The Insulin-Sensitive Hexose Transport System in Adipocytes

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I X

The Insulin-Sensitive Hexose Transport System in Adipocytes J . GLIEMANN AND W . D . REES Phvsiologv of Aarhus Aarhus. Denmark Institute of University

. . . . . . . . . . . . . . . . 339 Summary of the Present Status . . . . . . . . . . . . . . . . . . . . . Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 . . . . . . . . . . . . . . . . 342 Critical Steps in the Methodology 342 A. The Cell Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Measurement of Fluxes.. . . . . . . . . . . , . , . . . , . , . . . . , . . . . . . . . . . . . . . . . . . . . . 344 348 IV . Kinetic Approaches to the Study of Hexose Transport. , . . , . . A. General Concepts . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 B. Equilibrium Exchange Experiments _ . _ . . _ _ . . . . . . . . . . . 349 ... 35 1 C. Zero trans Experiments. . . . . . . . . . D. Infinite cis Experiments . . . . . . . . . . . , . . . . , . , , . , . , . , . . . . . . . . . . . . . . . . . . . . 353 354 E. Infinite trans Experiments. . . . . . . . . . , , . . , , . , , . . . . . . 355 V . Transport of Nonmetabolizable Sugars and Sugar Analogs in VI. The Requirements for D-GhCOSe Binding to the Adipocyte Hexose Transport System 359 360 VII. Nontransported Competitive Inhibitors of Transport. . . . . . . . . . . . . . VIII. Sugars Which Are Both Transported and Phosphorylated-Rate-Limiting Steps. . . . . 362 IX. Modulation of the Transport System by Glucose Metabolites . .. . . . . . . . . . . . . . . . . . 366 X. Mechanism of Insulin’s Ability to Increase V,,,, , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 367 . . . . . . . . . . . . . . . . 37 1 XI. Human Adipocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 1 XII. The Transport System in Obesity and Diabetes . . . . . . . . . . . . . . . . . . . . XIII. Reconstitution of the Hexose Transporter . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 XIV. Concluding Remarks, . . . . . . . . . . . . . . . . . , . . , , , , , . , . . , . , . . . . . . . . . . . . . . . . . . . . 373 373 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

11. 111.

1.

SUMMARY OF THE PRESENT STATUS

The plasma membranes of adipocytes are equipped with special structuresoften referred to as carriers-which greatly facilitate the transfer of hexoses 339

Capyright 0 1983 by Academic F’rerr. Inc All rights 01 reproduction i n any form reserved. ISBN 0- 12- I533 18-2

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(e.g., D-glucose) from the extracellular fluid to the cytosol. This mediated transport allows a nonmetabolizable sugar analog with an affinity similar to that of glucose (e.g., methylglucose, 3-0-methyl-~-glucose) to equilibrate across the plasma membrane at a much higher rate (up to lo4 times) than it would do by simple, nonmediated diffusion alone. The transport system shows saturation and can be described kinetically using equations analogous to those applied in enzymology. The K , for glucose is about 8 mM at physiological temperature, i.e., about twice the fasting plasma glucose concentration in mammals. Glucose metabolism is vital for the adipocyte; for example, fatty acids could not be esterified and triglycerides could not be stored in the absence of production of a-glycerophosphate. At low glucose concentrations, the rate of transmembrane glucose transport limits the rate of glucose metabolism and the transport step is therefore a major point of regulation. Several hormones, most notably insulin, influence the permeability of the plasma membrane. The properties of the transport system are also modulated when the cells metabolize glucose at a high rate. Insulin causes an approximately 10-fold increase in V,,,, without influencing K , significantly. Recent work strongly suggests that the insulin-induced increase in V,,, is brought about by a translocation of transporters from a site in which they are nonoperative (presumably intracellular) to the plasma membrane. Some nontransported sugar analogs inhibit transport of sugars only from the extracellularly facing side of the membrane, whereas others inhibit only from the inside. Such studies have helped clarify the orientation of the glucose molecule during transport in both human red blood cells and rat adipocytes. In both systems, the C-1 end of the glucose molecule is initially bound to the extracellularly facing site and the C-4/C-6 end to the intracellularly facing site. In addition, the spatial and hydrogen bonding requirements are similar although not identical. On the other hand, the transport systems of the two cell types are different in the sense that transport of glucose and 3-0-methylglucose exhibits markedly asymmetric parameters in human erythrocytes, whereas these parameters are symmetrical in adipocytes.

II. HISTORICAL BACKGROUND The development leading to the present view as summarized above has taken place over about four decades. Early studies on sugar transport were carried out in human red blood cells using a photometric method developed by 0rskov (1935) to measure volume changes. At that time the mechanism of permeation was thought of as nonmediated diffusion even though clear deviations from Fick’s law were noted (Bang and 0rskov, 1937; Meldahl and Idrskov, 1940). Later, several groups clearly demonstrated saturability, specificity, and a number

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of other features incompatible with nonmediated diffusion (see for instance LeFevre, 1948). Furthermore, the hexose transfer system of human red cells shows an asymmetric behavior as first reported by Wilbrandt (1954). Further detailed studies of this phenomenon have led to the suggestion of various models for the mechanisms involved. The rate of glucose metabolism in human red blood cells is very low as compared to the rate of unidirectional glucose transport at all glucose concentrations, and the very high transport capacity of the red cell serves no obvious physiological function in adult humans. The present knowledge about this system was recently excellently reviewed by one of the founders of the field (Widdas, 1980). The basic concepts and methods of analysis developed through the studies of the red blood cell system have been of great importance for studies of sugar transport in other cell systems, including adipocytes. The concept that the transport of glucose across the plasma membrane in skeletal muscle might limit the rate of metabolism was first put forward by Lundsgaard (1939), who demonstrated that muscle cells contain very little free glucose. Lundsgaard inferred that insulin must act primarily on the transfer of glucose into the cell. Independent of the work of Lundsgaard, the membrane hypothesis was put forward by Levine et a / . (1949) on the basis of the finding that insulin increases the galactose space in the extrahepatic tissue of dogs. Morgan et al. ( 1964) demonstrated glucose-induced countertransport of 3-0methylglucose in striated muscle and concluded that glucose is transported by carrier-facilitated diffusion. It was further shown that insulin increases the activity of the transport system. The interest in adipose tissue was aroused when it became clear in the 1940s and 1950s that it was not only a storage site but actually possessed a very high metabolic turnover. For instance, D-glucose is rapidly metabolized in adipose tissue, such as rat epididymal fat pads, and this process is markedly enhanced by insulin. Crofford and Renold (1965a,b) showed that glucose is mainly transferred across the cell membranes of adipose tissue by carrier-mediated (facilitated) diffusion and that insulin acts on this step in analogy with the results obtained with skeletal muscle. It is difficult or impossible to obtain a quantitative evaluation of the hexose transport system using pieces of tissue. One problem is that diffusion of substrate in the interstitial fluid may be rate limiting for its entry or exit into the cytosol. Another problem is cellular heterogeneity; for example, the adipocytes appear to possess less than half of the intracellular water in adipose tissue (Gliemann e t a / ., 1972). For these reasons, it seems obvious to study the transport system using suspended cells. In 1964, Rodbell prepared isolated fat cells by treating adipose tissue with crude collagenase and showed that the cells were metabolically active and insulin responsive. This was an important milestone and the preparation is now widely used as a model system. A further advantage is that only adipocytes

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have a density of less than 1 and the cell preparation is therefore homogeneous. However, some technical problems had to be solved before the insulin-sensitive hexose transport system could be characterized.

111.

CRITICAL STEPS IN THE METHODOLOGY

A. The Cell Preparation General procedures are given by Rodbell (1964), Gliemann ( 1967), Vega and Kono (1979), and Foiey et al. (1980b,d). The permeability to 3-O-methylD-glucose of isolated epididymal rat adipocytes incubated in the absence of hormones is about 7 X lOW7 cm sec- I (Whitesell and Gliemann, 1979). However, this number, and consequently the “basal” rate of glucose metabolism, is subject to large variations because a number of factors related to the preparation and incubation of the cells may increase the permeability. This phenomenon was first noted in early work on glucose metabolism (Gliemann, 1967) and it has been shown by Vega and Kono ( 1 979) for methylglucose transport. It has been generally observed in our laboratory that cell preparations giving a high “basal” glucose metabolism will always give a high “basal” methylglucose transport. Thus, the first and necessary condition is that the cell preparation is as responsive to hormones, particularly insulin, as the tissue from which it was prepared. As a rule of thumb, epididymal adipocytes from 130- to 200-g rats fed standard chow ad libitum should be at least about 10-fold stimulated by insulin at a high concentration. If this is not the case, the cells probably exhibit an increased permeability to start with. This may be caused by chemical or mechanical stimuli. Crude collagenase, produced by Clostridium histolyticum, which is used to disintegrate the fat tissue, may sometimes contain proteins with “insulin-like” properties. Highly purified collagenase does not disintegrate the tissue and the combined action of collagenase and an acid protease is necessary (Kono, 1969). Even though it might be possible to purify the active components, all workers in the field appear to use the crude commercial preparations. Everybody seems to compare a given batch of collagenase with an old batch which is known to produce “good” cells, i.e., with a low “basal” permeability. If the new batch is suitable, a good supply is bought, since the crude collagenase is stable for years at -20°C. The directions supplied by the manufacturer (for fat cells, for liver cells, etc.) seem of little value based on our experience and it would be a considerable advantage if more exact chemistry was applied to the active components of the collagenase preparations.

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The presence of albumin at a high concentration is necessary to maintain cell integrity (Gliemann, 1967). The bovine serum albumin (fraction V) which is usually used may be contaminated with proteins causing insulin-like effects. Small amounts of insulin itself may be present in the albumin preparation. This is easily checked by the addition of antiinsulin antibodies and is usually not a problem. Other contaminating proteins may lose their insulin-like effect by treatment of the albumin preparation with trypsin (Jordan and Kono, 1980). These contaminations may be due to bacterial growth in the albumin preparation since several microorganisms produce proteases with “insulin-like activities. ” For example, this applies to the ubiquitous Bacillus subrilis. Such contaminations may also occur, of course, in the laboratory if the buffer has been standing on the bench too long before use. The mechanical treatment involves shaking or swirling of the tissue with crude collagenase followed by filtration and repeated washings. Such treatments may increase the sugar permeability (Gliemann, 1967; Vega and Kono, 1979). It is difficult to define exactly what strain the cells can tolerate because, for example, one metabolic shaker may not perform mechanically in the same way as another even when set at the same number of cycles per minute. The general recommendation is to cany out all procedures as gently as possible and to make sure that the geometry and surface qualities of the plastic ware are suitable. The “insulinlike” effect of vigorous mechanical treatment, i.e., an increase in the membrane permeability due to carrier-facilitated diffusion, was difficult to understand for many years. However, part of the mechanism has been elucidated recently. Thus, Kono et al. (198 1) have observed that hard centrifugation causes a shift in the distribution of transporters from a nonfunctional (probably intracellular) site to a functional site in the plasma membrane. This mechanism is similar to that observed when the cells are treated with insulin (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; see below). Vega and Kono (1979) have reported that an increased “basal” transport in freshly prepared cells can be reduced by incubation for about 30 minutes with glucose. We have not observed this phenomenon in our laboratory, perhaps because the permeability was not enhanced to start with. It should be noted that an increased permeability, as caused by preparation artifacts, is not necessarily reflected in changes in morphology or an increased release of intracellular enzymes. More vigorous mechanical traumas may, of course, lead to cell rupture. However, this is easy to recognize because the preparation becomes greasy due to triglyceride droplets in the medium. The easiest way to monitor that problem is perhaps to spin a concentrated cell suspension (about 40% v/v) in a hematocrit centrifuge for 30 seconds (Gliemann et d., 1972); there should be no visible or only a thin film of free triglycerides on top of the cell layer.

