adipocyte + buffer should be 600 μL).

     
  8. 8.

    Pipet 200 μL in triplicate in 15-mL flat-bottomed tubes already containing [125I]-iodinated protein A (200,000 cpm/tubes, 100 μL), and incubate for 1 h at 25°C, with gentle shaking.

     
  9. 9.

    Place 270 μL adipocyte suspension on 100 μL diisononylphthalate in a 400-μL microtube (Beckman type).

     
  10. 10.

    Centrifuge 1 min at 6000g in a microcentrifuge at RT, to separate cells from the medium see Note 15 ).

     
  11. 11.

    Cut the tubes through the dinonylphthalate layer, and push the fat cell cake (with a yellow tip) into a 1.5-mL Eppendorf tube containing 300 μL of Laemmli buffer. Vortex, and boil for 30 min.

     
  12. 12.

    Put the tube in a γ-counter to count the radioactivity.

     
  13. 13.

    Centrifuge 1 min in a microcentrifuge at RT.

     
  14. 14.

    Transfer the aqueous phase, below the oil that is at the top of the tube, using a syringe, into new Eppendorf tubes.

     
  15. 15.

    Normalize the radioactivity by measuring the protein concentration in each sample by bicinchoninic acid assay (Pierce) see Note 25 ).

     
  16. 16.

    Substract nonspecific binding from all the values see Note 26 ).

     

3.3 Immunodetection of GLUT4

Translocation of GLUT4 can be determined following fractionation of adipocytes into plasma membrane and low-density microsomes (see  Chapter 5). The amount of GLUT4 (and possibly GLUT1) in each fraction can be quantified by Western blotting. The immunoblotting is described for GLUT4, but is identical for GLUT1 immunodetection, except that a specific anti-GLUT1 Ab is used.

  1. 1.

    Separate proteins (50–80 μg) from each fraction on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) (13), and transfer proteins to membranes (nitrocellulose or PVDF membranes) see Note 27 ).

     
  2. 2.

    Incubate the membrane in blocking buffer, with gentle shaking at RT for 1–2 h see Note 28 ).

     
  3. 3.

    Incubate the membrane with gentle shaking overnight at 4°C with anti-GLUT4 Ab (diluted 1∶500–1∶1000 in blocking buffer) see Notes 28 ).

     
  4. 4.

    To remove any unbound anti-GLUT4 Ab, wash the membrane 3× (10 min each) with washing buffer see Note 29 ), with gentle shaking at RT.

     
  5. 5.

    Incubate the sheet with either [125I]-protein A (500,000 cpm/mL blocking buffer) see Notes 5 and 28 ) or with HRP-conjugated antirabbit immunoglobulin G (IgG) for ECL detection (freshly diluted to 1∶5000 in TBS-Tween−20), with gentle shaking at RT for 1 h.

     
  6. 6.

    Discard the [125I]-protein A or the secondary Ab and wash the sheet as in step 4.

     
  7. 7.

    When using [125I]-protein A detection, make an autoradiography of the blot. For ECL detection, use the protocol of manufacturer.

     

4 Notes

  1. 1.

    To avoid calcium/phosphate precipitation, calcium chloride must be dissolved separately and added at the end to the salt solution.

     
  2. 2.

    BSA may contain some contaminants with insulinomimetic action. In accordance, highly purified BSA (Cohn fraction V) must be used, and several batches must be tested to select a batch that gives low basal values.

     
  3. 3.

    CAUTION: KCN is highly toxic, and must be handled with care.

     
  4. 4.

    Ig, from rabbit, against the Myc epitope must be used, and is commercially available (Santa Cruz). Do not use mouse Ig, Because protein A does not bind very well to mouse Ig.

     
  5. 5.

    [125I]-iodinated protein A is commercially available (Amersham, ICN) or, alternatively, protein A can be labeled by the chloramine-T method.

     
  6. 6.

    Adipocytes are very sensitive to the nature of the plastic, and may break if wrong tubes or beakers are used. It is important to use the tubes and the vials mentioned in the materials.

     
  7. 7.

    An adequate electroporator is mandatory for successful transfection of adipocytes. An electroporator able to make double electric shock is required. Successful transfection of adipocytes has been obtained using the Easyject electroporator system (11,14).

     
  8. 8.

    The EtOH-water stock solutions of the labeled compounds should be preferred to the aqueous solutions, which are more prone to microbial contaminations.

     
  9. 9.

    Cytochalasin B binds to glucose transporters, and inhibits the glucose transport (3).

     
  10. 10.

