Sep 28, 1992 - facially disposed transporters with 2-N-4-(1-azi-. 2,2,2-trifluoroethyl)benzoyl-lY3-bis(~-mannos-4- yloxy)-2-propylamine allowed calculation of ...
Vol. 268, No. 12, Issue of April 25, pp. 8514-8520, 1993
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Printed in U.S.A.
Kinetics of GLUT1 and GLUT4 GlucoseTransporters Expressed in Xenopus Oocytes* (Received for publication, September 28, 1992)
Haruo Nishimura$Q, Federico V. Pallardon(1, Glen A. Seidnerll, SusanVannucci$**, Ian A. Simpson$, and MorrisJ. BirnbaumT$$ From the $Experimental Diabetes, Metabolismand Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Instituteof Health, Bethesda, Maryland20892 and the TDepartmentof Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts02115
The predominant mechanism by which insulin actiInsulin stimulates glucose transport activity in hormonevates glucose transport inmuscle and adipose tissue is responsive tissues primarilyby inducing the redistributionof by affecting the redistribution of the facilitatedhexose the facilitated hexose carrier isoforms, G L U T l and GLUT4, carriers, GLUTl and GLUT4, from an intracellular memsite to the plasma membrane. A quantitative analysis from a n intracellular compartment(s) to the plasma brane (1-3). GLUTl is widely distributed whereas GLUT4 is of this process has been hampered by the lack of reliable determinations for kinetic constants catalyzed byexpressed exclusively in those tissues in which insulin proeach of these isoforms. In order to obtain such infor- ducesa markedincreasein glucose transport activity, i.e. mation, each transporter was expressed in Xenopus heart, skeletal muscle, and white and brown adipose tissue (4, oocytes by the injection of mRNA encoding rat GLUTl 5). In the latter, GLUT4 is the predominant glucose transor GLUT4. Equilibrium exchange 3-0-methylglucose porter species (6; for review, see Refs. 7, 8). a twouptake was measured and the data fitted to A persistent, controversial issue in the study of glucose compartment model, yielding K,,, = 26.2 mM and V,,, transport has been therelative contributions of alterations in = 3.5 nmol/min/cell for GLUTl andK,,, = 4.3 mM and cell surface carrier number and intrinsic catalytic activity to Vmpr= 0.7 nmol/min/cell for GLUT4. Measurement of the regulation of substrate flux (8, 9). Several years ago, the the abundanceof cell surface transporters wasaccomplished by two independent protocols: photolabeling lack of correlation between the change in plasma membrane withtheimpermeant hexose analog2-N-4-(1-aziglucose transporter, as assayed by cytochalasin B binding, 2,2,2-trifluoroethyl)benzoyl-1,3-bis(~-mannos-4and hexose uptake appeared to resolved, be first by the cloning yloxy)-2-propylamine and subcellular fractionationof of cDNAs and preparationof antisera specific to each of the oocytes. Data obtained by either technique revealed transporter isoforms, and subsequently by the development that the ratioof plasma membrane GLUTl toGLUT4 of impermeant photoaffinity labels of the transporter (4-6, was about 4; this paralleled the relative maximal velocities for hexose transport, indicating that the turn- 10). Nonetheless, the extrapolation of measurements of surover numbers for the two isoforms were the same. face transporter, obtained either by Western blot or photoMoreover, measurement of the concentration of exo- affinity labeling, t o predictrates of hexoseflux requires knowledge of the kinetic parameters of each of the isoforms facially disposed transporters with 2-N-4-(1-azi2,2,2-trifluoroethyl)benzoyl-lY3-bis(~-mannos-4involved. Such measurements have been difficult to obtain yloxy)-2-propylamine allowed calculationof the turn- due to the lack of mammalian cells expressing exclusively