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6. Measurement of Fluxes

Measuring the intracellular waterspace is a necessary prerequisite for measuring the fluxes of transportable, nonmetabolizable sugars or sugar analogs which equilibrate across the plasma membrane. In adipocytes, the intracellular waterspace is only about 2% of the cell volume. In early work, the intracellular waterspace was measured using a filtration technique (Crofford et al., 1966), but the trapped extracellular volume was about I0 times larger than the intracellular volume and the latter was therefore determined with low precision. We introduced an oil flotation method (Gliemann et al., 1972) using dinonylphthalate, which has a density between that of buffer and adipocytes, or a silicone oil with a similar density. Upon centrifugation, the cell suspension is separated into three layers with the cells on top followed by an oil layer and then buffer (Fig. 1A). Using a microfuge tube it is easy to cut through the oil layer and thereby obtain the packed cells separate from the buffer. The space between the cells is largely filled with oil, and the trapped extracellular water volume is reduced to about one-third of the intracellular water. The large size of the adipocytes (small surface-to-volume ratio) is actually an advantage since pellets of smaller cells, such as hepatocytes or thymocytes obtained by centrifugation through an oil with a density slightly higher than that of buffer, contain a considerably larger volume of extracellular buffer per volume of cell pellet (Andreasen et al., 1974). In this way the intracellular waterspace could be measured quickly and with sufficient precision using appropriate markers for the total and the extracellular waterspaces. The distribution space for tritiated water was indistinguishable from that of methylglucose. Centrifugation through oil did not influence the ability of the cells to metabolize glucose, and efflux experiments showed that whereas tritiated water was lost almost immediately from the cells, about 30 seconds was required before half of the methylglucose had passed from nonstimulated cells into the medium (Gliemann et af., 1972). However, it soon became clear that the method was not suitable for measuring methylglucose fluxes in insulin-stimulated cells which were expected to show an approximately 10 times lower efflux half time at low methylglucose concentrations. The reason is that it takes 3-4 seconds to obtain separation between cells, oil, and medium, and, more importantly, that the cells first carry a large amount of extracellular water into the oil phase and this is replaced by oil during the centrifugation in the following 30 seconds (Thorsteinsson et af., 1976). Therefore, transport of sugar into or out of the cells cannot be regarded as being stopped when the centrifuge is turned on or even 4 seconds later. Nevertheless, the method may well be used to determine qualitatively whether a given batch of cells responds to insulin (Kono er al., 1977). The method has been used to measure the flux of slowly transported sugars such as D-allose (Loten et al., 1976) and L-arabinose (Foley et af., 1978).

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However, these sugars are transported slowly because their Michaelis constants are high (50 mM or more), and it is therefore difficult to measure transport at concentrations higher than K,. The method was consequently modified to measure fluxes of rapidly transported sugars and sugar analogs such as 3-0-methylglucose. Figure IB shows the principles of an efflux method used to measure equilibrium exchange (Vinten ef al., 1976), which implies that the sugar concentration is equal on the two sides of the membrane at any given time. The cells are first incubated with unlabeled methylglucose for a time sufficient to ensure equilibration of the external sugar. The volume is then reduced and the concentrated cell suspension incubated with [ 14C]methylglucosefor a shorter time. At the end of this period, the intracellular sugar concentration should be the same as the extracellular concentration although the specific activity is not necessarily the same. The reason for this twostep procedure is first, that incubation of concentrated suspensions for long times may damage the cells (incubating dilute suspensions with tracer would be too expensive!), and second, that the procedure minimizes the metabolism (and irreversible trapping) of trace contaminations which are sometimes present in the labeled methylglucose preparations. The cells are then centrifuged lightly through silicone oil in a slender tube, ejected into a large bath which, in equilibrium exchange experiments, contains unlabeled methylglucose at the same concentration as that used for the equilibration. Aliquots are transferred to oilfilled microfuge tubes at appropriate time intervals. It is sufficient to centrifuge the suspension for about 4 seconds because the dilution of extracellular buffer is essentially infinite. It should also be noted that backflux of methylglucose is negligible since the intracellular water volume of the adipocytes is very small as compared to the volume of the bath. Using this method, efflux of [‘4C]methylglucose can be followed until the intracellular concentration is a few percent of the starting concentration, as shown in Fig. 2. The efflux of a labeled nonmetabolizable sugar from a population of identical cells with well-mixed extracellular and intracellular compartments should be monoexponential under equilibrium exchange conditions irrespective of the nature of the transport system. The curve (Fig. 2) actually deviates slightly from an exponential course and this is probably due to cellular heterogeneity with a range from “fast” to “slow” cells (Vinten et al., 1976; Gliemann and Vinten, 1974). This minor deviation is neglected in the analysis of net transport data as described below. Figure IC shows the principles of a method used to measure uptake of rapidly equilibrating sugars (Whitesell and Gliemann, 1979). A small volume of concentrated adipocyte suspension is squirted onto a droplet of buffer containing the labeled sugar to ensure mixing. This is followed by the addition of a large volume of 0.3 mM phloretin, a potent and rapidly acting competitive inhibitor of sugar transport. This acts as a stopping solution and arrests efflux of the sugar

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I4-I

A

3Omin

J. GLIEMANN AND W. D.REES

Density (glcm3) : cells : 0.915 di nonyl phthalate : 0.99 buffer :

1.012

2 - 2Omin

600~11

30MG

plus labeled 3.0M G

-

Equilibrium Exchange

Unlabeled calls

Decrease in spec. activity in medium

> LOO

B

40pl

12y1

C

4

Loop1

FIG. I . Oil flotation methods. (A) For slowly equilibrating sugars (or other ligands). (B) Efflux method for rapidly equilibrating sugars. (C) Uptake method for rapidly equilibrating sugars. 3-OMG. 3-0-rnethylglucose; 3-OMG*, 3 - 0 4 14C]methyl-~-glucose;3HTG, [3H]triglyceride, for sample volume correction. For further explanation, see text.

HEXOSE TRANSPORT IN ADIPOCYTES

347

Fic. 2. Efflux of 3 - 0 4 ''C]methyl-o-glucose under equilibrium exchange conditions at 37°C in adipocytes treated with a maximally stimulating insulin concentration (0.7 p N ) . The 3-0-methylglucose concentration was 30 mM. A, denotes the intracellular amount of sugar at time t . (Reproduced with permission from Vinten et al., 1976.)

taken up into the cell. Finally, the cells are centrifuged through an oil layer either in the incubation tube or after transfer to a microfuge tube. These two alternatives give the same experimental results. Trapping of the extracellular mixed incubation buffer is of the order of 0.02%, i.e., insignificant. Timing is aided by a metronome, and uptake can be measured precisely and reproducibly at intervals down to 1 second. Since the shortest half-time reported for uptake of methylglucose at tracer concentration in insulin-stimulated adipocytes is about 2 seconds, and since methylglucose is the fastest transported sugar we know of, initial velocities of sugar uptake can be measured or calculated with a good approximation under all conditions. At equilibrium exchange conditions (and in net uptake experiments when the substrate concentration is very low) the uptake curve is nearly exponential as was previously found for the efflux curve (Fig. 2 ) . This is merely a technical control since the theory demands that equilibrium exchange is identical whether efflux or influx of radiolabeled sugar is followed. (This is what theory demands, independent of the model. Flux into the cell and out of the cell is by definition the same under equilibrium exchange conditions.)

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IV. KINETIC APPROACHES TO THE STUDY OFHEXOSETRANSPORT

A. General Concepts The combination of the transported hexose with a membrane protein to form a transporter-substrate complex leads to a facilitated diffusion system exhibiting saturation kinetics. This system differs from the familiar model of an enzymic reaction with one free enzyme form E (Scheme a), in that two separate binding sites (E, and E,) are available for the substrate (S) on each face of the membrane. Scheme b shows the most widely used model for facilitated diffusion, the carrier model. The subscripts 1 and 2 refer to the two sides of the membrane, and transport is measured as the movement of substrate from side 1 to side 2 or side 2 to side 1.

SCHEME a

SCHEME b

Kinetic analysis of the carrier model for facilitated diffusion will thus show half saturation constants (K,’s) and maximum velocities (Vmax’s) which have different interpretations depending on the direction of flux (i.e., movement from 1 to 2 or 2 to I) and the substrate concentrations on each side of the membrane (S, and S2). Eilam and Stein (1974) showed that for any of the protocols described below the rate of flux (v) will be given by an equation of the MichaelisMenten form (1)

V,,,) for a facilitated It should be noted that the kinetic constants (K, and V,,,,,) diffusion system are analogous to but not identical with those determined for an enzymic reaction. Other transport models (for review see Naftalin and Holman, 1977) can be described by more complex forms of Eq. (I). During facilitated 1977) diffusion the substrate molecule remains unchanged and, therefore, in the absence of metabolism, the substrate will equilibrate to equal concentrations in both bulk solutions as the substrate runs down its electrochemical gradient. As described above, the main technique for measuring transport rates is to follow the flux of a nonmetabolized radiolabeled substrate from the bulk solution at one face of the membrane [the cis face using the nomenclature of Eilam and (1974)] to the bulk solution at the opposite face (the trans face). This Stein (1974)]

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HEXOSE TRANSPORT IN ADIPOCYTES

nomenclature shall be used whenever possible. The cis to trans flux of substrate can be measured either into (entry or influx) or out of (exit or efflux) the cell. The intracellular waterspace of the cell is very small (Gliemann ef af., 1972) relative to the external medium so that, in an entry experiment, the substrate concentration in the internal solution will change rapidly. On the other hand, the substrate concentration in the external medium will remain essentially unperturbed. In an entry experiment the radiolabeled substrate will accumulate in the internal (trans) solution. Backflux then occurs as part of this radiolabel, then returns in the trans to cis direction, and this reduces the rate of net flux. In an exit experiment backflux of substrate trans to cis (i.e., from the external solution into the cell) is minimal due to the large dilution of the radiolabeled substrate once it enters the bulk solution on the trans face. The small cis volume in exit experiments leads to a rapid drop in substrate concentration at the cis face as the substrate leaves the cell. Eilam and Stein (1974) in their review of the principles of the measurement of facilitated diffusion processes described integrated rate equations for the characterization of such a system. We shall include the appropriate integrated rate treatment for each protocol as adopted from Eilam and Stein (1974).

