The effects of insulin on the level and activity of the GLUT4 present in human adipose cells

Diabetologia, Jun 1995

Human adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine, ATB-BMFA. The rates of 3-O-methyl-D-glucose transport and the cell-surface level of GLUT4 are very similar in the human and rat adipocyte in the basal state. The Vmax for 3-O-methyl-D-glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of GLUT4 suggests that this low transport activity is associated with a low GLUT4 abundance (39·104 sites/cell; 19.9·104 sites at the cell surface). The turnover number for human adipose cell GLUT4 (5.8·104 min−1) is similar to that observed for GLUT4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50% of the GLUT4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient GLUT4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of GLUT4 that is available to respond to insulin is lower in the human than in the rat adipocyte.

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The effects of insulin on the level and activity of the GLUT4 present in human adipose cells

Diabetologia The effects of insulin on the level and activity of the GLUT4 present in human adipose cells I.J. Kozka 0 A . E . Clark 0 J. P. D. Reckless 0 S.W. Cushman 0 G.W. Gould 0 G.D. Holman I 0 0 School of Biology and Biochemistry,University of Bath, Bath, UK 2School of Postgraduate Medicine, Royal United Hospital, University of Bath, Bath, UK 3Experimental Diabetes, Metabolism and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland , USA 4Department of Biochemistry,University of Glasgow , Glasgow , UK Summary H u m a n adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-l,3-bis-(D-mannos-4-yloxy)-2-propylamine, ATB-BMlZA. The rates of 3-O-methyl-Dglucose transport and the cell-surface level of G L U T 4 are very similar in the h u m a n and rat adipocyte in the basal state. The Vmax for 3-O-methyl-I> glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of G L U T 4 suggests that this low transport activity is associated with a low G L U T 4 abundance (39.104 sites/cell; 19.9.104 sites at the cell surface). The turnover number for human adipose cell G L U T 4 (5.8 9 10 4 min-1) is similar to that observed for G L U T 4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50 % of the G L U T 4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient G L U T 4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of GLUT4 that is available to respond to insulin is lower in the human than in the rat adipocyte. [Diabetologia (t995) 38: 661-666] Glucose transport; h u m a n adipocytes; photolabelling 9 Springer-Verlag1995 In isolated rat adipose cells, glucose transporter isoform 4 (GLUT4) constitutes approximately 90 % of the total cellular glucose transporter, the remaining portion being GLUT1 [ 1-3 ]. Immunocytochemical studies on brown [ 4 ] and white [ 5 ] adipose cells and studies utilizing the cell-impermeant photoaffinity compound, 2-N-(4-(1-azi-2,2,2,-trifluoroethyl)benzoyl) - 1,3 - bis - (D-mannos-4-yloxy) - 2 - propylamine (ATB-BMPA) in white rat adipose cells [ 2,6 ], have suggested that very little of the cellular G L U T 4 is present at the cell surface in the absence of insulin. Following insulin stimulation, the intracellular GLUT4 is rapidly translocated to the cell surface where its level is increased by approximately 20-fold compared with basal cells [ 4-6 ]. The G L U T 4 isoform also appears to be the principal glucose transporter isoform in human adipose cells and is thought to be responsible for mediating the major portion of the insulin-stimulated glucose transport in these cells [ 7-9 ]. P e d e r s e n a n d G l i e m a n n [10] s h o w e d t h a t t h e i n c r e a s e in g l u c o s e t r a n s p o r t activity t h a t is a t t a i n a b l e o n insulin s t i m u l a t i o n o f h u m a n a d i p o s e cells is m u c h less t h a n in r a t a d i p o s e cells. To f u r t h e r c h a r a c t e r i z e this