Modulation of Basal Glucose Transporter K, in the Adipocyte by Insulin and Other Factors*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 261, No. 32, Issue of November 15, PP ,1986 Printed in U.S.A. Modulation of Basal Glucose Transporter K, in the Adipocyte by Insulin and Other Factors* Richard R. Whitesell$ and Nada A. Abumrad (Received for publication, September 12, 1985, and in revised form, April 21, 1986) From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent K, of approximately 35 mm. Under those conditions insulin stimulated transport predominantly by decreasing the transport K,,, (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, ). Our findings were in contrast with earlier reports that the K,,, of basal glucose transport was low (3-5 mm) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 "C, cell agitation for 10 min prior to the transport assay decreased the basal K, from 35 to 12 mm. Deprivation of metabolic substrate produced a further reduction down to 2 mm. Refeeding starved cells with 1 mm glucose returned the K, back up to 12 mm in agitated cells and to 40 mm in stabilized cells. The effects of agitation to lower and of glucose to raise the basal K, were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vm, of basal transport and were without effect on both K, and Vm,, in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 "C caused both basal K, and V, to drop proportionately from 25 to 5 mm, and 13 to 3.6 nmol/(rl*min), respectively. In insulin-stimulated cells, only the Vm,, the room was maintained at this temperature, * 2 "C. was decreased (K, went from 3.5 to 3 mm, V, from Transport Assay-For transport assays cells, pipettes and tubes 45 to 17 nmol/(pl-min)). The results support the con- were maintained at the same temperature. To study the effect of cept that experimental conditions can produce large agitation, a magnetic stirrer was used at about 200 rpm and placed changes in the K, of basal glucose transporters. Fur- in the incubator for experiments at 37 "C. For net uptake studies thermore they explain why, under certain assay con- samples were taken from the stirred cells and pipetted vigorously into ditions (with temperatures around 23 O C or with dep- tubes containing isotopically labeled sugar and then agitated 1-2 s rivation of metabolic substrate), the effect of insulin further. The volumes and concentrations in the assay were: 30 p1 of on transport K,,, is not observed. cells (40%, v/v) and 9 pl of isotopically labeled sugar at 3 X the final concentration desired. After the incubation (12 to 36 s for basal, 1 to Our data also suggest that basal transport character- 10 s for insulin-stimulated cells): ice-cold buffer containing phloretin istics do not persist in insulin-treated cells. We would (200 FM) was added in two rinses of 130 pl each and transferred to propose that one of the actions of insulin (in addition 400-p1 microfuge tubes containing silicone oil, which were centrifuged to raising V,,,,,) is to change the characteristics of basal transporters by overriding metabolic factors which keep the K, high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores. * This work was supported by Institutional Grant BRSG RR and a grant from the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. Recently, in a study of basal and insulin-stimulated transport in adipocytes under experimental conditions which made them particularly responsive to insulin, we reported that the action of insulin was predominantly to alter the K, of transport and to a quantitatively lesser extent, the Vmax (1). On the other hand, quite a few other reports (2-4), including some recent ones (5-7), argue convincingly for the converse, that insulin induces up to a 15-fold increase in V,,, with no change in the K,. In this report we extend our original observation to describe in detail how experimental conditions may lead to different conclusions. We have reconciled most of the differing reports, based on striking effects of temperature, agitation, and substrate deprivation on the kinetics of the basal carrier. We present additional evidence that the K, of basal glucose transport is profoundly affected by insulin and other factors. MATERIALS AND METHODS Adipocyte Preparatwn-Isolation of adipocytes was as described earlier (l), except that Krebs bicarbonate buffer was used during collagenase digestion and for the first two washing steps. The cells were washed two more times and maintained in the Hepes' buffer described. Bovine serum albumin (BSA, 2%) and glucose (1 mm) were included in all buffers unless specified otherwise. Where indicated, cells were treated with insulin, 10 nm at a cell concentration of 40% (v/v) for 10 min at 37 'C. The assay temperature was controlled throughout the experiment to 37 * 2 "C by the use of an infant incubator with a modified thermostat. Where 23 "C was indicated, The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum albumin; MeGlc, 3-0- methyl glucose; DNP, dinitrophenol. * The estimation of initial fluxes in kinetic studies is based on one or several assumptions regarding the transport process, such as symmetry, compartmental homogeneity, etc. In practice, initial fluxes are most easily estimated from short uptakes of isotope into cells previously equilibrated with a similar concentration of sugar, that is, under equilibrium exchange conditions. Most reports, including Refs. 1 and 6, cite only initial rate measurements from s samples for all kinetic determinations for basal cells, including net influx. We found that kinetic coefficients could be determined from such meas urements quite precisely for basal cells. The use of curve-fitting protocols for basal and stimulated cells did not change the conclusions from the data.