B. Equilibrium Exchange Experiments In these experiments the unidirectional flux of radiolabeled substrate is followed when there is no concentration gradient across the membrane, i.e., the substrate concentration in the cis solution is equal to that in the trans solution. Under these conditions there is no net flux of a nonmetabolizable substrate such as 3-O-methylglucose, and the rates of unidirectional influx and efflux are the same. As described above (Fig. 2) the exit curve is nearly exponential, and follows with close approximation the equation A, = A,ePkt

where A, and A, denote the amount of intracellular radioactivity at time zero and time t , respectively. These values are corrected for the small amount of radiolabeled sugar remaining in the cells at infinite time. The rate constant k is the fraction of intracellular radioactive sugar lost per unit time, i.e., the transport velocity v (moles sec- X liters intracellular water- I ) divided by the substrate concentration S (moles/liter). Thus Eq. (2) assumes the form k = viS = In( 1 - f,>/f

(3)

where f , (“fraction remaining”) = A,/A,. The equilibrium exchange influx curve follows (with the same approximation

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as the influx curve) the equation A, = A,(l

- eckt)

(4)

where A, denotes the amount of intracellular radioactivity at infinite time [equivalent to A, in Eq. ( l ) ] , A, and A, are corrected for the extracellular radioactivity in the cell pellet at zero time. As with Eq. (2), Eq. (4) can be rewritten as k = v1S = ln[l/(l - f , ) ] / t

(5)

where& (“fraction inside”) = A,/A,. Thus, for equilibrium exchange experiments plots of vlS vs t should be linear at any given substrate concentration. The half time for the efflux of 3-O-methyl-~-glucoseat tracer concentrations is approximately 2 seconds in insulin-stimulated adipocytes at 37°C. Therefore, from Eq. (3) k = vIS = 0.6912 = 0.345 sec-’

In equilibrium exchange experiments the transport of sugar from the extracellular compartment to the intracellular (or vice versa) may be measured as the initial velocity from time zero to a given time t . In experiments in which the labeled sugar is present only on the extracellular side, this implies that efflux of labeled sugar from time zero to time t is neglected. This does not introduce any serious error when only up to about 20% of the intracellular compartment is equilibrated. The limitations of the method may be illustrated using cells with maximal permeability to 3-0-methylglucose (half-time 2 seconds). By 1 second, 34.5% would be equilibrated if no backflux occurred (see above). However, the actual equilibration is 29.2% [cf. Eq. (4)]. Alternatively, once uptake has been shown to be exponential, Eq. (3) or ( 5 ) (for efflux or influx, respectively) can be used to calculate initial uptake rates. Then the values of vlS at different substrate concentrations can be plotted using the transformations of Eq. (1) which are used in enzymology. For example, the Slv vs S plot (Hanes’ plot) transforms Eq. (1) to It should be noted that some models for sugar transport in erythrocytes predict more than one K,, (Holman, 1980; Holman et al., 1981a). As wide a range of substrate concentrations as possible should therefore be used in determining the saturation kinetic parameters. The equilibrium exchange K,, and V,,, (Kee and Vee using Eilam and Stein’s terminology) reflects the properties of both the internal and external sites of the transport system when substrate is being transported in both directions. The equilibrium exchange experiment is therefore well suited to the determination of inhibition constants for inhibitors of transport. The inhibition of tracer flux can

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HEXOSE TRANSPORT IN ADIPOCYTES

be measured using a range of inhibitor concentrations and vlS values determined using Eq. (3) or (5). The inhibition constant is then given by the relationship

vdv

= 1

+ (I/Ki)

(7)

where vo is the uninhibited rate, v is the inhibited rate, and 1 is the inhibitor concentration. Ki can be determined from a plot of v,/v vs I (Rees and Holman, 1981). As with the equilibrium exchange experiment more than one K , may be apparent (Holman et al., 1981a).

C. Zero trans Experiments In these experiments the flux of substrate from cis to trans is followed when there is initially no substrate in the trans solution. Thus for a zero frans entry experiment substrate and radiolabeled substrate are added to the external solution and the uptake is measured. Substrate enters the cell rapidly and therefore over practical time courses there will be a finite substrate concentration in the trans solution and backflux into the cis solution. It is therefore necessary to calculate the initial rate of uptake from a net entry experiment in order to estimate the unidirectional zero trans influx. The uptake (or progress) curve is nonexponential except when the substrate concentration is very low as compared with K , for transport. Consider a system with symmetric transport of a given sugar, i.e., K , is the same for transport from the outside and in and from the inside and out. The rate constant for entry (and therefore the permeability) of the external sugar is reduced when its concentration is significant as compared with K, [Eq. (l)]. On the other hand, the permeability will be higher for sugar molecules that have entered the cytosol because the sugar concentration will initially be low in this compartment. Consequently, the sugar leaves the cell more rapidly than it would do under equilibrium exchange conditions and the progress curve of net entry becomes “flatter” than the exponential equilibrium exchange curve (Whitesell and Glieniann, 1979). This means that the error on the apparent initial velocity measured at a given time will be larger and corrections should be applied using an integrated rate equation (see below). Taylor and Holman (1981) reported that a 1 second uptake of 1 mM 3-0-methylglucose by insulin-stimulated adipocytes (net entry experiment) underestimated the initial rate of entry by 24%. Eilam and Stein [Eq. (70) in their 1974 paper] described an integrated rate equation for the net entry experiment. Their equation assumed a carrier model for transport (such as the simple model of Scheme b) but the parameters obtained can readily be reinterpreted in terms of other models for transport. The equation of Eilam and Stein contains a term correcting for volume changes due to the presence of the substrate. However, this may be neglected when the substrate con-

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D. REES

centration does not exceed about 40 mM. Using Eilam and Stein's equation, as described by Ginsburg and Stein (1975), the initial rate of a net entry experiment at a given substrate concentration can be obtained by plotting vs -[ln(l - CIS,) + C/S,]/C (8) where t is the time of uptake, C is the internal substrate concentration, and So is the external substrate concentration. Note that C/S, is the fractional filling of the internal sugar space, i.e., equivalent tofof Eq. (5). This plot therefore shows the initial rate as a function of the internal concentration as described by the carrier model IEq. (17) in Eilam and Stein, 19741. By extrapolating the corrected integrated rate replot to zero internal concentration, the initial rate of influx under zero trans conditions is obtained. This process is analogous to the procedure used for calculating initial rates from the exponential equilibrium exchange progress curve as described above. An example of the use of the integrated rate replot is shown in Fig. 3. The initial rate measured at different substrate concentrations can then be replotted in order to obtain the K, and V,, for zero trans entry. The zero trans entry K, (KT;) measures the K, for the outside site and the zero trans entry V,,, (VT$>is likewise the V,,, for unidirectional influx from 1 to 2. In a zero trans exit experiment, the cells are loaded with substrate and radiolabeled substrate until equilibrium is achieved. The extracellular substrate is then rapidly diluted with a large quantity of buffer (usually at least IW-fold), and the loss of radiolabel from the cells is followed. This method does not give true zero trans conditions but the small amount of substrate in the trans solution relative to the cis sohion Ieads to a minimal error. The practically infinite volume on the trans face of the membrane in a zero trans exit experiment leads to minimal flC

C mM

4.0

'

2.0

u 0.1

Tlma

imesl

0.2

0.3

0.4

In~l-C/s,J+C/&,

C

FIG. 3 . (a) A time course for net entry (zero trans entry) of I mM 3-O-methyl-~-glucosein cells pretreated with 10 nM insulin at 37°C. The apparent initial rate calculated from the I second measurement was 0.16 Mlsecond. (b) An integrated rate equation replot of I mM 3-0-methylD-glucose net entry. The initial rate V , = 0.210 &/second. (From Taylor and Holman, 1981.)

353

HEXOSE TRANSPORT IN ADIPOCYTES

backflux of the substrate trans to cis, and for the simple carrier model the rate of exit will be given by the Michaelis-Menten equation. However, there is a rapid change in the substrate concentration at the cis face as efflux proceeds so that an integrated rate equation treatment is required. The data from zero trans exit experiments can be conveniently analyzed by an integrated Michaelis-Menton equation (Karlish eruf., 1972) which can be rearranged (Baker and Naftalin, 1979) to give

-In(S,/S,)/(So - S,)

=

- S,)] - 1/K,

V,,,,t/[K,(S,

(9)

where S, is the starting substrate concentration inside the cell and S, is the internal substrate concentration at time t . This equation is equivalent to a form of the Lineweaver-Burk transformation of the Michaelis-Menton equation 1/S = (VmaX/K,,,)(l/v0) - 1/K,

(10)

Thus, by plotting -ln(S&)/(S,

-

S,) vs

r/(S, - S,)

a value of - I/Knl is obtained from the intercept on the ordinate and a value of l/Vmxxis obtained from the intercept on the abscissa. By comparing Eqs. (9) and (10) it can be seen that when -ln(SI/So)/(So - S,)

=

I/S,

then t/(S, - S,) is equal to I/V,, i.e., the reciprocal of the initial rate at a given substrate concentration. Therefore, one can alternatively calculate the initial rate of exit at a number of internal substrate concentrations and plot the data using Eq. (6). This procedure has the advantage of giving a more faithful reflection of the experimental error. The K , measured by the zero trans exit experiment (K$ ) measures the K, at the internal face of the membrane. The zero trans exit V,,, (Vf, ) measures the V,, for unidirectional efflux from side 2 to side 1. It should be noted that this analysis is based on a carrier model such as Scheme b and assumes that there is a single operational affinity (single K,) for substrate at the inner face of the membrane. There is now evidence for two operational affinities at the inside face of the human erythrocyte hexose transporter (Ginsberg and Stein, 1975) and this may lead to deviations from the curve predicted by Eq. (9) (see Holnian, 1980).