d i f f e r e n c e , we h a v e c o m p a r e d t h e 3 - O - m e t h y l - D - g l u c o s e t r a n s p o r t activity w i t h t h e levels o f c e l l - s u r f a c e G L U T 4 as d e t e r m i n e d b y p h o t o l a b e l l i n g w i t h t h e A T B - B M P A p h o t o l a b e l . Studies o n G L U T 1 a n d G L U T 4 p r e s e n t in 3 T 3 - L 1 cells [ 11 ] a n d e x p r e s s e d in X e n o p u s o o c y t e s [ 12 ] h a v e s h o w n t h a t this a p p r o a c h c a n b e u s e d to assess b o t h t h e n u m b e r o f c e l l - s u r f a c e t r a n s p o r t e r s a n d also t h e i r c a t a l y t i c t u r n o v e r . Materials and methods Materials. 3-O-methyl-D-[U-14C]-glucose was from Amersham International, Little Chalfont, Bucks., UK. ATB-[2-3H]BMPA was synthesised as described previously [ 13 ]. Collagenase was from Worthington, Freehold, N.J., USA. Bovine serum albumin was from Sigma, Poole, Dorset, UK and was extensively dialysed and filtered. Insulin was kindly provided by Dr. R. Chance, Eli Lilly, Indianapolis, Ind., USA. Nonaethyleneglycol dodecylether (C12E9) was from Boehringer, Lewes, East Sussex, UK. Patients and adipose tissue. Specimens of human subcutaneous adipose tissue were obtained, with consent, from the abdominal region of female patients undergoing elective gynaecological surgery. The patients were from 20 to 50 years of age, their body mass index (BMI) was 26 + 1 kg/m2, and none suffered from endocrine disorders. For comparison with the female tissue, one specimen was also obtained from a 51-year-old male patient (BMI 23.5 kg/m2) undergoing gastroenterological surgery. The patients were fasted for about 12 h. General anaesthesia was induced with a short-acting barbiturate and was maintained with a mixture of halothane, nitrous oxide and oxygen. For each experiment 10-30 g of specimen tissue was excised at the beginning of surgery and placed in HEPES buffer [(in retool/l) 140 NaC1, 4.7 KC1, 1.25 Mg2SO 4, 2.5 CaC12, 2.5 NaH2PO4, 10 HEPES, pH 7.4] and containing 5 mmol/1 glucose and 1% albumin. The tissue was transported to the laboratory in a thermos flask within 15-30 rain. Preparation of adipose cells and membranes. Adipose cells were prepared according to the methods previously described for rat [ 14-16 ] and human adipose cells [ 17, 18 ]. The tissue was cut into small fragments free of connective tissue and clotted blood. About 2 g of tissue was placed in 25 ml polystyrene tubes containing 3.5 ml of 4 % albumin in HEPES buffer supplemented with 5 mmol/1 glucose and containing 0.7 mg/ml collagenase. The tissue was minced with fine scissors and the adipocytes were isolated by incubation for approximately 30 min at 37 ~ with shaking. The cell suspension was filtered through a nylon mesh with a pore size of 400 ~tm. The cells were washed four times in the 4 % albumin/HEPES buffer without glucose and finally resuspended at 40 % cytocrit. Cells were then incubated in the absence or presence of 20 nmol/1 insulin for 40 min at 37 ~ and further subjected to either transport or ATB-BMPA labelling assays. Membrane fractions were isolated following homogenization and differential centrifugation as described previously [16]. Transport activity assays in human adipose cells. Glucose transport activity was determined by measuring the initial rates of uptake of 50 9mol/1 3-O-methyl-D-glucose at 37~ as previously described for the isolated rat adipose cells [ 14-16 ]. Briefly, 50 ~tl of adipose cells at 40 % cytocrit were rapidly pipetted into 10 ~tl HEPES buffer containing 3-O-methyl-D-[Ut4C]-glucose and unlabelled 3-O-methyl-D-glucose to give a final substrate concentration of 50 ~mol/1. The transport rates were slow compared with rat adipose cells and consequently longer uptake times were required for determination of rate constants. At appropriate times, usually 30 or 90 s for basal cells and 30 s for insulin-stimulated cells, the glucose uptake was terminated by the addition of 3 ml of HEPES buffer containing 0.3 mmol/1 phloretin. The cell-associated radioactivity was determined in the cells recovered after centrifugation through an approximately 0.5 ml layer of silicone oil. The uptake at these times was compared with the equilibrium filling of the cells at 5 min and from these fractional fillings the uptake rate constants were determined as described previously [ 14 ]. The equilibrated radioactivity associated with the cells was also used to calculate the intracellular water space by using the estimate of 8 . 