a over number to be about 20,000 min”. single isoform and the inability to definitively establish the These data indicate that, at low substrate concentranumber of cell surface transporters. Studies to evaluate the tions, the catalytic efficiency of GLUT4 is significantly greater than GLUT1. Extrapolationto mammalian hexose transport kinetics of the different carrier isoforms systems suggests that GLUT4 is responsible for vir- have included experiments using human erythrocytes,which tually all of the hexose uptake in insulin-responsive possess only GLUTl (for review, see Ref. l l ) , insulin-stimutargets, particularly in the presence of hormone. lated adipocytes, in which the major plasma membranespecies is GLUT4 (8,12,13), andXenopus laeuis oocytes, which have low rates of endogenous uptake but inwhich, in principle, any * This work was supported by Grant DK39519 from the National cloned transporter can be expressed (14-16). Using the latter Institutes of Health and a grant from the Juvenile Diabetes Foun- system, several groups have measured the apparent K , for dation (to M. J. B.). The costs of publication of this article were equilibrium exchange of 3-0-methyl-D-glucose as about 20 defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 mM for G L U T l and found theK, for GLUT4 either 2 mM or impossible to determine due t o low levels of expression (14U.S.C. Section 1734 solely to indicate this fact. 3 Present address: Dept. of Endocrinology and Metabolism, Shi- 16). However, the difficulty in establishing the number of cell zuoka General Hospital, 4-27-1Kita-ando, Shizuoka 420, Japan. surface transporters prevented a reliable determination of the 11 Supported by a North Atlantic Treaty Organization fellowship. transporters), the other Present address: Dept. de Fisiologia, Facultad de Medicina, Univer- turnover number (V,,./numberof important parameter for estimating “intrinsic activity.” In sidad de Valencia, Avenue Blasco IhaEz 17, Valencia 46010,Spain. ** Present address: Dept. of Pediatric Neurology, Milton Hershey the experiments presented below, we determined the apparent Medical Center, PennsylvaniaState University, Hershey, PA 17033. K, and Vmaxfor rat GLUTl and GLUT4 catalyzed 3-0$$ To whom correspondence should beaddressed Dept. of Cellular 25 Shattuck St., methyl-D-glucose transport activity in Xenopus oocytes by and Molecular Physiology, Harvard Medical School, analysis of a two-compartment model and utilized the exofaBoston, MA 02115.
8514
Kinetics of GLUTl and GLUT4 in Xenopus Oocytes
8515
1%sodium dodecyl sulfate (0.5 ml) was added to each vial, which were incubated at room temperature with shaking for at least 2 h prior to liquid scintillation spectroscopy. Measurement of 3-0-Methylglucose Equilibrium ExchungeMATERIALS ANDMETHODS Groups of 25-30 RNA- or water-injected oocytes were incubated for Injection of Oocytes-Stage VI oocytes were prepared from gravid approximately 18 h at 19 "cin varying concentrations (0.3-100 mM) X.laeuis females (Nasco, Fort Atkinson, WI) as described previously of 3-0-methyl-D-glucose (3-0-MG).Cells were divided into subgroups (5). For isolation of plasma membrane complexes, the oocytes were of threeto four oocytes in 0.5 mlof modified Barth's solution dissected manually without exposure to collagenase. The day followand the appropriate ing harvest, oocytes were injected with 50 nl of either autoclaved containing 2 pCi of [3H]-3-O-methyl-D-glucose water or RNA(0.4 mg/ml) prepared from rat GLUTl or GLUT4 equilibrium concentration of 3-0-MG. "Zero time" uptake was detercloned into pSP64T, linearized, and transcribed with SP6 polymerase mined by adding D-glucose (200 mM, final concentration) prior to in the presence of cap analog (5, 17, 18).Oocytes were