0. Infinite cis Experiments The zero trans experiments provide a means of measuring the kinetic constants of one side when no substrate is available on the opposite side. The K,,, of one side can also be measured when the opposite side is saturated with substrate. Thus, for

354

J. GLIEMANN AND W. D. REES

the infinite cis experiment the net flux cis to trans is measured when the substrate concentration in the cis solution is at a saturating concentration, that is for practical purposes at least 10 times the zero trans K,. The cis side of the transport system is saturated with substrate, and therefore the rate of unidirectional flux from cis to trans will be maximal. On the other hand, the backflux process is dependent on the substrate concentration in the trans solution, and K , on the trans side can therefore be determined by following the rate of net flux into solutions containing different substrate concentrations. In other words, the net flux (cis to trans minus trans to cis) is reduced as the substrate concentration on the trans side increases and the trans to cis flux depends on the K , on the trans side. The infinite cis entry experiment is most easily performed by measuring the time course for the net uptake of a single high concentration of substrate. As the uptake proceeds, the internal substrate concentration will increase from zero until it is finally at equilibrium with the external solution. Thus, the substrate concentration in the trans solution is changing with time, and this allows the determination of the infinite cis entry parameters. Eilam and Stein (1974) showed that their integrated rate equation for net entry could be applied to the infinite cis experiment (see also Ginsburg and Stein, 1975) so that a plot of

ttc vs

ln(l

+ CIS,) + CIS, C

[cf. Eq. (8)l

yields a straight line giving I/Vmax for infinite cis entry as the intercept on the ordinate. The intercept on the abscissa will be -K,ISi ( I + S o h ) , where 7~ is the effective osmotic concentration of the buffer, and K i can thus be calculated (Ginsburg and Stein, 1975; Holman, 1979). K k will be the K , for the internal side. In order to perform infinite cis exit experiments [also known as the Sen and Widdas experiment (Sen and Widdas, 1962)] the cells are first loaded with a saturating concentration of substrate (e.g., 40 mM) and the net efflux of this substrate into solutions containing different concentrations of substrate is followed. With a saturating concentration of substrate in the cis solution the net exit rate remains linear until the internal substrate concentration ceases to be saturating. In adipocytes this means 10-20 seconds even when the cells are treated with insulin. Thus, the rate of net efflux can easily be measured without the need for integrated rate equations. A plot of I / V vs S gives minus the infinite cis exit K, ( K g ) as the intercept on the abscissa. Kt;' measures the K,, for the external side. The infinite cis experiments are technically simpler to perform than the zero trans experiments since they offer the advantage of longer time courses.

E. Infinite trans Experiments In these experiments (which are equivalent to counterflow experiments) the substrate concentration in the trans solution is at a saturating concentration.

HEXOSE TRANSPORT IN ADIPOCYTES

355

These experiments allow an additional measure of the K , for one side (cis) when the other side is saturated with substrate. The infinite trans entry experiment will therefore measure the K , for the external site while the infinite trans exit experiment will measure the K , for the internal site. It is possible to formulate rejection criteria for different kinetic models by comparing the results of the different experimental protocols with the predictions of the models (Lieb and Stein, 1974a,b). Thus, using these criteria, Hankin et al. (1972) were able to show that the simple carrier model can be rejected as a model for hexose transport in the human erythrocyte.

V.

TRANSPORT OF NONMETABOLIZABLE SUGARS AND SUGAR ANALOGS IN THE ADIPOCYTE

Since D-glucose entering the adipocyte is rapidly metabolized, it is impossible to study its transport directly. When the rate of D-glucose transport is rate limiting for metabolism, a measure of its transport rate can be obtained through the use of indirect measurements such as the rate of D-glucose incorporation into lipids or CO,. The value of this approach is limited and it does not allow a full kinetic characterization of the transport system. Before considering the results obtained with nonmetabolizable sugars, it is important to note that the sugar permeability in the isolated adipocyte due to nonmediated diffusion is negligible under most conditions. This conclusion is in part derived from the finding that L-glucose at a tracer concentration exhibits an equilibration half time of about 60 minutes under conditions where the half time is 2-3 seconds for 3-O-methyl-~-glucose. The half time for equilibration of L-glucose is further increased to several hours in the presence of 40 mM methylglucose (Whitesell and Gliemann, 1979). In addition, Vinten (1978) found that the total methylglucose permeability, which could not be inhibited by a large concentration of cytochalasin B, was only a small fraction of the total permeability. The transport of the nonmetabolizable C-3 epimer of D-glucose, D-allose, was studied by Loten et al. (1976) who showed it to be transported slowly. Foley et al. (1978) reported that L-arabinose (a D-galactose derivative lacking the C-6 hydroxymethyl group) was also transported slowly by the adipocyte and not metabolized. Both analogs are transported by the glucose transport system since their transport is competitively inhibited by glucose and the rate of transport is stimulated by insulin. These sugars are transported slowly due to their high K,’s for the transport system. D-Allose has a K , of about 270 mM (Rees and Holman, 1981), and L-arabinose has a K , > 50 mM (Foley et al., 1978). These high K,’s limit the usefulness of D-allose and L-arabinose in a conventional kinetic characterization of the transport system since saturation of the transporter with these sugars requires very high concentrations, which are outside a practical concentration range. The low affinity, and hence slow transport of these sugars does,

356

J. GLIEMANN AND W. D. REES

however, offer a distinct advantage in inhibition experiments allowing uptakes to be followed over a period of several minutes as opposed to seconds with a high affinity sugar. 3-O-Methyl-~-glucoseis a rapidly transported D-glucose analog which is not metabolized (Czaky and Wilson, 1956), and Gliemann et al. (1972) showed that it has a distribution space not different from that of tritiated water and urea. Equilibrium exchange experiments carried out as demonstrated in Fig. 1B showed only one K , value of about 5 mM, and insulin caused a marked increase in V,,, without changing K , (Vinten et al., 1976). Similar data were obtained by Vinten (1978) using the same method and by Whitesell and Gliemann (1979) and Taylor and Holman (1981) using the influx method shown in Fig. 1C. An experiment of this type is shown in Fig. 4. It should be noted that the 3-0methylglucose concentration range used in these experiments was fairly narrow (up to about 20 mM). However, experiments have been carried out with substrate concentrations up to 60 mM (with the correction for nonmediated diffusion, which is necessary under these conditions) and this gave a K , value of 4.5 ? 0.6 mM at 37°C in cells stimulated maximally with insulin (G. D. Holman and W. D. Rees, unpublished data). It should be mentioned at this point that the reason for the insulin-induced increase in V,,, is probably that more transporters are available in the plasma membrane after treatment of the cells with insulin. This hypothesis is a result of the work of Kono, Cushman, and their co-workers, and will be discussed below.

S Methylglucose (mM) FIG. 4. The concentration dependence of 3-@methyl-~-glucoseequilibrium exchange at 37°C in the absence of insulin and in cells pretreated with 10 nM insulin. K,, was 4.4 mM and not significantly different for “basal” and insulin-treated cells. V,,,,, was 0.08 mM s e c - 1 for “basal” cells and 1.12 mM sec-1 for insulin-pretreated cells.

HEXOSE TRANSPORT IN ADIPOCYTES

357

It implies that one is probably looking at the same species of transporters whether or not the cells are treated with hormone. The properties of the transporter will be discussed in the light of this hypothesis. Adrenalin increases the rate of 3-0-methyl-~-glucosetransport in adipocytes by approximately twofold (Ludvigsen et al., 1980), and this stimulation also occurs through an increase in the V,,,, with no change in the K,. As with insulin, the adrenalin effect is preserved in isolated plasma membranes and it is tempting to speculate that insulin and adrenalin stimulate transport through a common mechanism. The effect of adrenalin appears to be mediated through preceptors (Ludvigsen er al., 1980). Glucocorticoids cause a marked decrease in hexose transport rate. However, no kinetic characterization has been carried out of the transport system in glucocorticoid-treated cells (Foley et al., 1978). It is also possible to measure the transport of hexoses in isolated plasma membranes even though the permeability due to nonmediated diffusion is much higher than in intact adipocytes. Since the membrane preparation does not metabolize D-glucose, the transport of D-glucose can be studied directly without the need for nonmetabolizable analogs. Ludvigsen and Jarett (1979, 1980) reported that the K , for D-glucose uptake was 9-26 mM in plasma membranes isolated from insulin-stimulated adipocytes. The value of the measured K , , was dependent on technical details in the preparation of membranes. The question of whether adipocytes show asymmetric transport parameters was first approached by Whitesell and Gliemann (1979), who measured the net entry of 20 mM 3-0-methylglucose (i.e., “almost” an infinite cis experiment). The progress curve did not deviate significantly from that predicted by symmetrical transport parameters. It was concluded that the system was probably symmetrical and that any asymmetry, if present, was certainly not like that described in human red blood cells. Taylor and Holman (1981) carried out a complete kinetic analysis following the principles of Eilam and Stein (1974) as outlined in the preceding section and found no evidence for kinetic asymmetry of 3-0methylglucose transport. Table 1 shows the transport parameters obtained using different protocols. It should be noted that some authors have reported much lower V,,,, values, particularly in insulin-treated cells. The reason may be that the initial velocities were underestimated. These values are not given in Table I [for discussion, see Whitesell and Gliemann ( 197911. Table 11 shows for comparison the kinetic parameters of the most intensively studied hexose transport system, that of the human erythrocyte. It appears that this transporter shows marked asymmetry as recently reviewed by Widdas ( 1980) in Volume 14 of this series. The data are generally obtained using glucose but the results using 3-0-methylglucose also show marked asymmetry ( G . D. Holman, unpublished observations). The kinetic constants vary depending on the experimental protocol, and the most marked asymmetry is observed in the zero trans experiments with zero trans entry showing a low K , and V,,,, while zero trans

TABLE I KINETICPARAMETERS FOR 3-0-METHYL-D-GLUCOSE TRANSPORT IN

THE

RATADIFQCYTE

K, (mM) Experiment Equilibrium exchange Zero trans entry (measures outside site)

Zero trans exit (measures inside site) Infinite cis entry (measures inside site) Infinite cis exit (measures outside site)

Reference

Basal

Vinten el a/. ( I 976) Whitsell and Gliemann (1979)" Taylor and Holman ( I98 1) I Whitesell and Gliemann ( 1979)" Taylor and Holman (1981)' Taylor and Holman (1981) r Holman and Rees ( 1982) Taylor and Holman (1981F Taylor and Holman (1981)c

About 5 2.5-5 4.22 2 1.24 2.5-5 5.41 2 0.98 4.09 5 1.05

9.03 5 3.28 4.54 2 1.32

Plus insulin About 5

2.5-5 4.45 5 0.26 2.5-5 6.10 5 1.65 2.66 5 0.26 5.65 5 2.05 6.51 t 0.83 3.60 5 1.33

V,,,

(mM sec - 1)"

Basal

Plus insulin

0.07-0.2 0.058 0.058 ? 0.001

1.6- I .9 0.8 0.84 t 0.002

-

0.034 5 0.034 0.153 2 0.023

1.20 -C 0.19 1.19 t 0.07

-

-

0.066 2 0.013 0.106 2 0.026

0.98 2 0.09 1.76 t 0.63

Equivalent to millimolesiliter intracellular waterkcond. Range of values obtained. 2 SE (from regression analysis). Values from Whitesell and Gliemann (1979) were obtained at 22"C, and the other values at 37°C