    Collagenase dissociation of adipocytes is described in  Chapters 5,  10,  15, and  24 of this book. Approximately 2 mL adipocyte suspension are usually obtained from rats weighing 170–200g. The weight of the rat must not exceed 220g, since adipocytes prepared from larger rats are more brittle and do not respond well to insulin stimulation.

     
  11. 11.

    Adipocytes contain a large amount of triglycerides, and float. To wash adipocytes, clamp a syringe, pour the adipocyte suspension, wait for sufficient time (2–5 min) to allow all the adipocytes to float at the top of the syringe, open the syringe to eliminate the washing solution, and repeat. Centrifugation should be avoided.

     
  12. 12.

    The adipocyte concentration must be between 10 and 30% (v/v), because a higher concentration could be responsible for a lower insulin response.

     
  13. 13.

    To accurately pipet adipocytes, the suspension should be kept homogenous by handshaking of the beakers or tubes during pipeting. Further, the tips of the automatic pipets should be cut to increase their diameter.

     
  14. 14.

    Uptake of DOG in these conditions is linear during the first 5 min following the addition of the medium containing labeled deoxy-d-glucose.

     
  15. 15.

    Because of its density, dinonylphthalate forms a layer between the adipocytes and the medium, and can thus be used to rapidly and efficiently separate adipocytes from the medium (centrifugation for 1 min at 3000–5000g is sufficient).

     
  16. 16.

    Nonspecific transport is determined in the same conditions, except that cytochalasin B (10 μL 1.5 mM stock solution) is added to the labeled medium before the cell addition.

     
  17. 17.

    Since 3-O-methyl-d-glucose can go in and out of the cells, it rapidly reaches an equilibrium. Thus, the uptake of 3-O-methyl-d-glucose is linear during a very short period of time (30 s for basal conditions, 5 s for insulin stimulation). A metronome can be used to time the assay.

     
  18. 18.

    Use a nonaqueous scintillation cocktail such as toluene/butyl PBD to extract radiolabeled lipids from the cells (8). The cell suspension in the labeled medium is carefully mixed with the scintillation fluid by vortexing, to allow for the lipid extraction in the toluene phase containing the scintillant. It is then necessary to wait for 2–12 h before counting the radioactivity, to ensure a complete separation between the aqueous phase and the toluene phase (8). The aqueous phase, which contains the nonincorporated 3H-radiolabeled glucose, although present at the bottom of the scintillation vial, will not be counted. It should be noted that [14C]-glucose cannot be used in this assay instead of [3H]-glucose, because 14C is more energetic than 3H, and 14C-glucose present in the aqueous phase would be counted.

     
  19. 19.

    Blank values are obtained by adding toluene/butyl PBD scintillation cocktail to the cells, before the medium containing radiolabeled glucose

     
  20. 20.

    For each condition (i.e., basal or insulin) three 400-μL aliquots of adipocyte suspension are submitted to electroporation.

     
  21. 21.

    The sequence of GLUT4 has been modified in order to insert a Myc epitope tag in the first extracellular loop of GLUT4 (10,11). Following insulin stimulation, the Myc epitope is exposed toward the extracellular medium, and the amount of GLUT4 at the cell surface can be estimated by the binding of an anti-Myc Ab (10). pCIS2 is an expression vector with a cytomegalovirus promoter and enhancer (15). All the described experiments (10,11) have been performed with this vector, but any vector with a cytomegalovirus promoter gives a high level of expression in adipocytes (11,15,16).

     
  22. 22.

    It is crucial to add gentamicin to the medium to avoid bacterial contamination during the duration of the culture.

     
  23. 23.

    Adipocyte should not be maintained in culture for more than 16–24 h, since the insulin response falls dramatically thereafter.

     
  24. 24.

    KCN, which causes a depletion of cellular adenosine triphosphate (2), is added to prevent the redistribution of GLUT4 during the time of the incubation with the Myc Abs and the protein A.

     
  25. 25.

    Five microliters of the sample are sufficient to measure the protein concentration with bicinchoninic acid (BCA reagent, Pierce), using the microassay protocol. For this volume of sample, SDS at the concentration used in the Laemmli buffer (3%) does not interfere with protein measurement.

     
  26. 26.

    Nonspecific binding of Abs obtained with cells transfected with empty pCIS2 alone, represents 20% of the total binding observed in cells transfected with pCIS GLUT4-Myc in the absence of insulin stimulation.

     
  27. 27.

    SDS-PAGE is not described here. The detailed technique has been described in ref. 17.

     
  28. 28.

    For [125I]-protein A detection, use the following blocking buffer: PBS-5% BSA (w/v) or PBS-5% fat-skimmed milk. For ECL detection, use TBS-Tween-20 containing 5% fat-skimmed milk as a blocking buffer.