2 and cut for counting as described before (1). For equilibrium exchange studies, a different procedure was followed. Cell suspensions at 50% (v/v) were mixed with unlabeled sugar, 3 x final concentration, to give a suspension at 40% (v/v) which was allowed to stand for 1 h. The assay was started by adding 30 pl of cells to 9 pl of isotopic MeGIc, 1 X concentrated. The temperature dependence of basal transport was carried out in one case using an equilibrium exchange efflux design which eliminated complications due to the slow increase in transport activity induced by lowered temperature (8). The effects of an "instantaneous" change in the temperature from 37 to 23 "C on the kinetics of transport were therefore studied by diluting samples of cells, which were previously preloaded at 37 "C, with a 20-fold excess of new buffer at 23 "C. A control experiment was done with all buffers at 37 "C. The dilution buffer contained MeGlc at the loading concentration and no BSA. The dilution and incubation were performed in 400-pl microfuge tubes containing silicone oil which were maintained at the stated temperature for the time periods desired. After incubation times of 0 (zero-time samples were diluted in ice-cold phloretin buffer in place of efflux buffer), 10, 20, 60, or 90 s, during which the tubes were capped and inverted several times, efflux was halted by centrifuging the tubes for 6 s in a Fisher microfuge rapidly run up to its 9 setting. After correction for the water space of the pellet (determined as described earlier (l)), the logarithm of the sugar space remaining after each incubation time was plotted against time to determine the time constants. To test the temperature dependence of insulin-treated cells, the equilibrium exchange uptake design was used instead of the equilibrium exchange efflux assay described above and sampling was at I, 2, and 3 s (timed by a metronome). The cells were maintained at the two desired temperatures before and during the assay. In another series of experiments the temperature dependence of transport in both basal and insulin-stimulated cells was tested using the net uptake assay. In this case DNP, 1 mm, was added to basal and stimulated cells for 5 min at 37 "C. To cool, the DNP-treated cells at 40% (v/v) were removed from the incubator and allowed to sit on the bench min before pipetting onto isotope. In some experiments, uptake of a low concentration of [U-"C] glucose into insulin-treated cells was measured. The assay was as described for MeGlc with the following modification. A layer of corn oil was included with the silicone oil to overlay the cells following centrifugation. The pellets were kept on ice, cut within 2-5 min and dispersed in 0.5 ml, ph 9, 10 mm Hepes. No significant loss of 14C02 occurred during this measurement as shown by the following observations. The rate of decrease in pellet radioactivity as the tubes were left on ice was 0.2 f l%/min and 2%/min if the pellets stood at room temperature. A typical assay takes about 4 min. The radioactive content remained stable after addition to the ph 9 buffer. All the data shown in the figures were drawn from typical experiments which were repeated two to five times with good reproducibility. RESULTS Effects of Agitation on Basal Transport Characteristics in the Absence or Presence of Metabolic Poison-The experimental conditions used to measure kinetic coefficients of glucose transport have varied considerably. Some workers have measured fluxes of methylglucose into cells which were previously deprived of metabolizable substrate3 and agitated by the considerable mixing and stirring required to keep large buoyant cells in homogeneous suspension. Different assay temperatures have been employed as well. Agitation and substrate deprivation can cause elevation of basal rate, as can prolonged incubation at temperatures below 37 "C (8). We describe here the effects of such manipulations on transport kinetics. Fig. 1 shows a time course at 37 "c of uptake of 0.2 mm MeGlc in basal cells stabilized with substrate and then agitated by a stirring bar for 10 min before the assay. The assay By substrate we mean the provision of 2 mm pyruvate or 1 mm glucose in the incubation buffer. The direct interaction of substrate with the glucose carrier can be defined. Pyruvate was without any effect, whileglucose inhibited with a Ki of5-10 mm for insulintreated, and mm for basal cells (1). In general, results were similar whether glucose or pyruvate were provided, and either substrate was used as noted in the legend to each figure. Basal Transport