1 0 s cells per ml of 40 % cells [ 17 ]. For the kinetic studies, the competing sugars were placed together with the radioactive tracer at the bottom of the tube in a final volume of 20 ~tl,into which 50 ~1 of cells were pipetted. In the experiments in which ATB-BMPA was used as a transport inhibitor, the cells were first preincubated with the compound for 5 rain. A 50-bd aliquot of cells was then withdrawn and pipetted into the radioactive sugar mixture. A TB-BMPA photolabelling of glucose transporters in human adipose cells. Following stimulation with insulin, and where appropriate a 5-rain preincubation with the competing sugars, 1 ml of cells in 4 % albumin in HEPES buffer was added to 500 ~tCi of ATB-[2-3H]-BMPA in 0.5 ml of HEPES buffer in 35-ram polystyrene dishes and irradiated for 1 rain in a Rayonet photochemical reactor as described previously [ 2,19 ]. Following irradiation the cells were rapidly washed three times with 1% albumin in HEPES buffer at 18 ~ and solubilized in I ml of detergent buffer containing 2 % C12E9 in 5 retool/1 sodium phosphate buffer at pH 7.2 and with the proteinase inhibitors antipain, aprotinin, leupeptin, pepstatin A, each at 1 ~tg/ml. To estimate the levels of the transporters present in the total cellular pool, cells were permeabilized with 0.025 % digitonin in the presence of 500 ~tCi ATB-[2-3H]-BMPA for 10 rain at 18 ~ and then irradiated [ 20, 21 ]. These cells were then, without washing, directly s01ubilized in 2 ml of detergent buffer. For all samples, the non-solubilized material was removed by centrifugation at 20,000 - g for 20 rain. Immunoprecipitation and Western blotting of GLUT4. Rabbit antisera against the GLUT1 and GLUT4 glucose transporters were prepared using synthetic C-terminal peptides as described previously [ 2,13 ]. To immunoprecipitate the photolabelled transporters, 100 ~tl of each antiserum was conjugated to 20 ~1 of protein A-sepharose by mixing for 2 h in 5 retool/1 phosphate buffer at 0-4~ The conjugates were washed with 5 mmol/1 phosphate buffer. The solubilized cell material in detergent buffer was then added to the antiserum-protein A conjugates and mixed at 0-4~ for 2 h with the appropriate antibody. In most cases, the first immunoprecipitation was with anti-GLUT4 antiserum and this was followed by the immunoprecipitation with anti-GLUT1 antiserum. In each case the immunopellets were washed four times with i ml of the detergent buffer containing 1% C12E9and once with detergent free buffer. Finally, the labelled proteins were released from the conjugates with electrophoresis buffer containing 10 % (w/v) sodium docecylsulphate (SDS), 6 mol/1 urea and 10% (v/v) mercaptoethanol. The proteins were resolved on 7 % SDS-PAGE and gel lanes were then separated and sliced. The radioactivity in the gel slices was extracted as previously described [2]. For Western blotting, proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 3 % albumin in 154 mmol/1 NaC1, 10 mmol/1 TRIS-HC1, pH 7.4 containing 0.1% Tween and incubated in the same buffer but containing 1% albumin with affinity purified GLUT4 antibody (4 ~tg in 10 ml). The bound antibody was localized by incubation with 12SI-proteinA and autoradiography. Results Glucose transport activity in human adipose cells. Insulin stimulation typically led to an approximately 3fold increase in the rate constant for u p t a k e of a non-saturating 50 ,umol/1 concentration of 3-O-methyl-D-glucose into h u