THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 246, No. 8, Issue of April 25, pp. 2472-2479, 1971

Printed in U.S.A.

Glucose Transport in Fat Cell Membranes

(Received for publication, October lB, 1970)

GENNARO ILLIANO* AND PEDRO CUATRECASAS$

From the Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland SO014

SUMMARY

The properties of D-glUCOSe transport by isolated fat cell membrane preparations have been studied by measuring directly the rates of sugar accumulation and efllux. Obser- vations are made under conditions in which zero order ki- netics prevails and during which no detectable metabolic alteration of glucose occurs. The initial velocities measured are proportional to the amount of membrane material present. This is a relatively simple model system which ,permits di- rect study of glucose transport and insulin action.

The rate of D-glucose accumulation is a temperature-de- pendent process. In contrast to L-glucose accumulation, D-glucose transport rapidly reaches a steady state, displays saturation kinetics with respect to substrate, is inhibited by 3-O-methyl glucose but not by L-glucose, is markedly sup- pressed by low concentrations of phloretin, and is enhanced by insulin. The transport process is not coupled to energy- producing metabolic processes, and no accumulation occurs against a chemical gradient.

The unidirectional efflux of D-glucose from fat cell mem- branes has been studied separately. This process is also temperature-dependent, energy (metabolic)-independent, stereospecific, saturable with respect to the concentration of glucose inside the membrane, and sensitive to the presence of insulin or phloretin in the medium external to the mem- brane. The kinetic rate constants for efllux (Kr, 0.2 mM;

3.8) differ from those calculated from the influx data rzy3.3 mM; v,,,, 12.3). Although these differences may reflect differences in the biological function of the membrane surfaces, they do not constitute definitive proof of asymmetry of the transport structures or processes.

Evidence for the operation of a mobile carrier mechanism of facilitated diffusion was obtained by detecting accelerated exchange diffusion. The unidirectional D-glucose flux from the inside of the membranes into a medium containing negligible glucose is accelerated by the presence of 3-O- methyl glucose in the medium. This suggests that the rate of transfer of free carrier from the outside to the inside phases of the membrane is a limiting process in the outward movement of the D-glucose-carrier complex.

* Recipient of a North Atlantic Treaty Organization Interna- tional Postdoctoral Fellowship, on leave from the Institute of Biological Chemistry, Medical School, University of Naples.

$ Present address, Departments of Medicine, and Pharmacology and Experimental Therapeutics, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.

Considerable attention has been directed toward the elucida- tion of the mechanisms of glucose transport and insulin action in adipose tissue (l-20). Extensive studies in this hormone- sensitive tissue have been performed on intact fat pads (1, 2, 4, 13), isolated fat cells (3, 6, 17, 18), and fat cell ghosts (21). Unfortunately, in all these studies only inferential interpretations of glucose transport have been made since direct measurements of glucose uptake have not been possible (12, 13, 22, 23). In these studies metabolism of glucose has been examined by determining the conversion of radioactive glucose to CO:! or to lipids, generally.over a period of hours. These measurements are believed to accurately reflect glucose transport since there is evidence that the rate of glucose entry into cells is, under certain conditions, the rate-limiting step of glucose metabolism in adipose tissue (1, 2, 5, 8). The accumulated data of these investigations support the view that glucose crosses the cell membrane by carrier-mediated facilitated diffusion (2, 5).

The present report describes a simple system for measuring accurately the rates of glucose transport across insulin-responsive isolated fat cell membrane preparations. The uptake of glucose by the membranes is measured directly over an early time period in which zero order kinetics prevails and no demonstrable me- tabolism occurs. The unidirectional efflux of glucose can also be studied selectively under similar conditions. The basic properties of this transport system indicate that it may serve as a simplified model to study and may characterize the funda- mental processes involved in sugar translocation across mam- malian cell membranes. This transport system may also be ideally suited for studying the interaction of insulin with the cell membrane, which appears to be the elementary process involved in the biological function of this hormone (24).