359

HEXOSE TRANSPORT IN ADIPOCYTES

TABLE I1 K I N ~ I I CPARAMETERS . FOR

D-GLUCOSEIN T H E HUMANERYTHROCYTE

Experiment

Reference

K , (mM)

Equilibrium exchange Zero trans entry (measures outside site) Zero trans exit (measures inside site) Infinite cis entry (measures inside site) Infinite cis exit (measures outside site)

Naftalin and Holman (1977) Lacko el a!. (1972)

34 1.6

6.0 0.6

Karlish et a / . (1972)

25.0

2.15

Hankin et (I/. (1972)

2.8

-

Lacko et a / . (1972)

1.8

-

V,,,

(mM sec

~

1)

exit shows a high K,,, and V,,,,,. However, when the Kn,’s of the inner and outer sites are measured by the two infinite cis procedures, both show symmetrical low K,’s. Equilibrium exchange experiments have until recently been reported to have a high K,,, and V,,,,,. Holman et af. (1981a) have presented evidence for negative cooperativity in the equilibrium exchange of D-glucose in the human erythrocyte, showing nonlinearity in reciprocal plots which reveal two apparent K,’s of 2 and 26 mM. Other cell types also show different kinetic constants for equilibrium exchange and zero trans entry. Whitesell et al. (1977) reported that sugar in the trans solution increased the rate of uptake in thymocytes. Plagemann et af. (1981) have reported similar results with a range of cultured cell types. On the other hand, hepatocyte preparations are similar to the adipocyte with symmetrical zero trans transport parameters for 3-0-methylglucose (Craik and Elliot, 1979). On the basis of kinetic studies it is thus possible to identify at least two classes of mammalian sodium-independent facilitated diffusion systems for hexoses, those which show symmetric, kinetic parameters, and those which show asymmetric parameters.

VI. THE REQUIREMENTS FOR D-GLUCOSE BINDING TO THE ADIPOCYTE HEXOSE TRANSPORT SYSTEM

From the K,,, values for transported substrates it is apparent that the relative affinities decrease in the order 2-deoxy-~-glucose (deoxyglucose) > 3-0methyl-D-glucose > (n-glucose) >> L-arabinose > D-allose. To characterize further the binding of D-glucose to the transporter, Rees and Holman (1981) studied the inhibition of D-allose transport by a range of D-glucose epimers, deoxy sugars, fluoro sugars, and other D-glucose analogs [cf. Eq. ( 7 ) ] . From the

360

J. GLIEMANN AND W.

D.REES

relative Ki’s of these analogs it was possible to determine which atoms of the glucose molecule are important for its binding to the transporter. These experiments revealed that the D-glucose molecule binds to the adipocyte transporter and is transported by it in a pyranose ring form. Hydrogen bonds are directed toward the ring oxygen and the oxygen atoms of the hydroxyls at C-1, C-3, and to a lesser extent C-6. The role of the C-4 hydroxyl is not as clear as the other positions but appears to be more important in the absence of a hydroxyl at C-6. There is no requirement for a gluco-configuration C-2 hydroxyl as is the case for the sodium-dependent active sugar transport systems of the intestine and kidney (Crane, 1960; Silverman, 1976). However, it should be noted that these hydrogen bonds need not form simultaneously, but that all are formed at some stage of the transport process. in solution the hexoses will be hydrogen bonded to water and changes in this hydration shell with different analogs may also influence the binding of the molecule to the transporter. The hydrogen bonding requirements of the adipocyte hexose transporter are very similar to those reported for the human erythrocyte by Kahlenberg and Dolansky (1972) and Barnett et af. (1973a), with only slight differences in the affinities of C-4K-6-modified sugars between the two systems. Similar hydrogen bonding requirements have also been reported for hexose transport across the blood-brain barrier (Betz ef af., 1975) and the sodium-independent system of the basal lateral membranes of the small intestine (Wright ef at., 1980).

VII. NONTRANSPORTED COMPETITIVE INHIBITORS OF TRANSPORT Not all competitive inhibitors of transport are transported by the transport system, apparently since spatial restrictions prevent them from passing through the membrane. The use of alkylated D-glucose analogs and disaccharides (Holman et d . , 198I b) has revealed that D-glucose binds to the external site of the transporter through the reducing part of the molecule (C-I) with the nonreducing part of the molecule (C-4) facing the external solution. There is a close approach of the molecule at C-l and C-2 with little space being available around these hydroxyls. There is rather more space around the C-3 hydroxyl which accounts for the transport of 3-O-methyl-~-glucose.Glucose molecules with bulky hydrophobic substitutions at C- 1 or C-416, for example, 4,6-0-ethylidene-~-glucopyranose (4,6-O-ethylidine-~-glucose), n-propyl, or n-butyl-P-~-glucosides,are not transported by the insulin-sensitive hexose transporter, but these compounds are able to enter the cell through an alternative route, probably by nonmediated diffusion (Holman and Rees, 1982). These analogs show asymmetric side-specific competitive inhibition of 3-U-methylglucose transport; 4,6-U-ethylideneD-glucose is an inhibitor on the outside of the cell but does not inhibit on the

HEXOSE TRANSPORT IN ADIPOCYTES

361

inside, while the alkyl-@-r>-glucosidesare effective inhibitors at the inside of the cell membrane but do not inhibit at the outside. A similar situation exists in the human erythrocyte. Baker and Widdas (1973) reported that 4,6-O-ethylidene-~-glucosewas a much more effective inhibitor of the outside site than the inside site. Barnett et al. (1973b, 1975) showed that 6-0alkyl derivatives of D-glucose were inhibitors only outside the membrane while alkyl-@-~-glucosideswere inhibitors only at the inner face of the membrane. On the basis of the results from both the human erythrocyte and the rat adipocyte, similar models for the mechanism of D-glucose transport have been put forward (Fig. 5). The glucose molecule in the external solution binds to the transporter through the C-1 end of the molecule. The transporter protein is then proposed to undergo a conformational change and the glucose molecule is transferred to the inner site with C-1 facing the internal solution. Inhibitor studies have thus revealed an asymmetry of the inner and outer binding sites of the adipocyte glucose transport system which is not shown by the kinetics of hexose transport. In this context it is interesting to note that the

FIG.5 . The proposed structure of the transporter. (a) In the absence ofthe substrate the system is closed. (b) Binding to the external site destabilizes the interface between subunits. Sufficient spacc is available to accommodate a bulky group at C-4. (c) Binding to the internal site opens the internal subunit interface. Sufficient space is available to accommodate a bulky group at C-I. i, 0, Inner and outer sites, respectively. (From Holman and Rees, 1982.)

362

J. GLIEMANN AND W. D. REES

inhibition constants of both the inside and outside-directed side-specific analogs are very similar in both the erythrocyte and the adipocyte. There is no evidence for the 10-fold asymmetry of affinities between the inner and outer sites of the human erythrocyte, which is evident in the zero trans experiments, when the inhibition constants of these analogs are compared in the two cell types. Models such as the asymmetric carrier (Geck, 1971; Regen and Tarpley, 1974) predict such as asymmetry and this observation may provide a further reason for rejecting such models. The observation that D-glucose may inhibit transport by binding to the transporter on the cytoplasmic facing side through the nonreducing part of the molecule should also be considered in experiments with isolated plasma membranes. The currently favoured membrane preparation (McKeel and Jarett, 1970) uses a sucrose-containing buffer for the isolation procedure. If sucrose can gain access to the inner site of the transporter (the inner face of the membrane) as in a membrane preparation it may cause competitive inhibition of transport through the free C-4/C-6 part of the glucose molecule. If so, this will lead to an increase in the apparent K , for transport and may explain the reported K,, of 26 mM for D-glucose (Ludvigsen and Jarett, 1980). It has been suggested that cytochalasin B inhibits hexose transport through binding to the inside facing site in the human erythrocyte (Basketter and Widdas, 1978), and it might therefore by analogy also bind to the inside site of the adipocyte transporter. Therefore, sucrose may also compete for the cytochalasin B binding site reducing the apparent binding in a competitive manner.

VIII.

SUGARS WHICH ARE BOTH TRANSPORTED AND PHOSPHORYLATED-RATE-LIMITING STEPS

2-Deoxy-~-glucoseis phosphorylated by the hexokinase and is not believed to be metabolized any further to any major extent (Wick et a l . , 1951). 2-Deoxyglucose phosphate is trapped in the cells and the total rate of 2-deoxyglucose uptake might therefore be taken as a measure of the rate of 2-deoxyglucose transport when the transport step is rate determining. Olefsky (1 978) used 2-deoxyglucose (3-minute uptakes) in an attempt to characterize the transport system and found a K,, for deoxyglucose of about 1.2 mM (Fig. 2 of the reference) as well as an inhibition constant (Ki) for glucose of about 2 mM. However, it turns out that the hexokinase becomes partially rate limiting for the uptake of 2-deoxyglucose at deoxyglucose or glucose concentrations as low as about 50 pM (Foley er al., 1980b). This shift in the rate-limiting step from transport at a trace sugar concentration to hexokinase at higher sugar concentrations is particularly evident in insulin-stimulated cells due to the high sugar permeability of the plasma membrane. Therefore, the measured inhibition

HEXOSE TRANSPORT IN ADIPOCYTES

363

constants of phosphorylated sugars will depend on the time of incubation: at short times (a few seconds) the measured inhibition constants will reflect mainly K , on the transport system and at infinite time mainly Ki of the hexokinase. At infinite time the apparent Ki for deoxyglucose and glucose is of the order of 100 @ in insulin-stimulated cells; on the other hand, the inhibition constant of deoxyglucose on the initial velocity of methylglucose uptake ( 1 second measurements) is about 5 mM (Foley et d.. 1980b). Recent results have revealed some surprising characteristics of the 2-deoxyglucose transport (Foley and Gliemann, 1981a). In the presence of 2-deoxyglucose at a very low concentration (7 pM) the hexokinase should act as a sink and the uptake should continue at a linear rate in the absence of efflux of 2deoxyglucose phosphate (or 2-deoxyphosphogluconate which is a minor metabolic product of 2-deoxyglucose phosphate). In fact, the uptake curve was linear for only about 10 minutes and this was caused by a slow efflux of free deoxyglucose. Furthermore, a high fraction of the intracellular sugar was present in the free form. Time course studies showed that the intracellular concentration of free deoxyglucose remained essentially zero for about 1 minute. The ratio of intracellular deoxyglucose concentration to extracellular concentration (accumulation ratio) exceeded unity by 3-5 minutes and then continued to increase. By 60 minutes, the intracellular deoxyglucose concentration had exceeded the extracellular concentration by 50-fold. In other words, free deoxyglucose was markedly accumulated in the cell against its concentration gradient. This accumulation was absent in cells depleted of ATP by treatment with dinitrophenol. The mechanism of the accumulation is in part explained by the phosphorylation of newly transported 2-deoxyglucose followed by dephosphorylation. However, it remains to be explained why the 2-deoxyglucose generated by dephosphorylation does not equilibrate with the extracellular medium within seconds. One possihility is that the transport rate of free 2-deoxyglucose out of the cell is much slower than the inward transport. However, this seems highly unlikely, first because the transport system is symmetric with respect to methylglucose and second because the internal deoxyglucose concentration is so low when the accumulation starts that it is difficult to understand how it could exert any inhibition of the transport system. Therefore, it seems necessary to postulate a diffusion barrier between the site of dephosphorylation and the transporter (Fig. 6 ) . In this connection it is worth noting that deoxyglucose phosphate, generated by phosphorylation of deoxyglucose in a tumor cell line, appears to be located in a compartment of a much lower pH (6.4) than that of inorganic phosphate (cytosol, pH 7.1) (Griffith et al., 1981). Other time course experiments (Foley and Gliemann, 1981a) showed that at higher deoxyglucose concentrations, the accumulation of intracellular free deoxyglucose started earlier whereas the steady-state accumulation ratio de-