m a n adipose cells (from 0.32 + 0.04min -1 to 0.96+0.07min-1; from 15 experiments). A t equilibrium, 50 ~tmol/1 3-O-methyl-D-[U14C]-glucose distributed into an intracellular volume of 2.9 + 0.2 ~tl/106 cells. To determine the kinetic characteristics of 3-O-methyl-D-glucose transport in the insulin-stimulated state for comparison with the results from photolabelling the cell surface G L U T 4 , the initial rates of uptake of 3-O-methyl-[U-14C]-Dglucose in the presence of increasing concentrations of unlabelled 3-O-methyl-D-glucose were determined. Figure 1 shows a single experiment in basal cells, with K m of 3..6 mmol/1 and Vmax of 0.8 mmol/1 min-1', and a representative experiment from three experiments on insulin-treated cells. In the insulinstimulated state, the Km and Vmax values are 4.7 + 1.1 - mmol/1 and 3.3 + 0.8 mmol/1 9min -1 (from three experiments). These results are consistent with those of Pedersen and G l i e m a n n [ 10 ] who showed that insulin does not m a r k e d l y alter the K m for 3-O-methyl-D-glucose transport activity in h u m a n adipose cells. Previous studies on rat adipose cells have shown that the affinity of G L U T 4 for A T B - B M P A is approximately 200 vmol/1 [ 2,11 ]. In h u m a n adipocytes, A T B - B M P A inhibits 3-O-methyl-D-glucose transport with a K i of approximately 200 ~tmol/1 both in the basal and insulin-stimulated states (Fig. 2). A TB-BMPA labelling o f cell-surface and total-cellular GLUT4 and GLUT1. To assess the cell-surface levels of G L U T 4 and G L U T 1 , these transporters were labelled in intact h u m a n adipose cells with the ATB-[2-3H]-BMPA. The labelled transporters were i m m u n o p r e c i p i t a t e d and resolved on SDS-PAGE. A representative gel profile of the i m m u n o p r e c i p i t a t e d G L U T 4 is shown in Figure 3. Insulin increased the cell-surface exposure of G L U T 4 to the i m p e r m e a n t photolabel by 2-3-fold. B o t h G L U T 4 and G L U T 1 isoforms were labelled by A T B - B M P A . However, the cell-surface level of G L U T 1 was less than 10 % "~ E > I 2.0 I 4.0 1 6.0 I 8.0 I 10.0 3 - O - M e t h y l - D-Glucose (mmol/I) Fig.2 Inhibition of 3-O-methyl-D-glucose by ATB-BMPA. Human adipose cells were incubated in the absence (e) or presence (A) of insulin and then the initial rates of uptake of 50 ~tmol/1 3-O-methyl-D-[U-14C]-glucose were determined in the absence (Vo) or presence (v) of the indicated concentration of ATB-BMPA. The data points shown are the means from two separate experiments I O x E s 700 600 500 400 300 200 Gel slice number Fig.3 ATB-BMPA photolabelling of the cell-surface GLUT4 present in human adipose cells. 1-ml suspensions of human adipose cells treated either with (o) or without (o) 20 nmol/1 insulin for 40 min at 37 ~ were photolabelled by irradiation for 1 rain in the presence of 500 ~tCi of ATB-[2-3H]-BMPA. The cells were washed in 1% albumin/HEPES buffer and solubilized in C12E9 detergent buffer as described in "Methods". Photolabelled material was immunoprecipitated by antiGLUT4-C-terminal peptide antiserum and was then analysed by SDS-PAGE of that of G L U T 4 and no attempt was m a d e to quantify the insulin responsiveness of this isoform. Figure 4 shows the results from a series of experiments in which the levels of G L U T 4 at the cell surface were c o m p a r e d with the total cellular levels. The total cellular G L U T 4 was estimated by labelling the transporters in the presence of digitonin, a comp o u n d which permeabilizes the cells, t h e r e b y allowing the normally i m p e r m e a n t A T B - B M P A access to the intracellular transporter stores [ 20,21 ]. Labelling in the digitonin-treated cells also suggested that G L U T 4 was m u c h m o r e a b u n d a n t than G L U T 1 and constituted m o r e than 80 % of the total cellular glucose transporter pool. In this series of experiments, the proportion of G L U T 4 at the cell-surface was 27.2 + 3 . 