Characteristics of Rat Adipocytes I.O[ 0 30 s after isotope FIG. 1. The effect of stirring to increase transport activity in basal cells and its prevention by DNP. Adipocytes were stabilized at 37 "C by incubating 500 pl of cell suspension in a 13 X 55-mm polypropylene tube for 10 min without stirring in the presence of 2 mm pyruvate. In some cases 1 mm DNP was added for 5 min after stabilization. Finally, a 2 X 5-mm stirring bar was added to the incubation vial and the cell suspension was stirred (at 200 rpm) for 5-10 min prior to the transport assay. The assay was carried out at 37 "C as described under "Materials and Methods," by the vigorous addition of an aliquot of the stirred cell suspension to medium containing 0.2 mm ["CIMeGlc. The uptake was stopped and sampled at the times indicated as seconds (s) after isotope. Open symbols, no additions; solid symbols, DNP was added before stirring commenced; X, DNP was added following stirring for 5 min at 37 "C. Similar results were obtained when 1 mm glucose instead of pyruvate was provided as a substrate (data not shown). was started by pipetting the cells from the stirring solution vigorously onto isotope (see "Materials and Methods"). This procedure gave analogous results to those reported by Vega and Kono (8), although in that case the agitation wasby repeated centrifugation and resuspension. The transport rates for agitated cells were rather high (tllz about 30 s). When dinitrophenol (DNP) was added before stirring commenced, uptake rates were low (til2 about 2 min) and in agreement with our published data for stabilized cells (1). DNP was itself without effect when it was added after stirring had already raised transport activity. Insulin-treated cells were not affected by this procedure (Fig. 2). Fig. 3 shows the consequences of the above-mentioned experimental manipulations on the kinetic coefficients K, and Vm,,. The water spaces used to normalize the data were determined after each run to control for cell loss through breakage, and/or volume changes, which were less than 25% in all cases. Stirring (Fig. 3 and Table I) decreased the high K, of sitting cells without significantly altering the V,,,,,. The effect on the K, was blocked by DNP. The decreases in K, without Vmax changes that we record after cell agitation would be reflected as an increase in transport activity ( Vmax/Km) in line with what was noted by Vega and Kono (8). The effect of DNP suggests that the operation of agitation to reduce the K, is dependent on ATP metabolism. Furthermore, this effect of DNP argues against the involvement of mixing artifacts in the K, change observed with agitated versm stabilized preparations. Effect of Substrate Deprivation on Kinetics of Basal Transport-Although stirring lowered the basal transport K, to 12 mm, that was still 3-4-fold higher than the K, for insulintreated cells incubated under the same conditions. Since others have measured no insulin-induced K, differences in stirred cells at 37 "C, we investigated the possibility that substrate deprivation as practiced in some of those studies

3 15092 Basal Transport Characteristics of Rat Adipocytes TABLE I Summary of transport coefficients of basal cells with various experimental conditions Conditions" Prestabilized at 37 "C, DNP. treated, assayed at 23 "Cb mm nnwl/pl/min s after isotope FIG. 2. Lack of effect of stirring on transport activity of insulin-stimulated cells. Incubations at 37 "C were carried out as described in the legend to Fig. 1, except that insulin, 100 nm, was present during the stabilization period and throughout the experiment. Open symbols, no additions; solid symbols, DNP was added before stirring commenced; x, DNP was added following stirring for 5 min at 37 "C. The same result was obtained when the cells were fed 1 mm glucose, or 2 mm pyruvate (the substrate of the experiment shown), or when no metabolizable substrate was provided mm MaGlc FIG. 3. Effects of stirring and DNP on the kinetics of basal transport. The net uptake rates of several concentrations of MeGlc are shown replotted in order to determine K,,, and V-. Initial rates were estimated from 0.2-, 0.4-, and 0.8-min uptake of ["CIMeGlc by the integrated rate equation method as applied by Taylor and Holman (6). Open symbols, no additions; closed symbols, DNP was added before stirring commenced. The transport coefficients are summarized in Table I. The results were similar whether glucose or pyruvate were used as metabolic substrates. could itself have a K, lowering effect. The effects of starvation on transport kinetics are shown in Fig. 