EXPERIMENTAL PROCEDURES

Male Sprague-Dawley rats (120 to 140 g) were used in this study. They had free access to Purina laboratory chow and water.

Reagents and Enzymes-n-Glucose-6-3H and D- and L-glucose- U-14C were purchased either from New England Nuclear or from ICN, Irvine, California. n-Glucose-6-phosphate-U-14C was purchased from Amersham-Searle Corporation, Arlington Heights, Illinois, and inulin-14C-carboxyl was from New England Nuclear. D-Glucose, 3-0-methylglucose, ATP, DPN, TPN, D-

and L-lactate, and ouabain were obtained from Calbiochem. n-Glucose was obtained from Pfanstiehl Chemical Company, Waukegan, Illinois, dinitrophenol was from Eastman, phloretin was from K and K Laboratories, Plainsview, New York, crystal-

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line pork zinc-insulin was from Lilly, crude bacterial collagenase was obtained from Worthington, and bovine albumin (Fraction V) was from Armour.

Preparation of Fat Cell Membranes-Fat cell ghosts were pre- pared from isolated epididymal fat cells essentially by the pro- cedures described by Rodbell (20). The epididymal fat pads from 20 rats were cut into several small pieces and distributed in 15 vials, each containing 3 ml of Krebs-Ringer-bicarbonate buffer (3) with 3% bovine albumin and 3 mg of collagenase. These vials were incubated at 37” for 40 min with vigorous shaking (130 to 150 cpm). The membranes (ghosts) were pre- pared by treating the isolated fat cells with the hypotonic me- dium described by Rodbell (20), except that it contained no ATP, DPN, or TPN. The inclusion of these in the hypotonic medium did not measurably affect the basic glucose transport properties of the membrane preparations. The fat cell mem- branes were freshly prepared for each experiment; they were kept at 4’ until used. The membrane material obtained from the fat cells of 15 rats was sufficient for about 100 transport incuba- tion samples. About 657, of the glucose transport capacity was retained by membranes stored in liquid nitrogen for 3 days; after 1 month in liquid nitrogen they retained about 40% of the transport activity. The amount of membrane material used was represented as milligrams of protein, estimated by the method of Lowry et al. (25) with crystalline bovine serum al- bumin as the reference standard.

In&x Experiments-The usual incubation mixture consisted of about 200 pg of membrane protein in oxygenated Krebs- Ringer-bicarbonate buffer (3) containing 3% bovine serum albumin and titrated to pH 7.4 with 0.1 M NaOH. All incuba- tions were performed in glass test tubes (100 x 11 mm). The

total volume of the incubation mixture varied from 0.1 to 0.2 ml. The reaction was initiated by the addition of the radioactive sugar. The incubation was stopped by adding 3 ml of ice- cold Krebs-Ringer-bicarbonate buffer, followed rapidly by filtra- tion with suction on H-A Millipore filters. The filters were immediately washed with 15 ml of the same cold buffer. The dilution, filtration, and washing steps consumed about 10 sec. A multiple filtration manifold apparatus (Instrument Division, National Institutes of Health) containing 45 filtration channels was used. After washing, the filters were immediately removed and placed in glass scintillation counting vials which contained 1 ml of 10% sodium dodecyl sulfate. The vials were shaken at room temperature for 15 min. Radioactivity was determined at 50% (3H) or 94% (‘“C) efficiency in the presence of a scintilla- tion fluor consisting of 10 ml of TLA toluene Fluoroalloy (Beck- man) and 2 ml of Biosolv Solubilizer BBS-3 (Beckman). All samples were performed in triplicate or quadruplicate, and con- trol samples lacking membranes were obtained for each condi- tion which was tested.