     
  29. 29.

    For [125I]-protein A detection, use PBS-1% Nonidet-P40 (v/v). For ECL detection, use TBS-Tween-20.

     

References

  1. 1.
    Rea, S. and James, D. E. (1997) Moving GLUT4. The biogenesis and trafficking of GLUT4 storage vesicles. Diabetes 46, 1667–1677.PubMedCrossRefGoogle Scholar
  2. 2.
    Simpson, I. A. and Cushman, S. W. (1986) Hormonal regulation of mammalian glucose transport. Annu. Rev. Biochem. 55, 1059–1089.PubMedCrossRefGoogle Scholar
  3. 3.
    Gould, G. W. and Holman, G. D. (1993) The glucose transporter family: structure, function and tissue-specific expression. Biochem. J. 295, 329–341.PubMedGoogle Scholar
  4. 4.
    Rodbell, M. (1968) Metabolism of hormones on glucose metabolim and lipolysis. J. Biol. Chem. 239, 375–380.Google Scholar
  5. 5.
    Olefsky, J. M. (1975) Effects of dexamethasone on insulin binding, glucose transport and glucose oxidation of isolated rat adipocytes. J. Clin. Invest. 56, 1499–1508.PubMedCrossRefGoogle Scholar
  6. 6.
    Livingston, J. N. and Lockwood, D. H. (1975) Effect of glucocorticoids on the glucose transport system of isolated fat cells. J. Biol. Chem. 250, 8353–8360.PubMedGoogle Scholar
  7. 7.
    Vinten, J., Gliemann, J., and Østerlind, K. (1975) Exchange of 3-O-methylglucose in isolated fat cells. J. Biol. Chem. 251, 794–800.Google Scholar
  8. 8.
    Moody, A. J., Stan, M. A., and Stan, M. (1974) A simple free fat cell bioassay for insulin. Horm. Metabol. Res. 6, 12–16.CrossRefGoogle Scholar
  9. 9.
    Kashiwagi, A., Verso, M. A., Andrews, J., Vasquez, B., Reaven, G., and Foley, J. E. (1983) In vitro insulin resistance of human adipocytes isolated from subjects with noninsulin-dependent diabetes mellitus. J. Clin. Invest. 72, 1246–1254.PubMedCrossRefGoogle Scholar
  10. 10.
    Quon, M. J., Butte, A. J., Zarnowski, M. J., Sesti, G., Cushman, S. W., and Taylor, S. I. (1994) Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells. J. Biol. Chem. 269, 27,920–27,924.PubMedGoogle Scholar
  11. 11.
    Tanti, J.-F., Grémeaux, T., Grillo, S., Calleja, V., Klippel, A., Williams, L. T., Van Obberghen, E., and Le Marchand-Brustel, Y. (1996) Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J. Biol. Chem. 271, 25,227–25,232.PubMedCrossRefGoogle Scholar
  12. 12.
    Robinson, L. J., Pang, S., Harris, D. S., Heuser, J., and James, D. E. (1992) Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: Effects of ATP, insulin, and GTPγS and localization of GLUT4 to clathrin lattices. J. Cell Biol. 117, 1181–1196.PubMedCrossRefGoogle Scholar
  13. 13.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  14. 14.
    Cormont, M., Bortoluzzi, M.-N., Gautier, N., Mari, M., Van Obberghen, E., and Le Marchand-Brustel, Y. (1996) Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes. Mol. Cell. Biol. 16, 6879–6886.PubMedGoogle Scholar
  15. 15.
    Quon, M. J., Zarnowski, M., Guerre-Millo, M., De La Luz Sierra, M., Taylor, S. I., and Cushman, S. W. (1993) Transfection of DNA into isolated rat adipose cells by electroporation. Evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture. Biochem. Biophys. Res. Commun. 194, 338–346.PubMedCrossRefGoogle Scholar
  16. 16.
    Tanti, J.-F., Grillo, S., Grémeaux, T., Coffer, P. J., Van Obberghen, E., and Le Marchand-Brustel, Y. (1997) Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 138, 2005–2010.PubMedCrossRefGoogle Scholar
  17. 17.
    Smith, F. S. and Titheradge, M. A. (1998) Detection of NOS isoforms by Western-Blot analysis, in Methods in Molecular Biology, Vol 100. Nitric Oxide Protocols (Tithradge, M. A., eds.), Humana, Totowa, NJ, pp. 171–180.Google Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Jean-François Tanti
    • 1
  • Mireille Cormont
    • 1
  • Thierry Grémeaux
    • 1
  • Yannick Le Marchand-Brustel
    • 1
  1. 1.Faculté de MédecineINSERM E99-11NiceFrance

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