364

J. GLIEMANN AND W.

D.REES

plasma membrane

5

Hexokinase

/

[14C] DO

l7pMI

(I

-;

rephosphorylation

a

Slow efflux of accumulated [14d DG from postulated compartment

FIG.6 . A model proposed to explain the accumulation of free 2-deoxyglucose against its concentration gradient in adipocytes. DG, Deoxyglucose; DGP, deoxyglucose phosphate. For further explanation, see text.

creased progressively. Thus, a maximum accumulation ratio of 3.5 was reached by 7 minutes using I mM and a ratio of about 1.6 was reached by 3 minutes using 10 mM extracellular 2-deoxyglucose. This phenomenon is probably related to the limited capacity of the hexokinase. It is difficult to predict the effect of intracellular deoxyglucose on the measured transport parameters of other sugars since the accumulation ratios are not necessarily indicative of the internal concentration at the transport site. Recent experiments have shown that high concentrations of phloretin cause a rapid drop in the ATP level of adipocytes and that this is associated with a dephosphorylation of 2-deoxyglucose phosphate (Wieringa et al., 1981). Phloretin was not used in the experiments cited above showing intracellular accumulation of free deoxyglucose. However, the results of Wieringa et al. (1981) demonstrate that the ATP level may be important in regulating the adipocyte phosphatase activity. Marked accumulations of 2-deoxyglucose have previously been reported in mammalian cells, for example, hamster kidney cortex slices (Elsas and McDonell, 1972). However, in this system sugar transport is sodium dependent and active (uphill), and transport clearly precedes phosphorylation. On the other hand, Kleinzeller and McAvoy (1973) have found evidence for a slight accumulation of 0.5 mM deoxyglucose against its concentration gradient following sodium-independent transport across the basolateral membrane of flounder renal cells. Since dephosphorylation of deoxyglucose phosphate also occurs in this system, the mechanism of accumulation might be similar to that postulated for adipocytes. From a physiological point of view, D-glucose is of course the most interesting

HEXOSE TRANSPORT IN ADIPOCYTES

365

sugar. Its inhibition constant on the initial velocity of methylglucose transport is about 8 m M , and this is in agreement with experiments designed to measure uptake of glucose itself (Whitesell and Gliemann, 1979). Similar values have been reported for the inhibition constants of glucose on allose uptake ( 1 3 mM, Loten et al., 1976; and 9 mM, Rees and Holman, 1981) and arabinose uptake (8 mM, Foley et al., 1978). Using the “slow” sugars, the measured inhibition constant would be one of equilibrium exchange ifthe rapidly transported glucose was not metabolized. However, glucose is actually metabolized and transport is rate limiting at low concentrations (Foley et al., 1980d) but not at concentrations above 0.5 mM in insulin-stimulated cells (Gliemann, 1967, 1968). In fact, it has been shown that 2 mM glucose equilibrates across the membrane in insulinstimulated cells (Foley er al., 1980a). Therefore it seems unlikely that K , for net entry is different from that of equilbrium exchange. In other words, the system is probably symmetric with respect not only to 3-O-methyl-~-glucosetransport (as described above) but also to D-glucose transport. In view of the unexpected results with accumulation of free intracellular deoxyglucose, similar experiments were carried out with 7 pV glucose. However, we were unable to detect any accumulation of glucose, which is perhaps not surprising in view of the rapid further conversion of glucose 6-phosphate to metabolites (Foley and Gliemann, unpublished observations). However, this does not rule out that a phosphorylation-dephosphorylation cycle might occur in analogy to the findings with 2-deoxyglucose. The glucose concentration giving half-maximal glucose metabolism is about 1 mM in insulin-stimulated cells (Gliemann, 1968). This is the reason why insulin assays based on glucose metabolism are carried out at a low glucose concentration (Gliemann, 1967; Moody er al., 1974). However, from a physiological point of view this seems paradoxical considering that the plasma glucose concentration varies roughly between 4 and 8 mM. The low “metabolism Km” agrees neither with experiments on epididymal fat pads (Gliemann, 1968) nor with insulin action in vivo. It seems likely that interstitial diffusion gradients exist not only in incubated pieces of adipose tissue, as shown by Crofford and Renold (1965a,b) but also in vivo. Several other metabolizable sugars are transported via the insulin-sensitive glucose transport system but rather little information is available. Mannose at tracer concentration is transported at a slightly slower rate than glucose and is rapidly phosphorylated and metabolized (Foley et al., 1980~). Galactose is transported at about half of the rate of glucose but is phosphorylated at a much slower rate (Vega and Kono, 1978). Fructose is transported slowly by the glucose transporter discussed in this article. However, it should be stressed that the adipocytes possess a specific transport system for fructose which is not influenced by insulin (Schoenle et al., 1979). In “basal” cells, the fructose uptake is almost entirely accounted for by transport via its specific system, whereas the

366

J. GLIEMANN AND W.

D.REES

insulin-sensitive system accounts for about half of the total fructose uptake in insulin-stimulated cells.

IX. MODULATION OF THE TRANSPORT SYSTEM BY GLUCOSE METABOLITES Using 2-deoxyglucose as a probe, it has been found that a high rate of glucose metabolism modulates the transport system. The ability of 2-deoxyglucose to inhibit the initial velocity of 3-0-[ 14C]methylglucoseis slightly greater than that of methylglucose (Foley et af., 1980~).Therefore, it would be expected that 2deoxyglucose was transported at least as rapidly as methylglucose. It is, however, transported at only about one-third of this rate indicating some resistance to the transfer of 2-deoxyglucose across the membrane after its initial binding. Incubation of the insulin-stimulated adipocytes with 10 mM glucose for 30 minutes at 37°C increases the permeability of deoxyglucose to the same level as that of methylglucose (Foley et a/., 1980~).Mannose, which is transported rapidly, phosphorylated, and further metabolized to glucose intermediates, has the same effect. Sugars which are either transported or metabolized slowly have no effect. Thus, a high rate of glucose metabolism modulates the transport system and removes the resistance to transfer of 2-deoxyglucose at a tracer concentration. It is possible that the effect is caused by a feedback of an intermediate of glucose metabolism but no direct evidence is available. It also remains to be clarified whether a high rate of glucose metabolism affects v,, or K,, for transport of 2-deoxyglucose. A model for the hexose transport system would have to account for this phenomenon and we have proposed that shown in Fig. 7. The initial sugar binding occurs at step 1 and here deoxyglucose and methylglucose have equal affinities. The putative glucose metabolite is presumed to act at the cytoplasmic side of the membrane, i.e., at step 3; in the absence of glucose the resistance of step 3 is higher for 2-deoxyglucose than for 3-0-methylglucose, whereas a high rate of glucose metabolism causes a modification of step 3 to give the same resistance for the two sugars. The transmembrane distance between the two points of solution contact of an intrinsic protein or assembly of proteins is rather large as compared with the size of a hexose molecule, and for this reason a diffusive step (step 2) is proposed between the two “discriminators” or “microcarriers” (steps 1 and 3 ) . The properties of the “microcarriers” would be as described by Holman and Rees (1982): the C-1 region of glucose binds to the extracellularly facing (at step 3 pore facing) side; this induces a conformational change and the sugar is let loose on the other side (cf. Fig. 5). Conversely, the C-4 region binds to the cytosolic facing (at step 1 the pore facing) side followed by a conformational change and transfer of the sugar. This model of two re-

HEXOSE TRANSPORT IN ADIPOCYTES

367

Step i 0

FIG. 7. A model of the glucose transporter in adipocytes. The model was proposed to explain the acceleriltion of glucose metabolism on the initial velocity of 2-deoxyglucose uptake. Each of the microcamers is proposed to function as illustrated in Fig. 5. (From Foley and Gliemann, 1981b.)

sistances in series separated by a pore (Fig. 7) eliminates the necessity of a very large protein carrier moving the sugar molecule across the membrane. The kinetic transport parameters of a model of this type will be described by complex equations. The resistances at step 1 and step 3 and the pore volume will determine the flux through the model and detailed predictions depend on the value of these parameters. The model is not incompatible with the available data for transport of 3-0-methylglucose in the adipocyte but more refined experiments are necessary to assign values for the basic model parameters.

X.