Identification of Radioactive Cornpour& in Fat Cell Membranes -n-Glucose-U-r4C of high specific activity (90 mCi per mmole) was used in these experiments. Incubations were performed in quadruplicate as described for the influx experiments. Two of the filters were counted directly to determine total radio- activity. The radioactivity of the other two filters was ex- tracted by incubating three times with 1.5 ml of distilled water for 15 min at room temperature. The filters were counted after the extraction procedure to determine the residual, nonextract- able radioactivity. The three eluates from each filter were pooled and lyophilized. The residue was suspended in 60 ~1 of

distilled water and centrifuged. The radioactivity in a sample of the supernatant was determined, and a 10-J aliquot was applied to the origin of a Silica Gel F-254 thin layer plate (Brinkman). n-Glucose and n-glucose-6-phosphate were ap- plied to each plate as separate standards, as well as superim- posed on the unknown samples. The plates were developed on three different solvent systems: chloroform-methanol-water (60:70:26, v/v/v), ethanol-O.5 ivr ammonium acetate (5:2, v/v), and butanol-acetic acid-wate: (4:1:5, v/v/v). The thin layer plates were placed in contact with Eastman Kodak Royal Blue x-ray film for 24 to 48 hours.

Concentration Gradient-The ratio of the intramembranal glucose concentration to the concentration of glucose in the medium was determined after varying periods of incubation. A

membrane suspension containing about 3.5 mg of protein was centrifuged at 15,000 x g for 15 min. The volume of the pellet was recorded (total membrane volume) and sufhcient Krebs- Ringer-bicarbonate buffer was added to bring the final volume of the suspension to 5.5 ml. Aliquots of 0.33 ml, corresponding to 20 ~1 of total membrane volume and 200 pg of ljrotein, were incubated at room temperature in the presence of labeled 3H-~-

glucose or r4C-inulin. At varying times the incubation mixture was cooled in ice and centrifuged at 4” for 5 min at 37,000 x g. A 20.~1 aliquot of the supernatant was assayed for r4C and for 3H. The pellet was accurately drained and resuspended in 0.50 ml of water. The radioactivity in a sample of this sus- pension was determined. Identical procedures were performed for membranes incubated in the presence of Y-inulin and 3I-I-r)- glucose. The intermembranal space was calculated from the ratio of inulin present in the original supernatant and that pres- ent in the pellet suspension. The volume of extramembranal space was found to vary from 10 to 1374 of the total membrane volume. The true intramembranal space was calculated by sub- tracting the intermembranal space from the apparent or total membranal volume. The ratio between the %n-glucose present in a volume of the corrected intramembranal pool and the 3H-glucose found in a corresponding volume of the membrane- free medium was considered the concentration gradient.

Efluz Experiments-The fat cell membranes were loaded by incubating them for 4 min at 25” in a medium containing radio- active D- or L-glucose, as described for the influx experiments. The incubation mixture was then diluted with 3 ml of Krebs- Ringer-bicarbonate buffer kept at 25” or at 4”. The suspensions were then incubated at 4” or at 25” for varying periods of time. The reaction was stopped by filtering and washing with cold buffer as described for the influx experiments. The zero time value of glucose uptake (100% glucose content) was that found in the samples which were diluted with cold buffer and immedi- ately filtered, after incubation for 4 min at 25”. The results are

expressed as the percentage of glucose remaining in the mem- branes relative to this zero time value.

RESULTS

Under the experimental conditions described in this report the rate of uptake of glucose into fat cell membranes is linear during the initial 5 min of incubation (Fig. 1). This permits description of the transport processes in terms of initial velocities and thus it satisfies an important condition for proper kinetic analysis. The transport of glucose across these membranes is a stereospecific process, as indicated by the lo-fold faster trans-

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FIG. 1. Uptake by fat cell membranes of 3H-n-glucose at 4” (0-O) and at 25’ (O-O), and of 14C-L-glucose (O--D) at 25” as a function of time. Concentration of both sugars (L- and n-glucose) was 1 mM. The specific activity of 3H-D-glucose was 149 &i per /*mole and that of %J-L-glucose was 23 &i per &mole. Fat cell membranes, equivalent to 0.1 mg of protein, were incubated for varying times in 0.1 ml of Krebs-Ringer-HCOS buffer containing 37, bovine albumin and adjusted to pH 7.4. The reaction was initiated by the addition of the radioactive sugar. The incubation was stopped by adding 3 ml of ice-cold Krebs- Ringer-bicarbonate buffer to the incubation mixture followed rapidly by filtration on H-A Millipore filters as described in the text. Assays were performed in triplicate.

port of rj-glucose compared to L-glucose (Fig. 1). Furthermore, D-glucose transport appears to reach a steady state during the first 15 min of incubation, whereas L-glucose uptake is linear for over 40 min. The transport system is also sensitive to tem- perature since the rate of D-glucose accumulation at 25” is 5 times greater than at 4” (Fig. 1).