incubated in radioactive 3-0-MG. Uptake a t 22"Cwas terminated by quickly modified Barth's solution (19) at 19 "C for 4 days prior to assay. For washing three times with 8 ml of ice-cold phosphate-buffered saline 0.1 mM phloretin. Individual oocytes were solubilized and counted metabolic labeling, oocytes were incubated in the same solution containing 60 p~ methionine and 1 mCi/ml [35S]Translabel (ICN as described above. Preliminary experiments of equilibrium exchange Radiochemicals, Irvine, CA) for 2 days at 19 "C prior to cell fraction- 3-0-MG uptake indicated that, even after substantialperiods of time, the intracellular space had not completely equilibrated with extracelation. Preparation of Total Membranes-Oocytes were homogenized in lular hexose. As illustrated in Fig. L4, oocytes previously injected TES buffer (20 mM Tris, pH 7.4,l mM EDTA, 255 mM sucrose, 1p M with GLUTl mRNA or water had accumulated a t 2 h only about half aprotinin, 0.1 WM leupeptin, 1 p~ pepstatin, 50 p~ phenylmethylsul- the [3Hl-3-0-MG takenup at 20 h. This was interpreted as indicative fonyl fluoride) and crude membranes pelleted by centrifugation a t of the presence a second, more slowly filled, intracellular compart50,000 revolutions/minute in aBeckman 100.3 rotor. The supernatant ment. Therefore, data were analyzed by a two-compartment model, contained no glucose transporter detectable by Western blotting (data as follows. not shown). Membranes were washed two times by resuspension in When two intracellular compartments are present in series, the TES buffer and centrifugation as above. Plasma membranes were concentration of glucose at time t can be expressed as the following: prepared from insulin-stimulated adipocytes as described previously (20). Membranes were subjected to SDS-PAGE and Western blot Xl(t) = Xoe-klt (Es. 1) analysis with polyclonal antibodies raised against carboxyl termini of GLUTl and GLUT4 (21). The glucose transporters were visualized using '261-labeledprotein A and quantitated by cutting the nitrocellulose paper and placing in a y-counter. Preliminary experiments showed the counts/minute to be linearly related to the amount of transporter protein in the range of concentrations utilized. Plasma membrane complexes from oocytes injected with both GLUTl andGLUT4 mRNA were prepared as described by Wall and Pate1 (22), except that only two to three strokes were used in the where Xl(t) is the concentration of [3H]-3-0-MGin the extracellular initial homogenization. Purified membranes were solubilized in 0.4% medium, and Xz(t) and XJt)are the concentrations in the first and SDS, 20 mM Tris-HC1, pH 7.5, 100 mM NaCl, and 2 mM EDTA, 0.1 second compartmentin the cell. When each compartment has a volume 20% Triton X-100 added, and reacted overnight a t 4 "C with antisera specific for the carboxyl termini of GLUTl or GLUT4. The immune complexes were adsorbed with protein A-sepharose, washed three times with 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, GLUTl 0.1% Triton X-100, and 1mM EDTA, eluted into transporterloading buffer (23), and visualized by SDS-polyacrylamide electrophoresis and autoradiography. Radioactivity was quantitated using a Molecular Dynamics phosphorimager equipped with ImageQuant software. Photoaffinity Labeling of Glucose Transporters-Photoaffinity labeling of glucose transporters with [3H]ATB-BMPAwas performed according the method of Holman et al. (6) with modifications. Intact oocytes or adipocyte plasma membranes were incubated in 150 pl of modified Barth's medium with [3H]ATB-BMPA (500 pCi) in a 200pl fluorescence quartz cuvette at room temperature. The cuvette was irradiated six times for 30 s using a Rayonet photochemical reactor equipped with 300-nm lamps. Cells or membranes