5 % in the basal state and increased to 57.7 + 6.6 % in the insulin-stimulated state. Figure 4 also shows that the inclusion of 100 mmol/1 3-O-methyl-D-glucose reduced the level of surface labelling of G L U T 4 b y 82 % and the labelling of the total cellular pool by 74 % of the respective controls. We have to confirmed that the digitonin permeabilization p r o c e d u r e gives a reliable estimate of the proportion of G L U T 4 at the cell surface by Western Basal Insulin Insulin + 3 M G Fig.4 Distribution of GLUT4 in human adipose cells. The photolabelling of GLUT4 in human adipose cells in the basal and insulin-stimulated states was carried out in the presence ([]) and absence ( [ ] ) of 0.025 % digitonin (which allows the normally impermeant reagent access to the intracellularly located glucose transporters). The results show the mean + SEM from 4-9 separate experiments. The ability of 100 mmol/1 3-O-methyl-D-glucose to compete with the ATBBMPA in the photolabelling reaction is also shown (I + 3MG) blotting of plasma m e m b r a n e and total cellular membranes from insulin-treated cells. Figure 5 shows that the level of G L U T 4 is approximately the same in plasma m e m b r a n e and totalcellular m e m b r a n e samples. W h e n expressed as a percentage of the total m e m b r a n e s r e c o v e r e d these Western blot data show that approximately 50 % of the G L U T 4 is at the cell surface of insulin-stimulated rat and h u m a n adipocytes. If we convert the o b s e r v e d level of labelling by A T B - B M P A from dpm (Fig.4) into moles of label b o u n d then we can use the equilibrium affinity constant to obtain a value for the n u m b e r of G L U T 4 molecules at the cell surface (Bmax) according to E q u a tion 1: Bmax B . (KD + F) F Eqn. 1 w h e r e F is the free A T B - B M P A concentration (46 ~tmol/1), K D is the affinity constant (200 ~tmol/1) and B and Bmax are the moles of A T B - B M P A bound. B and Bmax can be expressed either in moles/cell or this unit can be converted to Fmol/1 using the intracellular water space of 2.9 ~1/106 cells. In the basal state the cell surface level of G L U T 4 was found to be 72 fmoles/106 cells or 24 nmol/1 corresponding to 4.3. 10 4 sites/cell. In the insulin-stimulated state the cell surface G L U T 4 was 165 fmol/106 cells or 57 nmol/1 corresponding to 9.9.104 surface sites/cell (39.104 sites/cell). As the Vmax for transport is expressed as mmol/1, min q, the catalytic turnover n u m b e r (TN) can be obtained by dividing the Vmax by the concentration of G L U T 4 according to Equation 2: TN Vmax [GLUT4] Eqn. 2 Comparing the Vmax for insulin-stimulated transport with the concentration of G L U T 4 at the cell surface gives a catalytic turnover n u m b e r of 5.8 9 10 4 min -1 at 37~ Discussion In h u m a n adipose cells, the glucose transport activity in the basal state is similar to that observed in rat adipose cells [ 14 ]. However, there is a major difference in the Vmax in these systems following insulin treatment. The Vm~x for 3-O-methyl-D-glucose is approximately 15-fold lower in h u m a n (this study) than in rat adipose cells [ 14 ]. We have assessed here whether the low transport activity in fully insulin-stimulated h u m a n adipose cells can be attributed to a low abundance of G L U T 4 at the cell surface.Application of the A T B - B M P A photolabelling procedure has shown that this effect is mainly due to a low abundance of G L U T 4 at the cell surface in the insulin-stimulated state, there being 9.9.10 4 sites/human adipose cell present in the plasma membrane. Using a cytochalasin B binding procedure, Simpson et al. [ 16 ] showed that in the plasma m e m b r a n e of rat adipose cells there were 195 9104 cell-surface sites per cell. The turnover n u m b e r of G L U T 4 in human adipose cells has not been previously estimated but several estimates of the turnover numbers of rodent G L U T 4 have been determined. Using cytochalasin B binding data the estimated catalytic turnover