4 using a net uptake experiment with cell stirring at 37 "C as described for Fig. 3. As shown, starvation (by maintaining cells in glucose-free medium for 1 h at 37 "C) had striking effects on basal cells. The K, was lowered from 12 to 2 mm. Refeeding with 1 mm glucose restored the K, to 12 mm within minutes in continuously stirred cell preparations and to about 40 mm when agitation was halted for 20 min (data not shown). The effect of glucose refeeding was blocked by DNP (Fig. 4). In addition, DNP treatment of stabilized cells blocked the decrease in K, upon subsequent removal of glucose (data not shown). Thus, if the cells are treated with DNP, the method of the assay (i.e. stabilization of cells uers'sus stirring and substrate deprivation) should not affect the conclusion as to K, and V,,, of the original preparation. But the similarity between the K,,, of starved-stirred cells and that of insulin-treated cells indicates that under these conditions the action of insulin to modulate the K, will not be seen. Transport V,,, was not affected by starvation, and it was much lower than that of Prestabilized at 37 "C, DNPtreated, assayed at 37 "C Stirred 10 min at 37 "C DNP-treated, then stirred 10 min at 37 "C Starved, stirred 10 min at 37 "C 0.4 Starved, DNP-treated, 2 mm pyruvate added, stirred 10 min at 37 "C ' d "- a Unless indicated by "starved,'' substrate was provided to the cells in the form of 2 mm pyruvate. Similar results were obtained with glucose (Fig. 4). Starvation was accomplished by maintaining cells in substrate-free buffer for 1 h at 37 "C. The assay procedure described under "Materials and Methods" for net uptake was used throughout. The table summarizes the results of two or more separate experiments which, unless noted otherwise, were performed under the same conditions and had the same controls as those shown in the figures. Values for insulin-treated cells were at 23 "C, K, = 3, V,. = 17, and V&Km = 6, and at 37 "C, K,,, = 3.5, V,, = 45, and V-JK,,, = 13 (Fig. 4C). The conditions described other than temperature were largely without effect on cells pretreated with insulin (Fig. 2 and data not shown). e The last four measurements of this series were made with the procedure modified so that 5 min after DNP was added the cell suspensions were washed 2-5 times with glucose-free buffer containing 1 mm DNP. This procedure had the effect of further stabilizing the behavior of basal cells and did not affect the kinetic parameters of insulin-treated cells in four out of five trials. The lower V, values obtained with the last two experimental conditions were within the range of V,, variations observed and do not indicate an effect of starvation to lower basal V-. As shown in Fig. 4 the control fedcell preparation had the same V,. as the starved. insulin-treated preparations (7 uersus 45 nmol/gl/min, Table 1). Effect of Temperature on the Kinetics of Basal and Znsulinstimulated Transport-It has been reported by Czech (9) and later by Whitesell and Gliemann (2) that glucose transport activity in basal cells was similar at 23 and 37 "C, while activity of insulin-treated cells was increased by 2-3-fold over the same temperature interval. This argued for somewhat differing characteristics of basal and insulin-stimulated glucose transport. On the other hand, Vinten, in an efflux study, found similar temperature dependencies of transport with or without insulin treatment (4). Ezaki and Kono (10) showed that a decrease in temperature maintained over a period of min had insulin-like effects. Furthermore, Ezaki et al. proposed that this effect of temperature explained the difference in previously published results. We have re-addressed

4 the question of the different temperature sensitivities of basal and insulin-stimulated carriers using experimental designs which measured the effect of an acute change in temperature on transport kinetics (see Materials and Methods ). This eliminated complications due to the gradual increase in transport activity induced by low temperature (10). Our results are shown in Fig. 5. We found that a drop in the temperature of -2 0 IO 20 mm MeGlc FIG. 4. Effects of substrate deprivation and refeeding on the transport kinetics of basal cells. Adipocytes were washed in substrate-free buffer and stirred 10 min before any additions were made. DNP was then added to half of the cell preparation. Five minutes later, 1 mm glucose (fed) was added as labeled on the figure. Net uptake of [l4c]meg1c was determined from min uptakes after 5-10-min further stirring as described under Materials and Methods