MECHANISM OF INSULIN’S ABILITY TO INCREASE V,,,,,

Two groups have recently reported important observations clarifying the cause of the insulin-induced increase in V,,, for 3-0-methylglucose transport. Cushman and co-workers (Wardzala et al., 1978) characterized a specific class of D-glucose inhibitable cytochalasin B binding sites in adipocyte plasma membranes and found that insulin treatment of the cells prior to preparation of the membranes caused a marked increase in the number of sites. The cytochalasin B binding site was taken to be a marker for the transporter and the insulin-induced increase in specific plasma membrane cytochalasin B binding sites should therefore be analogous to the insulin-induced increase in V,,, as shown in Fig. 4. Cushman and Wardzala ( 1980) also found glucose-displaceable cytochalasin B binding sites in a low-density microsomal fraction, and, moreover, treatment of the cells with insulin before the subcellular fractionation caused a marked shift in the distribution of binding sites so that the insulin-induced increase in plasma

368

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membrane sites was accompanied by a comparable decrease in the microsomal sites. This shift does not occur in cells depleted of ATP, and neither does the insulin effect on transport (Kono er al., 1977; Siege1 and Olefsky, 1980) or the reversal of the transport activity to the basal state in insulin-pretreated cells (Vega et al., 1980; Laursen et al., 1981). The increment in the plasma membrane cytochalasin B binding sites after treatment with insulin at a high concentration corresponds well to the insulin-induced increase in transport of 3-0methylglucose. Moreover, there is good quantitative agreement between the steady-state insulin dose-response relationships of the transport increase on the one hand and the appearance of cytochalasin B binding sites on the other, as well as between the time course of the two phenomena (Karnieli et af., 1981a). Suzuki and Kono (1980) used a modification of the method described by Shanahan and Czech ( 1977) to solubilize from isolated membranes components which catalyze stereospecific glucose transport. These authors found that insulin caused an increase in the transport activity derived from the plasma membrane and a decrease in the activity derived from a light microsomal fraction. Also the experiments of Kono and co-workers show adequate quantitative correlations between the insulin-induced increase in transport activity of the whole cell and the transport activity that can be extracted from the plasma membrane fraction (Kono et al., 1981). Taken together, these independent experiments provide convincing evidence that hexose transporters are in two pools, one (functional) in the plasma membrane and another (nonaccessible or nonfunctional) at some other location. Moreover, insulin causes a translocation of the transporters from the nonfunctional to the functional pool. The model proposed by Cushmann and co-workers is shown in Fig. 8. There is little doubt that an increase in the number of functional transporters in the plasma membrane is a major effect of insulin in adipocytes and the same mechanism has been proposed in striated muscle (Wardzala and Jeanrenaud, 1981). However, criticism has been raised (Carter-Su and Czech, 1980) and an alternative mechanism has been proposed in the adipocyte (Pilch er al., 1980). The translocation hypothesis explains an observation made by several authors and first by Martin and Carter (1970), namely, that insulin has no effect when added directly to plasma membrane vesicles retaining the stereospecific glucose transport system. This hypothesis is also in agreement with the identical K , values for transport of 3-0-methylglucose in basal and insulinstimulated cells using different experimental protocols (Taylor and Holman, 1981) and with identical K , values for a range of different sugars (Holman et al., 1981b). The question is whether the hypothesis explains the entire insulin effect. Some observations favor at first glance the proposal that insulin also increases the sugar transport across each individual transport unit. Thus, an increase in temperature from 20 to 37°C increases transport of 3-0-methylglucose in insulinstimulated but not in “basal” cells (Czech, 1976a; Whitesell and Gliemann,

369

HEXOSE TRANSPORT IN ADIPOCYTES Dissociation

(9 Translocation

“V

Glucose Glucose

-

0 + -. Transport

Glucose

\b

\\

\@Fusion

lntracellular Pool

I

d a n s l o c a tion

\-@Binding

7

Plasma

0 Association FIG.8. Schematic representation of a hypothetical mechanism of insulin’s stirnulatory action on 1981.) glucose transport in adipocytes. (From Karnieli et d,,

1979). However, Kono et al. (1981) have observed that decreasing temperature causes a shift in the steady-state distribution of transporters toward the plasma membrane pool, and this may explain the apparent difference in the behavior of transporters in the “basal” and insulin-stimulated state. Sonne et al. (1981) observed that the “basal” but not the insulin-stimulated transport rate increased with increasing pH. There is n o information as to whether this difference might also be due to a shift in the distribution between the pools of transporters. It also remains to be established whether sections of the plasma membrane are pinched off and transferred to an intracellular pool as depicted in the model of Cushrnan and co-workers (Fig. 8). It should be noted that insulin influences various transport systems to different degrees. Thus, transport of the nonmetabolizable amino acid a-aminoisobutyric acid is not stimulated by insulin in the adipocyte (Minemura ef al., 1970) and neither is transport of adenosine (W. D. Rees, unpublished observations). Also, as noted above, transport of fructose through the fructose transporter is not modulated by insulin. Therefore, a model of the type proposed by Kono, Cushman, and their co-workers seems to

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demand that some areas of the plasma membrane are specialized to contain the glucose transporters or that the transporters are able to cluster in such areas. In any case, the transfer, at least from the putative intracellular pool and to the plasma membrane, is surprisingly rapid since the maximal effect of insulin at a high concentration on 3-0-methylglucose transport is manifest by about 1 minute (Whitesell and Gliemann, 1979). At lower (physiological) insulin concentrations the rate-limiting step for activation or deactivation of the transport system appears to be the association or dissociation of insulin from the receptors (Karnieli et af., 1981a). This is in agreement with previous studies on the time course of insulin binding and insulin-induced activation of lipogenesis from glucose (Gliemann et al., 1975). The next question is whether insulin after binding to its receptor causes the formation of a chemical signal which in turn mediates the transfer of transport units from the inactive to the active pool. Larner et al. ( 1979) have extracted a heat- and acid-stable factor from muscle which inhibits cyclic AMP-dependent protein kinase and activates glycogen synthase phosphoprotein phosphatase. Jarett and Seals (1979) have shown that insulin activates pyruvate dehydrogenase in mitochondria provided that plasma membranes are present in the mixture. Later studies showed that insulin can induce the release of a material from adipocyte membranes which appears to mediate its action on pyruvate dehydrogenase and this material was indistinguishable from the “Larner material” (Kiechle et al., 1981). The factor seems to be a peptide with a molecular weight of 1000-4000 (Seals and Czech, 1981; Kiechle et al., 1981) and is perhaps produced from an endogenous substrate by a protease which becomes activated after binding of insulin to its receptor (Seals and Czech, 1980). The putative mediator is probably not a part of the insulin molecule since its release from adipocyte plasma membranes can be initiated by proteases such as trypsin in the absence of insulin (Seals and Czech, 1980). It should also be noted in this connection that irreversible binding of photoaffinity-labeled insulin to adipocytes appears to cause an irreversible activation of the transport system (Ushkoreit et al., 1981). The question remains open as to whether the “Larner material” has a function as a signal for the stimulation of hexose transport. Another possibility is that the intracellular concentration of calcium ions plays a critical role, even though several authors have noted that the effect of insulin on hexose transport is not influenced by extracellular calcium. Clausen and co-workers (Sorensen et al., 1980) have shown that insulin increases the efflux of radiolabeled calcium ion from preloaded adipose or muscle tissue with a similar time course as the increase in transmembrane sugar transport. Thus, insulin may increase the release of calcium ions from intracellular stores and thereby cause a shift in the distribution of transporters. Several other mechanisms have been proposed [see Gliemann et al. (1981) for review].

HEXOSE TRANSPORT IN ADIPOCYTES

XI.

371

HUMAN ADIPOCYTES

Transport of 3-0-methylglucose has been studied using the technique shown in Fig. IC (Ciaraldi et al., 1979; Pedersen and Gliemann, 1981). The transport system appears very similar to that of the rat adipocyte in that K , for net entry as well as for equilibrium exchange of 3-0-methylglucose is about 4 mM. In other words, the system seems to behave symmetrically with respect to 3-0-methylglucose transport. The inhibition constant of glucose on the initial velocity of 3-0-methylglucose uptake is about 8 mM as in the rat adipocyte. Transfer of glucose across the plasma membrane by nonmediated diffusion is also insignificant in the human adipocyte. The main difference between epididymal adipocytes from 200-g rats and abdominal subcutaneous adipocytes from normal weight adult humans is the relatively small response to insulin (two- to threefold) in the human cells. In the “basal” state, the permeability of the human cells is about half of that of the rat cells. Assuming that the turnover of sugar molecules is the same on each transporter from the two species, this indicates that the density of transporters in the human cell is about half of that in the rat cell. In the presence of insulin the permeability of the human adipocyte is about one-tenth of that of the rat adipocyte. It is likely, therefore, that the adipocyte of adult humans is able to recruit only a limited number of additional transport units when treated with insulin. The questions of the orientation of the glucose molecule in the transporter and the spatial and hydrogen binding requirements have not been studied in the human adipocyte but there is no a priori reason to expect any important differences between the species. As in the rat, the rate of metabolism of glucose appears to be limited by the hexokinase and not by transport when insulin is present and the glucose concentration exceeds a few millimoles per liter (Pedersen and Gliemann, 1981).

XII. THE TRANSPORT SYSTEM IN OBESITY AND DIABETES Results from our laboratory using 3-0-methylglucose have shown that the permeability (cdsecond) in the absence of insulin is about the same in small cells from small lean rats and large cells from large obese rats (Foley el al., 1980d). The number of transporters per unit surface area is thus probably quite independent of the cell size. On the other hand, after treatment with insulin the permeability is much smaller in cells from large rats than in cells from small rats. Therefore, it is likely that the cells from obese rats are able to recruit less transporters from the inactive pool after treatment with insulin. Recently, Cushman et al. (198 I ) showed that the number of cytochalasin B binding sites per unit

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D.REES

area of the plasma membrane was independent of the cell size whereas the insulin-induced increase in the number of binding sites in the plasma membrane was markedly reduced in cells from obese rats. This agrees with the transport studies of Foley et af. (1980d). Earlier studies have shown that the decreased insulin responsiveness with respect to glucose metabolism was related to the degree of obesity of the animals rather than to the adipocyte size per se (Gliemann and Vinten, 1974; Hansen et uf., 1974). These studies were carried out using a low glucose concentration (0.5 mM) and the same conclusion probably applies, therefore, to the glucose transport step. Other authors studying transport in small and large cells (Livingston and Lockwood, 1974; Czech, 1976b; Olefsky, 1976) have obtained different results, probably because 2-deoxyglucose uptake was taken as a measure of transport or because the initial velocity was missed in transport studies using 3-O-methylglucose (for discussion, see Foley et af., 1980d). Streptozotocin-induced diabetes in the rat is associated with a marked reduction in the ability to stimulate glucose transport and metabolism in the adipose cell (Kasuga et af., 1978; Kobayashi and Olefsky, 1979). Recent studies by Cushman and co-workers (Karnieli et af., 1981b) have shown that this, in fact, is associated with a depletion of the pool of cytochalasin B binding sites (and therefore probably hexose transporters) that can be recruited by insulin treatment.

XIII. RECONSTITUTION OF THE HEXOSE TRANSPORTER The reconstitution of intrinsic membrane proteins into an artificial phospholipid bilayer offers a powerful technique for the study of hexose transport. These techniques have been pioneered with the human erythrocyte hexose transporter (Kasahara and Hinkle, 1976) and have now been refined, giving a high efficiency of reconstitution [for recent reviews see Baldwin and Lienhard (1981) and Jones and Nickson (1981)l. Briefly, the results indicate that the purified transporter is a protein of 46,000 molecular weight (Gorga et af., 1979) which binds cytochalasin B in what Baldwin and Lienhard (1981) have suggested to be a 1:l ratio. Studies on the native membrane using radiation inactivation (Jung et al., 1980) suggest, however, that the transporter is larger with a molecular weight of 2 X lo5. Wheeler and Hinkle (1981) have shown that the reconstituted transporter-like the transporter of the intact erythrocyte-shows accelerated sugar transport when sugar is added to the trans side (accelerated exchange). The transporter is incorporated at random in the liposome and the reconstituted system is therefore not asymmetric. However, asymmetry becomes manifest when the liposomes are treated with trypsin which cancels transport in the transporters incorporated “upside down” (Wheeler and Hinkle, 1981).