were solubilized in 0 60 30 90 1201200 a detergent buffer (1%SDS, 1%Triton X-100, 0.4% deoxycholate, 66 mM EDTA, 50 mM Tris-HC1, pH 7.4) for 1 h at 4 "C, centrifuged a t 10,000 X g for 30 min., and the supernatant was diluted 10-fold with 1%Triton X-100, 66 mM EDTA, 50 mM Tris-HC1, such that the final concentration of SDS was 0.1%. The extract was incubated with a-GLUT4- or a-GLUT1-coated proteinA-Sepharose CL-4B for GLUT4 12 h at 4 "C. Immunoprecipitations were performed three successive times for each sample and thepellets pooled, resulting in adsorption of >90% of the transporter. The beads were washed, the immune complexes eluted with sample buffer and subjected to SDS-PAGE, and quantitatedby scintillation countingas described (6). Measurement of 2-Deoxyglucose Transport Activity-Groups of 10 oocytes were incubated in 2 ml of modified Barth's medium containing 0.1 mM 2-deoxyglucose and 1 pCi of [3H]2-deoxyglucosefor 30 min at 20 'C. Hexose uptake was linear for at least 30 min, as reported previously ( 5 ) . The reaction was terminated by washing the oocytes lime (min) three times with ice-cold phosphate-buffered saline, quickly aspirating the medium, and dispensing the oocytes to glass scintillation vials. FIG. 1. Time course of 3-0-methyl-D-glucose uptake into oocytes. Oocytes were injected with water or 20 ng of mRNA encodThe abbreviations used are: ATB-BMPA, 2-N-4-(1-azi-2,2,2-tri- ing GLUTl (A)or GLUT4 ( B )and 4 days later assayed for transport fl~0r0ethyl)benz0yl-l,3-bis(~-mannos-4-yloxy)-2-propy~a~in~; 3-0- of 3-0-MG. Oocytes were incubated for 18 h in non-radioactive 5 mM MG, 3-0-methyl-D-glucose; PAGE, polyacrylamide gel electrophore- 3-0-MG, tracer [3H]-3-0-MGwas added, and accumulation of radiosis; dpm, disintegrations/minute. activity measured at thetimes indicated.
cia1 affinity photolabel [3H]ATB-BMPA' t o simultaneously evaluate the numberof cell surface glucose transporters.
+
Kinetics of GLUTl and GLUT4 in Xenopus Oocytes
8516 volume V2and
V3,net
substrate uptake into thecell Si(t) is,
Si(t) =
V2XAt)+ V3X3(t)
(Eq. 4)
Thus, totalhexose uptake is expressed as a two-exponential equation, S i ( t ) = ae-klf
+ be-&+ c
(Es.5)
in which a, b, and c are constants. Because Si(0) = 0, the above equation can be transformed to,
Si(t)= ml(l - e+)
+ m2(1 - e&)
(Eq. 6)
in which ml and m2 are constants. The initial velocity, u(O), is the derivative of Si(t ) a t time 0.
4 0 ) = mlkl
+ mzh
2ooo-
(Eq. 7)
loo0
(Eq. 8)
0
For each concentrationof 3-0-MG, uptakea t five to eight time points was measured, and the radioactivity associated with water-injected cells subtracted. Typically, water-injected oocytes accumulated less than 10% the 3-0-MG of transporter-expressing calls, although a t the earliest time points this approached 50% (Fig. 1). Values were curve-fitted to Equation 6 using the program MLAB (Civilized Software, Inc., Bethesda, MD) with the Marquardt-Levenberg iterative curve fitting algorithm. Next,ml, kl, m2,and k2 were determined, and initial velocity was calculated using Equation 8. Calculations-Statistical significance was tested with one way analysis of variance followed by Duncan's multiple range test and unpaired Student's t test, as appropriate, and differences were accepted as significant at thep < 0.05 level.
-
1
15 5
10
20
Slim Number FIG. 3. Photoaffinity labeling of glucose transporters inoocytes. 10 oocytes were injected with water (Control, A), CLUTl or GLUT4 mRNA (0)and exofacially photolabeled with mRNA (0). 13H]ATB-BMPA.The cells were solubilized and immunoprecipitated with antisera directed against GLUTl (GLUT],Control) or GLUT4 (GLUT4). A gel profile is shown which is representative of a t least three independentexperiments. The molecular size standards are indicated in kDa.