and in the legend to Fig.3. The contribution of 1 mm glucose to the saturation of uptake in the refed cells was equivalent to 0.6 mm MeGlc, and the points on the graph were corrected for this by shifting them on the x axis. The correction changed K apparent from 13 to 12 mm and from 4 to 3 mm, respectively. The corrected values corresponded to values determined in cells fed pyruvate instead of glucose (data not shown). Basal Transport Characteristics Adipocytes of Rat basal cells from 37 to 23 C at the moment of initiation of an efflux assay decreased transport rate by lowering both K, and V,,, proportionately (Fig. 5A). The time the cells were kept at the lowered temperature was presumably too short to permit the activation effect described by Ezaki and Kono (10). Transport at a concentration much lower than K, remained virtually constant (the last, a very reproducible finding by us and others, see insert to Fig. 5A). Since an instantaneous temperature change could not be incorporated into uptake assays, DNP treatment was used to prevent the insulin-like effects of incubation at lowered temperature (15). The effects of temperature drop on transport K, and V, were reproduced in uptake assays, using cell preparations treated with DNP (Fig. 5B). This further distinguishes these effects from those described by Ezaki and Kono (10) who found that DNP prevented the insulin-like effect of low temperature. On that same basis the K, lowering effect of temperature can be separated from that produced by stirring which can also be prevented by DNP. Insulin-stimulated cells on the other hand did not exhibit a decrease in the K, as temperature was dropped from 37 to 23 C, but the V,, decreased by 50 to 70% and the transport of low concentrations of sugar, which is definable as V,JK,, was thus affected in a major way. These effects of temperature on transport in insulin-stimulated cells could be observed in preparations exposed to DNP following insulin treatment to mimic the condition of the uptake assay used for basal cells (see inset to Fig. 5B) as well as with preparations without DNP (Fig. 5C). Since a decrease in temperature lowered the K, of basal cells, one might expect that a kinetic study done at the lower A A 30 s 20 /t, frnin) IO 3-10 & Km= 25mM,L * 1 IO IO 20 30?m IO 15 mm MeGlc mm MeGlc mm MeGk FIG. 5. A, temperature effects on transport kinetics of basal cells. The plot shown was generated from equilibrium exchange of various concentrations of MeGlc with efflux of [ 4C]MeGlc. To avoid complications due to the gradual increase in transport activity reportedly induced by low temperature, the change in temperature was accomplished at the moment of the assay by diluting cells into a large volume of buffer maintained at the desired temperature (see Materials and Methods ). Two sets of data used to generate the points near the intercept are shown in the inset. The quantity S/u of the plot was generated by tl,z(1n2), where tllz was taken from the efflux time courses. In three experiments the K,,, and V,, at 37 C in mm and nmol/pl/s, respectively, were: 40 and 14; 100 and 35; and 70 and 25, respectively. K, and V,. at 23 C were 2-3-fold lower in each case. Insulin-treated cells gave a K, under 10 mm in experiments such as these (data not shown). The efflux procedure generally confirms the contention that stabilized basal cells have a higher K, than insulin-treated cells. The wider than usual range of the basal data may be due to the brief agitation of the cells and to dilution. B, temperature effects on kinetics of basal cells after poisoning with DNP. After stabilization at 37 C, 1 mm DNP was added and the incubations were stirred 10 min at the temperature indicated and transport was assayed by the net uptake procedure described in the legend of Fig. 3A and under Materials and Methods. Similar results were obtained with equilibrium exchange of MeGlc and influx of [ CJMeGlc(data not shown). The inset to the figure shows the result obtained with cells treated with insulin before DNP. The experiment in this case was camed out in the same way except that uptake rates were estimated from 1-, 2-, and 5-s measurements. C, temperature effects on transport kinetics of insulin-t~ated cells. The cell suspensions were pre-equilibrated with unlabeled sugar at 37 C and then equilibrated for 10 min at the temperature indicated before the assay but DNP was not added. The plot shown was generated from initial rates of equilibrium exchange of MeGlc with uptake of [ CC]MeGlc. Similar results were also obtained from net uptake studies (data not shown).