of G L U T 4 in rat adipose cells is 5.6.104. min-1 at 37~ [ 16 ]. Using the A T B - B M P A photolabelling procedure to determine G L U T 4 activity in the mouse 3T3-L1 cell line we have calculated a catalytic turnover of 7.9- 10 4. min -1 at 37 ~ [ 11 ]. A much lower value for the turnover of rat G L U T 4 expressed in oocytes has been obtained (1.7 9 10 4. min -1) at 22 ~ [ 12 ]. The catalytic turnover of the G L U T 4 would be expected to increase by about three-fold for a 15 ~ rise in temperature [ 22 ]. There are clearly many assumptions involved in using the A T B - B M P A photolabel to calculate the turnover n u m b e r [ 11,12 ] but the turnover calculated from application of this technique in h u m a n adipose cells (5.8.104. min -1) is strikingly similar to values obtained using other techniques and other cell types. Fig.5 Western blot analysis of the effect of insulin on the distribution of GLUT4 between the plasma membrane and the total-cellular membrane pool. Membranes were isolated from human and rat adipocytes following homogenization and differential centrifugation and proteins were resolved by SDSPAGE. GLUT4 was detected using affinity purified antibody raised against a synthetic GLUT4 C-terminal peptide. Results are from an experiment representative of three separate experiments As approximately half of the cellular G L U T 4 is at the cell surface of both h u m a n and rat adipose cells in the insulin-stimulated state (Figs. 4, 5) [ 6 ], the insulin-stimulated exocytosis process for G L U T 4 seems to be equally efficient in these systems. Since in the basal state, a higher proportion of the total cell G L U T 4 is at the surface (approximately 25 %) than in rat adipocytes (where only approximately 2 % is at the surface) it appears that the G L U T 4 sequestration and retention process [ 6, 23 ] is inefficient in hum a n adipose cells. However, the cell surface levels of G L U T 4 and the Vmax for 3-O-methyl-D-glucose are very similar in the h u m a n and rat adipocytes in the basal state [10,14 and present study]. The basal Vmax is presumably sufficient to provide the basal metabolic needs of the adipocyte. It therefore seems likely that in the basal state, the intracellular retention process has adapted to release a greater proportion of the lower total cellular pool of G L U T 4 to the cell surface of h u m a n adipose cells. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of G L U T 4 that is available to respond to insulin is lower in the h u m a n than in the rat adipocyte. A similar mechanism has been proposed for the adaptation of rat adipose cells to increased cell size. In large rat adipose cells, a depleted intracellular pool of transporters is associated with a greater proportion of the available transporters being distributed to the cell surface [24]. Levels of GLUT1 that are detected in h u m a n adipose cells using the photolabelling procedure are only about 10 % of the G L U T 4 levels and are not easily resolved from the background on SDS-gels. Because of the low abundance of GLUT1 and because Acknowledgements. We are grateful to the Medical Research Council (UK) for grant support and thank the surgeons and nursing staff of the Royal United Hospital, Bath, UK, for their generous help. 1. Zorzano A , Wilkinson W , Kotiar G , Thoidis G , Wadzinski BE , Ruoho AE , Pilch PF ( 1989 ) Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations . J Biol Chem 264 : 12358 - 12363 2. Holman GD , Kozka IJ , Clark AE et al. ( 1990 ) Cell surface labelling of glucose transporter isoform GLUT4 by bismannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester . J Biol Chem 265 : 18172 - 18179 3. Birnbaum MJ ( 1992 ) The insulin-sensitive glucose transporter . Int Rev Cytol 137A : 239 - 289 4. Slot JW , Geuze H J , Gigengack S , Lienhard GE , James DE ( 1991 ) Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat . J Cell Biol 113 : 123 - 135 5. Smith RM , Charron MJ , Shah N , Lodish HF , Jarett L ( 1991 ) Immunoelectron microscopic demonstration of insulin stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxy terminal epitope of intracellular GLUT4 . Proc Natl Acad Sci USA 88 : 6893 - 6897 6. Satoh S , Nishimura H , Clark AE et al. ( 1993 ) Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells: evidence that exocytosis is the critical site of hormone action . J Biol Chem 269 : 17820 - 17829 7. Pilch PF , Wilkinson W , Garvey, WT , Ciaraldi TR Hueckstaedt TR Olefsky JM ( 1993 ) Insulin responsive human adipocytes express two glucose transporter isoforms and target them to different vesicles . J Clin Endocrinol Metab 77 : 286 - 289 8. Garvey WT , Huecksteadt TP , Matthaei S , Olefsky JM ( 1988 ) Role of glucose transporters in the cellular insulin resistance of type II non-insulin-dependent diabetes mellitus . J Clin Invest 81 : 1528 - 1536 9. Garvey WT ( 1992 ) Glucose transport and NIDDM . Diabetes Care 15 : 396 - 417 10. Pedersen O , Gliemann J ( 1981 ) Hexose transport in human adipocytes: factors influencing the response to insulin and kinetics of methylglucose and glucose transport . Diabetologia 20 : 630 - 635 11. Palfreyman RW , Clark AE , Denton RM , Holman GD , Kozka IJ ( 1992 ) Kinetic resolution of the separate GLUT1 and GLUT4 glucose transport activities in 3T3-L1 cells . Biochem J 284 : 275 - 281 12. Nishimura H , Pallardo FV , Seidner GA , Vannucci S , Simpson IA Birnbaum , MJ ( 1993 ) Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes . J Biol Chem 268 : 8514 - 8520 13. Clark AE , Holman GD ( 1990 ) Exofacial labelling of the human erythrocyte glucose transporter with azitrifluoroethylbenzoyl-substituted bismannose . Biochem J 269 : 615 - 622 14. Taylor LR Holman GD ( 1981 ) Symmetrical kinetic parameters for 3-O-methyl-D-glucose transport in adipocytes in the presence and in the absence of insulin . Biochim Biophys Acta 642 : 325 - 335 15. Whitesell RR , Gliemann J ( 1979 ) Kinetic parameters of transport of 3-O-methylglucose and glucose in adipocytes . J Biol Chem 254 : 5276 - 5283 16. Simpson IA , Yver DR , Hissin PJ et al. ( 1983 ) Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells. Characterization of subcellular fractions . Biochim Biophys Acta 763 : 393 - 407 17. Pedersen O , Hjollund E , Lindskov HO ( 1982 ) Insulin binding and action on fat cells from young healthy females and males . Am J Physiol 243 : E158 - E167 18. Cushman SW , Salans LB ( 1978 ) Determination of adipose cell size and number in suspensions of isolated rat and human adipose cells . J Lipid Res 19 : 269 - 273 19. Kozka IJ , Clark AE , Holman GD ( 1991 ) Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes . J Biol Chem 266 : 11726 - 11731 20. Kozka IJ , Holman GD ( 1993 ) Metformin blocks down-regulation of cell-surface GLUT4 caused by chronic-insulin treatment of rat adipocytes . Diabetes 42 : 1159 - 1165 21. Yang J , Clark AE , Kozka IJ , Cushman SW , Holman GD ( 1992 ) Development of an intracellular pool of glucose transporters in 3T3-L1 cells . J Biol Chem 267 : 10393 - 10399 22. Joost HG , Weber TM , Cushman SW ( 1988 ) Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin stimulated rat adipose cells . Biochem J 249 : 155 - 161 23. Yang J , Holman GD ( 1993 ) Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells . J Biol Chem 268 : 4600 - 4603 24. Hissin PJ , Foley JE , Wardzala LJ et al. ( 1982 ) Mechanism of insulin-resistant glucose transport activity in the enlarged adipose cell of the aged, obese rat . J Clin Invest 70 : 780 - 790 25. Keller K , Strube M , Mueckler M ( 1989 ) Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes: comparison of kinetic parameters . J Biol Chem 264 : 18884 - 18889


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I. J. Kozka, A. E. Clark, J. P. D. Reckless, S. W. Cushman, G. W. Gould, Dr. G. D. Holman. The effects of insulin on the level and activity of the GLUT4 present in human adipose cells, Diabetologia, 1995, 661-666, DOI: 10.1007/BF00401836