GLUT4 were not detected by Western blot of t o t a l membranes of water-injected cells (Fig. 2). Quantitation of Glucose Transporters in Oocytes-To deterMeasurement of Surface Transporters by Labeling with mine the quantity of glucose transporters expressed in oocytes ATB-BMPA-Fig. 3 shows a typical profile of immunoprecipinjected with in uitrosynthesizedRNA, crude membranes itated transporter from oocytes injected with water, CLUTl were preparedfrom10 oocytes, yielding about 200 pg of mRNA, orGLUT4mRNAand labeled with ['HI ATBprotein/cell. Membranes were subjected to SDS-PAGE and BMPA. Water-injected oocytes contained little or no radioWesternblotusingantisera specific for the GLUTl and activity in immunoprecipitates using antisera directed against GLUT4 transporter isoforms. As a standard, plasma memeither transporter isoform. Oocytes injected with mRNA enbranes from insulin-stimulated rat adipocytes were included coding GLUTl or GLUT4 and exofacially labeled contained on the same immunoblot. This membrane fraction has been 1404 f 83 dpm/cell ( n = 3) and 460 2 66 dpm/cell ( n = 3) shown to contain22 pmol of glucose transporter/mg of protein associated witheachtransporter, respectively. Inparallel as determined by cytochalasin B bindingand a ratio of experiments, plasma membranes from insulin-stimulated fat GLUT4 to GLUTlof 1O:l as assayed by labeling with ATB- cells, which contain 2pmol of GLUTland 20 pmol of BMPA (6). A representative immunoblot is shown in Fig. 2. GLUT4/mg of protein (see above), were photolabeled in the There was no difference in the total level of expression of same concentrationof [3H]ATB-BMPAwith a geometry ideneach of the transporters: 1.8 f 0.4 pmol of GLUTl/cell and tical to that used for intact oocytes. Data from two independ2.4 f 0.6 pmol of GLUT4/cell ( p > 0.05, n = 3). GLUTl and ent experimentsrevealed that the incorporation of photolabel into adipocyte transporters were, respectively 563, 532 dpm/ 0.05 pmol of GLUT1/25 pg of protein and 5897, 5634 dpm/ GLUTl GLUT4 0.5 pmol of GLUT4/25 pg of protein. These results confirm kDa 1 .. . - .. that both transporters are labeled with identical efficiencies 200 of 1.1 X 10' dpm ATB-BMPA/pmol transporter. Measurement of Glucose Transporters in Plasma Membrane loo Complexes-Manually defolliculated oocytes were injected 68with both GLUTl and GLUT4 mRNA and incubated for 4 43days at 19 "C,during the last 2 days in the presence of [3sS] amino acids. Plasma membrane complexes were isolated, solubilized, and divided into 3 equal aliquots for immunoprecip28 itation with nonimmune sera, a-GLUT1, or a-GLUT4. Results of a typical experiment are shown in Fig. 4 A , in which immunoprecipitates from whole cell homogenates are also included. Immunoprecipitation of membranes from GLUT1FIG. 2. Expression of glucose transporters in Xenopus oo- or GLUT4-expressing oocytes with antisera directed against cytes. Oocytes were injected with water (Control)or 20 ng of in oitro the other carrier isoform or non-immune serum consistently synthesized GLUTl or GLUT4 mRNA. Total membranes were preyielded no detectable radioactive species (data not shown). pared and subjected to Western blot using antisera specific for GLUTl or GLUT4. As a standard, plasma membranes purified from The results from six independent experiments are summainsulin-stimulated rat adipocytes (Fat, 25 pg) was also included. rized in Fig. 4B; 7.6% of total cellular GLUTl and 2.0% of GLUT4 cofractionated with plasma membrane complexes. In Molecular mass markers are indicated in kDa. RESULTS