5 15094 Basal Tra~~rt C~a~acteris~ics of Rat A~i~ocytes temperature will find little or no Km-altering effect of insulin. In fact, cells stirred at 23 C both with and without substrate had a low K,,,, 2.3 mm (data not shown), which was actually slightly lower than that of insulin-trea~d cells. Also, at 37 C when basal cells were stirred and deprived of substrate, K, was similar to that of insulin-treated cells (Fig. 5 and Table I). The effects of temperature, substrate deprivation, and agitation underlie the differences between the report of Whitesell and Abumrad (1) and the report of Whitesell and Gliemann (2). Furthermore, the observation that transport V,,, could remain unaltered under conditions which changed the K,,, of transport implies that the apparent K,of the transporters themselves can change. Are K,,, Changes and ~ ~ T r a of ~ Transpor~ers~~ ~ ~ t Dis- ~ n tinct Processes?-A question which is left outstanding is whether basal and insulin-activated carriers represent two species which are distinct in their mode of activation. If so, the most active glucose carriers could be sequestered in the cell under basal conditions, appearing in the plasma membrane only during activation. The basal transport mechanism could persist in the membrane of stimulated cells but its contribution to the total transport activity would be insignificant at physiological concentrations of 3-5 mm glucose. A preliminary investigation of this possibility involves determining the relative homogeneity of initial rate transport kinetics in insulin-stimulated cells. To test for homogeneity we observed estimates of unidirectional tracer uptake rates at unlabeled sugar concentrations much higher than its low Km in stimulated cells. Under these conditions, the activity of the high K,,, basal transporter, if present, would be expected to become distinguishable. The data were then analyzed by curve-fitting to determine whether a higher K, component could be detected in insulin-stimulated cells. The experimental conditions used were those of equilibrium exchange (Fig. 6). These assays required preequilibration of cells with the nonmetabolizable sugar, MeGlc, at concentrations ranging up to 50 mm. The tracers used to measure influx were [ 4C]glucose(0.2 mm) (A) and [H3]MeGlc (B). Nonmediated diffusion of glucose estimated from uptake of ~-[~H]glucose in ins~in-stimulated cells is very low (about 0.1% of the permeability of D-glucose (see Refs. 1 and 2). This amount of carrier-independent permeability would cause only negligible bending of the S/v plots (Fig. 6A). On the other hand, the diffusion of MeGlc is apparently higher (up to 3% of D-glucose permeability in insulin-stimulated cells) which could lead to some ambiguity in distinguishing diffusion from a persistent high K,,, transport. (We defined diffusion as the uptake of label which was not inhibited by 20 I.LM cytochalasin B). From the uptake time courses used to generate the points of Fig. 6A, initial rates could be estimated both by exponential fitting and from 1-5-s uptake measurements with nearly equal results. Rates were more linear than exponential due to incorporation of YXabeled metabolites during uptake, which in any case was terminated at early time points comparable to those used with MeGlc (under con~tions designed to retain any metabolites for scintillation counting, see Materials and Methods ). The data with [ 4C]glucosewere fit wellby a st.raight line indicating negligible high K, or diffusive com- ponents and arguing against the persistence of the basal transporter in insulin-stimulated cells. The data with tracer MeGlc (B) is consistent with this conclusion although, as already mentioned, ambiguity is introduced by the higher diffusion observed with this sugar. The above interpretation of the data is justified in more detail below taking the experiment with tracer glucose (A). The equation for uptake used to test the hypothesis of two classes of carriers in insulin-treated cells was: v = u1 + u2 = GFg,/(l+ S/Kd + GFB/(l + S/K2), where S is the concentration of MeGlc, G the concentration of glucose, Fg the initial rate of clearance of isotope alone, and Kl and K2 the inhibition constants of MeGlc for each of the hypothetical classes of carrier. The equations relate to the traditional form of the I / / smond o/.., G S PREO?TEO T E N T BASAL WITH (sacod40 %.) ~ db, d subtracted, 0,,#:..-, o,~,.~..., CONTRIBUTION / O,I),..erve fit with 20. V = O.6nmol ls K 2.2 mm &O d=0.008plb I IO MeGlc, mm mm MeGic FIG. 6. Lack of persistence of a basal-like glucose transport component in insulin-treated cells. A, initial entry rates of 0.2 mm [ 4C]glucose were measured in cells equilibrated with a range of methylglucose concentrations. These conditions were optimal to measure the possible contribution of persistent basal tr~spo~rs (see text for more details). The dotted line is the best fit assuming persistence of basal transport. If the basal component had persisted and the transport of insulin-treated cells was hybrid, the data should have fit the dotted line. B, equilibrium exchange of MeGlc with influx of [14C]MeGlc was measured over a large range of MeGlc concentrations. The data were fit with the kinetic coefficients Vmsx, Km, and d (diffusion) shown on the figure (dotted line). The diffusion coefficient necessary to fit the data was similar to the rate of [l C]MeGlc uptake in the presence of 20,LM cytochalasin B under equivalent conditions (0.008 pl/s or 3% of total D-glucose permeabilit~~. The dashed line was calculated from the same coefficients V,, and K, used in the curve-fitting but with d subtracted.