373

HEXOSE TRANSPORT IN ADIPOCYTES

Reconstitution of the adipocyte hexose transporter has also been reported (Shanahan and Czech, 1977), but since there are few copies of the transporter in the adipocyte membrane, this system has presented greater difficulties. Carter-Su et al. (1980, 1981) have partially purified a protein from rat adipocyte membranes and report that an integral membrane protein can be reconstituted into liposomes which then show stereospecific glucose transport. The transport protein is-in contrast to the glycoprotein insulin receptor-not retained by column chromatography using immobilized concanavalin A (Carter-Su et al., 1981). This does not rule out the possibility that the two proteins are noncovalently associated within the native membrane. The molecular weight of the adipocyte transport protein has not been determined, but the molecule has been reported to have a Stokes radius of 60-80 A (Carter-Su er af., 1981). These molecular dimensions would be sufficient to allow the protein to span the membrane consistent with a model in which substrate binding sites are in contact with each solution. The molecule could therefore provide a channel through which the sugar can move (cf. Fig. 7).

XIV.

CONCLUDING REMARKS

Studies over the last decade have elucidated the kinetics of the binding of insulin to its receptor and the relation between insulin binding and biological effects such as the enhancement of glucose transport. Furthermore, the subunit structure of the insulin receptor has been clarified (for reviews see, for example, Czech, 1980; and Gliemann ef al., 1982). In addition, the characteristics of the insulin-sensitive glucose transporter have been elucidated using adipocytes as a model system. This transporter is similar to that of human erythrocytes with respect to substrate specificity but is different with respect to the kinetic properties of glucose transport. The insulin-induced increase in hexose transport seems to be brought about by a transfer of transporters to the plasma membrane from a storage pool. Future experiments may clarify the important questions of the precise nature of the transfer process and the properties of a possible chemical mediator. ACKNOWLEDGMENTS W. D. Rees is a recipient of a NATO-SERC overseas postdoctoral fellowship. The authors wish to thank Drs. S . W. Cushman, G . D. Holman, and J . Vinten for allowing us to reproduce their published figures. REFERENCES Andreasen, P . , Schaumburg, B . , Plsterlind, K., Vinten, .I.Gammeltoft, , S . , and Gliemann, J . ( 1974). A rapid technique for isolation of thymocytes from suspension by centrifugation through silicone oil. Anal. Biochcm. 59, 110-1 16.

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Baker, G . F., and Naftalin, R. J. (1979). Evidence for multiple operational affinities for D-glucose inside the human erythrocyte membrane. Biochim. Biophys. Acza 550, 474-484. Baker. G. F., and Widdas, W. F. (1973). The permeation of human red cells by 4,6-0-ethylidene-a-D-glucopyranose(ethylidene glucose). J . Physiol. (London) 231, 129- 142. Baldwin, S . A , , and Lienhard, G. E. (1981). Glucose transport across plasma membranes: Facilitated diffusion systems. Trends Biochem. Sci. 6 , 208-2 I 1. Bang, 0.. and (Zlrskov, S. L. (1937). Variations in the permeability of the red blood cells in man. J. Clin. Invest. 16, 279-281. Bamett, J. E. G., Holman, G . D., and Munday, K. A. (l973a). Structural requirements for binding to the sugar transport system of the human erythrocyte. Biochem. J. 131, 21 1-221. Bamett, J. E. G . , Holman, G , D., and Munday, K. A. (1973b). An explanation of the asymmetric binding of sugars to the human erythrocyte sugar transport system. Biochem. J. 135, 539541. Bamett, J: E. G., Holman, G . D., Chalkley, R. A., and Munday, K. A. (1975). Evidence for two asymmetric conformational states in the human erythrocyte sugar transport system. Biochem. J. 145, 417-429. Basketter, D. A., and Widdas, W. F. (1978). Asymmetry of the hexose transfer system in human erythrocytes. J. Physiol. (London) 278, 389-401. Betz, A. L., Drewes, L. R., and Gilboe, D. D. (1975). Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues. Biochim. Biophys. Acta 406, 505-5 15. Carter-Su, C.. and Czech, M. P. (1980). Reconstitution of o-glucose transport activity from cytoplasmic membranes. J. B i d . Chem. 255, 10382- 10386. Carter-Su, C., Pillion, D. J., and Czech, M. P. (1980). Reconstituted D-glucose transport from the adipocyte plasma membrane. Chromatographic resolution of transport activity from membrane glucoproteins using immobilized Con A. Biochemistry 19, 2374-2385. Carter&, C., Pillion. D. I., and Czech, M. P. (1981). Chromatographic resolution of the insulin receptor from the insulin insensitive D-glucose transporter of adipocyte plasma membranes. Biochemistry 20, 216-221. Ciaraldi, T. P.,Kolterman, 0.G., Siegel, J. A,, and Olefsky, J. M. (1979). Insulin stimulated glucose transport in human adipocytes. Am. J . Physiol. 236, E621LE625. Craik. J. D., and Elliot, K. R. F. (1979). Kinetics of 3-O-methy~-o-gh1cosetransport in isolated rat hepatocytes. Biochem. J. 182, 503-508. Crane, R. K. (1960). Intestinal absorption of sugars. Physiol. Rev. 40, 789-825. Crofford, 0. B., and Renold, A. E. (1965a). Glucose uptake by incubated rat epididymal adipose tissue. Rate limiting steps and site of insulin action. J. Biol. Chem. 240, 14-21. Crofford, 0. B., and Renold, A. E. (1965b). Glucose uptake by incubated rat adipose tissue. Characteristics of the glucose transport system and action of insulin. J. Biol. Chem. 240, 3237-3243. Crofford, 0. B., Stauffacher, W., Jeanrenaud, B., and Renold, A. E. (1966). Glucose transport in isolated fat cells. Procedures for measurement of the intracellular waterspace. Helv. Physiol. Pharmacol. Acta 24, 45-57. Cushman, S . W., and Wardzala, L. J. (1980). Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. J. Biol. Chem. 255, 4758-4762. Cushman, S. W.. Hissin, P. I., Wardzdla, L. J., Foley, J. E., Simpson, 1. A., Kamieli, E., and Salans, L. B. (1981). Mechanism of insulin resistance in the adipose cell in the aging rat model of obesity. Biochem. Sac. Trans. 9, 518-522. in the rat. Biachim. Czaky, T. Z., and Wilson, J. E. (1956). The fate of 3 - 0 4 14C]methyl-~-g~ucose Biophys. Acta 22, 185-186. Czech. M. P. (1976a). Regulation of the o-glucose transport system in isolated fat cells. Mol. Cell. Biochem. 11, 51-63.

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Czech, M. P. (1976b). Cellular basis of insulin insensitivity in large adipocytes. J. Clin. Invest. 57, 1523-1532. Czech, M. P. ( 1980). Insulin action and the regulation of hexose transport. Diabetes 29, 399-409. Eilam, Y., and Stein, W. D. (1974). Kinetic studies of transport across red blood cell membranes. In “Methods in Membrane Biology” (E. D. Korn, ed.), Vol. 2, pp. 283-354. Plenum, New York. Elsas, L. J., and McDonell, R. C. (1972). Hzxose transport and phosphorylation by hamster kidney cortex slices and everted jejunal rings. Biochim. Biophys. Acta 255, 948-959. Foley, J. E., and Gliemann, J. (1981a). Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes. Biochim. Biophys. Acru 648, 100-106. Foley, J. E., and Gliemann, J. (I981 b). Glucose transport in isolated adipose cells. Int. J . Obesity 5, 679-684. Foley, J. E., Cushman, S. W., and Salans, L. B. (1978). Glucose transport in isolated rat adipocytes with measurements of L-arabinose uptake. Am. J. Physiol. 234, El 12-El 19. Foley, J. E., Cushman, S. W., and Salans, L. B. (1980a). Intracellular glucose concentration in small and large adipose cells. Am. J . .Physiol. 238, E180-El85. Foley, J. E.. Foley. R.. and Gliemann, 1. (1980b). Rate limiting steps of 2-deoxyglucose uptake in rat adipocytes. Biochim. Biophys. Acfo 599, 689-698. . acceleration of deoxyglucose Foley, J. E., Foley, R., and Gliemann, J. ( 1 9 8 0 ~ )Glucose-induced transport in rat adipocytes: Evidence for a second barrier in sugar entry. J . Biol. Chem. 255, 9614-9677. Foley, J. E., Laursen, A. L., Sonne, O., and Cliemann, J . (1980d). Insulin binding and hexose transport as related to fat cell size. Diahetologia 19, 234-241. Geck, P. (1971). Properties of a carrier model for the transport of sugars by human erythrocytes. Biochim. Biophys. Acra 241, 462-47:!. Ginsburg, H., and Stein, W . D. (1975). Zkro-trans and infinite-cis uptake of galactose in human erythrocytes. Biochim. Biophys. Aria 382, 353-368. Gliemann, J. (1967). Assay of insulin-likc activity by the isolated fat cell method. I. Factors influencing the response to crystalline insulin. Diubetologin 3, 382-388. Gliemann, J . (1968). Glucose metabolism and response to insulin of isolated fat cells and epididymal fat pads. Aria Physiol. Scand. 72, 481-491. Gliemann. J., and Vinten, J. (1974). Lipcigenesis and insulin sensitivity of single fat cells. J. Physiol. (London) 236, 499-516. Gliemann, J., Osterlind, K., Vinten, J., and Gammeltoft, S. (1972). A procedure for measurement of distribution spaces in isolated fat c(:lls. Biochim. Biophys. Acra 286, 1-9. Gliemann, J., Gammeltoft, S., and Vinten, I . (1975). Time course of insulin-receptors binding and insulin-induced lipogenesis in isolated rat fat cells. J . B i d . Chem. 250, 3368-3374. Gliemann, J., Laursen, A. L., Foley, J. E., and Sonne 0. (1981). The insulin receptors: Looking at the present. In “Current Views on Insillin Receptors (D. Andreani, ed.), pp. 1-12. Academic Press, New York. Ghemdnn, J . , Foley, J. E., Sonne, 0..and Laursen, A. L. (1982). Insulin. In “Polypeptide Hormone Receptors (B. I. Posner. ed.). Dekker, New York, in press. Gorga, F. R., Baldwin, S. A,, and Lienhard, C.E. (1979). The monosaccharide transporter from human erythrocytes is heterogenously glycosylated. Biochem. Biophys. Res. Commun. 91, 955-961. Griffith, J. R., Stevens, A. N., Iles, R. P I . , Gordon, R. E., and Shaws, D. (1981). 31P-NMR investigation of solid tumors in the living rat. Biorci. Rep. 1, 319-325. Hankin, B. L., Lieb, W. R., and Stein, W. D. (1972). Rejection criteria for the asymmetric carrier and their application to glucose transport in the human red blood cell. Biochim. Biophys. Acta 288, I 14- 126.

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