-
Kinetics of GLUTl and GLUT4 in Xenopus Oocytes A
Total
PMC
1 oocyte
30oocytes
8517
A
m r
mM
kDa
- 116 84
15ooo
- 48.5
loo00
- 36.5 - 26.6
5ooo n
0
515
20
10 lime (min)
mM
B
c
l2
0
B
515
10 lime (min)
20
m -\
\
GLUTl
0 0 GLUT1 GLUT1
GLUT4
GLUT4
FIG. 4. Immunoprecipitation of metabolically labeled gluin oocyte plaema membrane complexes. Panel A, oocytes were injected with both GLUTl and GLUT4 mRNA and incubated with a mixture of [R5S]aminoacids. Either intact oocytes (Total) or plasma membrane complexes (PMC) were solubilized and immunoprecipitated with antibody specific for GLUTl or GLUT4. Immune complexes were eluted, subjected to SDS-PAGE, and visualized by fluorography. For total extract, transporterimmunoprecipitated from a single oocyte was loaded in each lane, whereas 30 oocytes provide the material for each lane of immunoprecipitate from plasma membrane complexes. The migration of molecular weight markera is indicated. Panel B, the data from six independent experiments such as that shown in p a n e l A are summarized as percent of t o t a l cellular transporter present in the plasma membrane complex fraction. Data represent the mean f S.E. co88 transporter
spite of significant variability among experiments in the recovery of glucose transport protein in the plasma membrane complexes, the ratio of GLUTl/GLUT4 in this fraction was remarkably consistent: 3.8 f 0.047 ( n= 6). 2-Deoxyglucose Uptake-Uptake of 0.1 mM 2-deoxyglucose was measured as 118 18 and 81 & 13 pmol/30 min/cell for GLUT1- and GLUT4-injected oocytes, respectively. Waterinjectedcells accumulated less than 5 pmol/30min/cell). These values were unaltered by insulin treatment for 30 min (data not shown). Kinetics of 3-0-Methylglucose Transport-Asdescribed above, the primary data from 3-0-MG uptake was best described by a two-compartment model, in which there is a rapidly filled compartment which equilibrates more slowly with a second intracellular compartment. Data from a representative 3-0-MG equilibrium exchange transport assay are
*
-I
0
0
0 0
50
100
150
I 200
v / [SI FIG. 5. Equilibrium exchange 3-0°C transport into oocytes expressing GLUT1 or CLUTI. Oocytes injected with water or mRNA encoding either GLUTl ( p a n e l A ) or GLUT4 ( p a n e l R ) were incubated for 4 days a t 19 ‘C,with the indicated concentration of 3-0°C present during the last18 h. Uptake was initiated by the addition of tracer 13H]-3-0-MGand theassay terminated at thetimes indicated. The accumulation of (‘HJ-3-0-MG in water-injected oocytes has been subtracted. Data are themean f S.E.of three to four oocytes from an experiment representativeof at least three independent experiments. Panel C, Woolf-Hofstee plot of the datafrom panepb A and B. Initial velocities were calculated as described under ”Materials and Methods.”
shown in Fig. 5. Oocytes injected with mRNA encoding either GLUTl or GLUT4were incubated to equilibrium with nonradioactive 3-0-MG and the assay initiated by the addition of radioactive tracer. Concentrations of hexose tested ranged from 0.3 to 30 mM for GLUT4 and 1 to 100 mM for CLUT1. By using Equations 6 and 8, initialvelocities were determined andplottedagainstsubstrateconcentrations according to
Kinetics of GLUTl and GLUT4 in Xenopus Oocytes
8518
Woolf, as advocated by Hofstee (24) (Fig. 5C). From a series of experiments of this kind, the kinetic constants derived were K,,, = 26.2 f 4.9 and V,, = 3491 k 448 pmol/min/cell ( n = 3) for GLUTl and K,,, = 4.3 f 0.6 mM and V,,, = 666 f 187 pmol/min/cell ( n = 3) for GLUT4-expressing oocytes (Table I).