6 Michaelis-Menten equation with competitive inhibition, v = ( VmaxG/Kg)/( 1 + G/Kg + S/KJ if Fg = V,,,,,/K,,, for glucose and G << Kg. Fgl was taken from the mean basal F, of many experiments. Kl was determined from two experiments where 0.2 mm [l4c]g1c plus a range of MeGlc concentrations influxed into basal cells pre-equilibrated with the same range of MeGlc concentrations. Under these conditions Kl was estimated to be 22 mm. The values FS and K2 were given unlimited freedom to give the best fit to the observed data, specifying only the K, and Fgl persist unchanged at 22 mm and pl/s. The fit shown by the dotted line in Fig. 6A with K2 at 3.6 mm and F@ at pl/s is only one of an infinitude of equally bad fits. DISCUSSION In our previous work we suggested that the discrepancy between the kinetic estimates we obtained for basal glucose transport and other reports could be related to experimental conditions. In this study we have described the effects of multiple factors on the kinetics of basal transport. Depriving the cells of metabolizable substrate, cell agitation, or decreasing the assay temperature from 37 to 23 "C all lowered the basal K, and obscured the difference in K, between basal and insulin-stimulated cells. The mechanism for the effect of agitation on transport K,,, was not considered within the scope of this study. One obvious speculation would be that such agitation may serve to keep BSA circulating around the cells, keeping intracellular fatty acids low and dissipating metabolites such as adenosine. The role of BSA, adenosine, and fatty acids in the regulation of glucose transport has been recently considered (18-20). Perhaps all the effects on K,,, are due to changes in levels of metabolites that may function as inhibitors of the transporter. This inhibition, however, would work through an energy-dependent mechanism, since DNP prevented the reversal of the K,,, changes. These considerations are no doubt further complicated by multiple inter-relation- ships. For example, insulin and perhaps other effectors of glucose transport are also capable of regulating the transport of fatty acids (21). The observations reported here reconcile most of the reports regarding the action of insulin on glucose transport kinetics. They further establish that large modulations of the K, of basal transport can occur. Reconciling the Results-Temperature change, agitation, and substrat,e deprivation each had very reproducible and dramatic effects on the kinetic coefficients of basal transport. K,,, of basal cells varied from 2 to 40 mm, and V, from 3 to 14 nmol/wl/s (Table I). These values cover the range reported by others (Table 11). In most instances, the basal transport coefficients could be reconciled broadly with the various values of Table I, depending on the experimental conditions used. None of the manipulations, however, affected the K, of insulin-treated cells to any significant extent. The various reports of kinetic parameters for stimulated preparations are similar to those reported here. Translocation of Glucose Carriers Versus Activation in Situ-In this report we have documented further the differing characteristics of basal and stimulated transport. We have described in detail their different sensitivities to the actions of temperature and metabolic feedback. These findings would suggest that insulin activates glucose transporters directly. However, insulin has been postulated to stimulate glucose transport by translocating intracellularly located carriers into the plasma membrane (12,13). An interpretation which would be consistent with all the above is that insulin recruits low K, carriers into the membrane to add to the high K, persist- Basal Transport Characteristics of Rat Adipocytes TABLE I1 Transport coefficients for basal cells compiled from the literature Conditions Activity Assay Km (Vm,/Km) method" mm nmol/rl/s Prestabilized at 37 "C (1) EE* Prestabilized, DNP-treated NDd ND 0.60 Tracer at 37 "C (10)' uptake Starved, stirred at 37 "C (4) EE Starved, stirred at 37 "C (5) EE Starved, stirred at 28 "C (4) EE Prestabilized at 23 "C (11) EE Prestabilized at 20 "C (10) ND ND 0.26 Tracer uptake Starved, stirred at 22 "C (2) Starved, stirred at 18 "C 4.6 (4) EE EE "The activities for equilibrium exchange and traceruptakeare directly comparable. Net transport data are not necessarily comparable to the exchange data and are not included in this table. EE, equilibrium exchange with influx of MeGlc. e These numbers were originally published as pl/g at 1 min. They were converted to the expression of pl/pl cell HZ0 by the factor of 1 g of fat equals approximately 20 pl of cell H20. ND, not determined. ent basal carriers. This possibility, however, is not consistent with the apparently homogeneous transport kinetics of insulin-treated cells (Fig. 6). Other hypotheses might be considered. For example, basal carriers could be internalized as new low K, carriers are added to the plasma membrane. This might fit the observation of Baly et al. (14) that glucose carriers are immunologically homogeneous in plasma membranes from insulin-treated cells but heterogeneous in intracellular membrane locations. Another possibility is that basal carriers are modified by insulin or other factors as new low K, carriers are added. This wouldbe consistent with our finding that modulation of the basal carriers can occur inde- pendently of V,,, changes which are peculiar to insulin. Finally, one has to consider the simplest case where basal carriers are activated in the membrane by insulin or other factors. The dependence on ATP of the changes we describe in basal transporter K,,, with starvation or agitation of cells suggests that such changes might be related to the phosphorylation of the carrier or of a regulatory ligand. A similar dependence on ATP has been shown for the effect of insulin and other factors to produce an apparent translocation of glucose transport activity (10). This might indicate that K,,, changes and carrier redistribution are two aspects of a single phenomenon. Metabolic substrates with or without cell agitation did not alter the transport activity of insulin-treated cells under our conditions. This could indicate that insulin's action to lower transport K, might be exerted by an antagonism of the &-raising effect of metabolic feedback. Thymocytes (16, 17) and rat erythrocytes4 show metabolic feedback regulation of glucose transport via K, changes but are unresponsive to insulin. Modulation by metabolizable substrate may constitute a general property of the glucose transport system. The effect of insulin to lower the K,,, in adipocytes may represent a superimposed regulatory process. In summary, the primary focus of this work has been related to factors producing K,,, changes in the basal transporter of adipocytes. It is reasonable to suppose that body fat is frequently exposed to minimal insulin levels. We postulate that under such conditions metabolism will feed back to regulate glucose uptake and intracellular glucose via K, changes of basal transport. This regulation will enable cells to adjust N. A. Abumrad and R. R. Whitesell, unpublished observations.