tain absolute plasma membrane abundance with the aim of deriving turnover numbers. The first approach utilized was exofacial labeling with the impermeantglucose analog, ATBBMPA.Thisreagent was developedby Holmanand his colleagues (6,25-27) and has provenuseful for measuring the number of cell surface GLUTl and GLUT4 transporters in mammalian cells. There now exists muchevidence that ATBDISCUSSION BMPA binds to both transporter isoformswith the same 100% efficiency (6, 25, The primary goals of these experiments were to determine affinity and photolabels with virtually the apparent K,,, and V,, for equilibrium uptake of 3-0-MG 27). Thus, the major theoretical drawback of this reagent is in Xenopus oocytes injected with mRNA encoding the GLUTl that it might not recognize transporters which reside on the or GLUT4 glucose transporters and under the same condi- cell surface but are catalytically silent. In this study, we have adapted the ATB-BMPA-labeling tions measure the numberof carriers on thecell surface. This be would permit, for the first time in regard to GLUT4, a realistic technique for Xenopus oocytes. The primary data can assessment of turnover number incells expressing exclusively converted into numbers of transporters in two ways. In the one transporter. Establishing apparent affinitieswas the ini- first, insulin-treated ratadipocyte plasma membranes, which served tial task and largely served to confirm previously published contain a measurable quantityof GLUTl and GLUT4, work (14, 15). Nonetheless, we found that the presence of a as standards and were photolabeled in precisely the same oocytes (6). Since about1.1X lo4dpm ATBsecond, slowly equilibrating compartment for distribution of manner as intact a picomoleof either transporter of the data, BMPA was incorporated into hexose confounded a simple linear transformation of GLUTl and GLUT4 which were instead analyzed by fitting to the equations de- in adipocyte membranes, the amount f 13 and scribed under “Materials and Methods.” The precise biochem- on the surfaceof the oocyte can be calculated as 128 ical nature of this apparent intracellular space remains un- 40 f 8 fmol/cell, respectively (Table I).Alternatively, one can clear, but might well represent the yolk platelets, which oc- take advantage of the fact that essentially all bound ATBcupy about 50% of thetotal volume of the oocyte (22). BMPA is covalently linked to transporter upon exposure to Utilizing such a two-compartment model eliminated the re- ultraviolet light toderive a value for total binding sites,Bmax Kd, for ATB-BMPA quirement for determining intracellular space andprovided a (27). By using the dissociation constant, relatively good fit for the data. Values obtained for the K,,, which is known to be about 150 p M (27), the concentrationof were 26.2 k 4.9 and 4.3 k 0.6mM for GLUTl and GLUT4, free ligand,F, which equals 333 p M , deriving the boundligand, respectively (Table I). The apparent affinity of GLUTl for 3- B, from the specific activity, 10 Ci/mmol, and the Michaelis 0-MG compares favorably with values previously reported, equation, whether determined in human erythrocytes or Xenopus oocytes (Table11).There has been one prior studywhich in the K,,, for GLUT4 expressed in oocytes was found tobe 1.8 mM, or about half of that reported here (14). Measurements of one derives the totalcell surface binding,B,,,, to be 103 f 13 fmol/cellfor GLUTl and 32 f 7fmol/cellfor GLUT4, in GLUT4’s affinity forhexose inmammaliansystemshave relied on the assumption that this isoform catalyzes themajor good agreement with the above values. As illustrated in Fig. portion of 3-0-MG uptake in insulin-stimulated adipocytes. 2, the total cellular transporter can be estimated from quanThe good correlation between estimates inadipocytes and the titative Western blot to be the same for GLUTl and GLUT4 current values (Table 11), as well as calculations described and the fraction expressed on the cell surface 7.2 and 1.8%, respectively (Table I). below based on the presently reported affinities and turnover A completelyindependent methodfor determining the ratio numbers of each of the isoforms, provide direct evidence that GLUT4 is catalytically dominant in hormone-treatedcells. fat of cell surface GLUTl to GLUT4 was to coinject mRNA encoding both carrier isoforms into the same population of Measuring cell surface transporter has proven a difficult task in past experiments. Because of the problems inherent oocytes and prepare a plasma membrane-enriched fraction. t o all current methodologies, we elected to utilize two inde- Wall and Pate1 (22) have described a method for the purifipendent techniques. It should be emphasized that the critical cation of “plasma membrane complexes” almost completely parameter required to calculate therelative “intrinsic activi- devoid of intracellular membranes, as determined by electron ties” of the two transporter isoforms is the ratio of their cell microscopy. Using this procedure, we measured the plasma surface expression. Nonetheless, we have attempted to ascer- membrane GLUTl and GLUT4 to be 7.6 and 2.0% of total TABLEI Summary of kinetics of hexose transport activity in Xenopus oocytes expressing rat GLUTl or GLUT4 2-Deoxyglucose uptake
Surface 3-0-MG transport
K,
V,,
(ATB-BMPA)”
Total (Western)
Surfaceftotal ATB-BMpAb PMC‘
Turnover no?