7 15096 Basal Transport Characteristics Adipocytes of Rat their substrate supply to match their energy needs. Regulation of basal transport by metabolic factors could be of special importance physiologically in cases of insulin lack or resistance as in prevalent forms of diabetes. Furthermore, insulin may act partially by over-riding the constraints of metabolic feedback to increase the K, of the basal carrier. The possibility that insulin's effect on V,, is independent of its effect on the K, remains likely. Large K, effects can be brought about without associated V, changes (Table I). In contrast we and others have shown that insulin under certain conditions can produce V, changes only. The V, changes reported were in some studies (5) 2-3-fold higher than those we observe in a typical experiment. This might be related to differences in the experimental protocol which remain to be elucidated. Ack~w~gments-We are grateful to Dr. Nagayasu Toyoda for introducing to us the use of the infant incubator for the control of ambient temperature during transport assays. We also are thankful to Drs. David Regen and Charles R. Park for helpful suggestions and to Patricia Perry for skilled technical assistance. REFERENCES 1. Whitesell, R. R., and Abumrad, N. A. (1985) J. Bwl. Chem. 260, Whitesell, R. R., Gliemann, J. (1979) J. Bwl. Chem. 254, Vinten, J., Gliemann, J., and Osterlind, K. (1976) J. BioL Chem. 251, Vinten, J. (1978) Biochim. Bwphys. Acta 511, Vinten, J. (1984) Bwchim. Bbphys. Acta 772, Taylor, L. P., and Holman, G. D. (1981) Biochim. Biophys. Acta 642, Kashiwagi, A., Huecksteadt, T. P., and Foley, J. E. (1983) J. Bwl. Chem. 258, Vega, E'. V., and Kono, T. (1979) Arch. BiOChem. Bwphys. 192, Czech, M. P. (1976) Mol. Cell. Biochem. 11, Ezaki, O., and Kono, T. (1982) J. Biol. Chem. 267, Mav. J. M.. and Mikuleckv. D. C.. (1982). J. BioL Chem isO Suzuki. K.. and Kono., T. (1980).. PFOC. Nutl. Acud. Sei. U. S. A. 77, Cushman. S. W.. and Wardzala. L. J. (1980).. J. Biol. Chem Balv. D. L.. I. A. Simmon. S. Matthaei. and R. Horuk (1985).~ Fid. PFOC: 69,1156 (ab&.) 15. Kono. T.. Robinson. F. W.. Sarver. J. A.. Vega. F. V.. and Pointer. R. H. (1977) J. B&l. Chem. 252,2226-2c Whitesell, R.R., and Lynn, W. S. (1979) Fed. Proc. 38, 914 (abstr.) 17. Whitesell, R.R., and Regen, D. M. (1978) J. Biol. Chem. 263, Gliemann, J., Bowes, S. B., Larsen, T. R., and Rees, W. D. (1985) Biochim. Biophys. Acta 845, Smith, U., Kuroda, M., and Simpson, I. A. (1984) J. Bwl. Chem. 269, Joost, H.G., Steinfelder, H. J. (1985) Biochem. Biophys. Res. Co~m~n. 128, Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. BioE Chem. 260,