m.w fmolfcell pmolfrninfcell prnolfcell % min” X I@ 2.7 & 0.5 7.6 % 3.2 7.2 f 0.9 128 f 17 1.8 & 0.4 3491 & 448 26.2 f 4.9 1.7 f 0.4 2.0 f 0.7 2.4 f 0.06 1.7 & 0.04 40 f 8 4.3 f 0.6 666 f 187 NS’ p < 0.05 p < 0.05 p < 0.01 NS p < 0.01 p < 0.05 NS Values calculated on the basis of ATB-BMPA labeling of intact oocytes, using adipocyte plasma membranes as standards. * Values are the number of surface transporters as determined by ATB labeling divided by total cellular transporter as ascertained by Western blot of crude membranes, using adipocyte plasma membranes as standards. Values represent the fraction of surface transporters, as determined by the immunoprecipitation of metabolically labeled transporters from plasma membrane complexes and from total oocyte homogenate. Calculated using the number of surface transporters determined by ATB-BMPA labeling. Comparison of values for GLUTl and GLUT4. NS, not significant. prnolf30 rninfcell
GLUTl GLUT4
118 f 18 90 & 13
Kinetics of GLUT1 a n d GLUT4 in Xenopus Oocytes
8519
TABLEI1 Summary of kinetic constants for GLUTl or GLUT4 3-0-MG equilibrium exchange transport GLUT4
GLUTl
Cell type
E
TN
fYmin"
X1
mM
Xenopus Xenopus Xenopus Xenopus
oocytes"
21 20 17 26
oocytes oocytes oocytes
Human erythrocytes
adipocytes
13.2 1
ND 2.7
-25
TN
mM
XI@ min"
"C
1.8 4.3
NDb ND ND 1.7
22 18 18 22
-5.0' ND 7.2d
ND 5.6' 7.Y 27
ND ND16
2.0-4.0
Rat Rat
37 3T3-Ll adipocytes
Temperature
E
37 7.2'
23d
20 37
Ref.
14 15
study This Reviewed in 11 Reviewed in 8
37
Data are derived from experiments inwhich mammalian transporterswere expressed in oocytes.
* Not determined.
These values are derived from experiments using insulin-stimulatedadipocytes and assume that all measurable transport is catalyzed by GLUTI. See "Discussion" for validityof this assumption. e
Values calculated fromthe displacement of ATB-BMPA. See Ref. 27 for an explanation of the derivation of these turnover numbers.
cellular transporter, respectively (Fig. 4). The striking correlation between the estimate of surface transporters as ascertained by ATB-BMPA photolabeling and subcellular fractionation provides strongsupport for the reliability of these measurements. Moreover, these data argue against the existence of a significant pool of plasma membrane transporters which are not labeled by ATB-BMPA in oocytes. Since the ratio of surface GLUTl/GLUT4 as determined in either of these series of experiments approximates the ratioof the V,, values, the turnover numbers of the two isoforms are essentially the same (Table I). It is possible to apply the kinetic constants obtained from oocyte experiments to measurements of GLUTl and GLUT4 in adipocytes. The velocity of transport into an adipocyte expressing both GLUTl and GLUT4 is, (Eq. 10)
in which the subscripts, and 4, indicate the constants pertaining to GLUTl andGLUT4, respectively. Since, V,,
= TN[GT]
(Eq. 11)
in which T N is the turnover number and [GT]the number of cell surface transporters, the relative uptake in the presence and absence of insulin, v+l/v-l, can be expressed as,
Km1 + [SI
.+
-~
- ---
K- + [SI
.
Moreover, since, as discussed above, TNl = TN,, under the usual assay conditions, i.e. [SI