The rate of D-glucose uptake into the fat cell membranes is related linearly to the membrane concentration over at least a 30-fold range of concentration (Fig. 2). The membrane trans- port system used here thus displays another fundamental prop- erty required of a reliable and sensitive kinetic assay system.

The dependence of the rate of uptake on the concentration of D-glucose indicates the presence of a saturable transport system (Fig. 3). The data for D-glucose obey classical Michaelis-Men- ten kinetics; the calculated apparent affinity constant (KT) for D-glucose is 3.3 mM, and the apparent rate constant (V,,,) is 12.3 mmoles per min per mg of protein (Fig. 3). In contrast, L-glucose uptake is related linearly to the sugar concentration over a broad range of concentration.

FIG. 2. Effect of membrane concentration on the initial rate of uptake of 1 mM 3H-D-glucose. The assay system is the same as described in Fig. 1. Incubations were performed at 25” for 3 min, during which time the rate of uptake follows zero order kinetics (Fig. 1).

zoz5 0 5 IO 15 D-GLUCOSE CONCENTRATION, mM

FIG. 3. Effect of concentration of L-glucose (O-O) and of n-glucose (0-O) on initial rates of uptake into fat cell mem- branes. Fat, cell membranes, equivalent to 160 pg of protein, were incubated for 3 min at 25” and assayed as described in Fig. 1. The concentration of the two stereoisomeric sugars was 1 aried as indicated. The specific activity of W-L-glucose was 23 &i per pmole and that of 3H-D-glucose was 149 &i per pmole (for concen- trations up to 5 mM) or 4OpCi perrmole (for concentrations greater than 5 mM). The double reciprocal plot for n-glucose, which is shown in the inset, indicates the KT to be 3.3 rnM and the V,,, (apparent) to be 12.3 mmoles per min per mg of protein; substrate concentration is expressed as millimolar. The rates of glucose uptake are described as initial velocities (Vi).

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FIG. 4. A, rates of n-glucose uptake by fat cell membranes in the absence (0-O) and presence of 10 mM (O---O) and 20 rnM (O-U) 3-O-methyl glucose. B, n-glucose uptake in the absence (0-O) and presence of 1 mM (0-O) and 20 mM (0-U) I,-glucose. The concentration of 3H-n-glucose was 1 mM. The fat cell membranes (0.1 mg of protein) were previously incubated at 25” in 0.1 ml of Krebs-Ringer-HCOs buffer with the nonradio- active sugar for 15 min. 3H-o-Clucose was added and the incuba- tion was stopped after 3 min at 25”.

0.6

0.6

“Vi

Ku L

0

/// 0

I I I IO 20 3c

3-O-METHYL-GLUCOSE, mM

FIG. 5. Dixon plot depicting competitive inhibition of the rate of uptake of n-glucose by3-O-methyl glucose in fat cell membranes. Two n-glucose concentrations (1 mrvr, O-O; 5 mM, O-O) were used in these experiments. Fat cell membranes (127 pg of protein) were incubated with the various sugars as indicated in Fig. 4 except that the incubation with 3H-n-glucose was carried out for 4f min. The Ki for 3-O-methyl glucose, indicated by the arrow on the abscissa, is 16.3 mM. The arrow on the ordinate points to the reciprocal of V mBI, which was obtained independently from Lineweaver-Burk plots (Fig. 3, inset) ; this point corresponds closely to the intersect of the two experimental lines. The rates of uptake are described as initial velocities, Vi (nanomoles of glucose per min per mg of protein).

r , I I

MINUTES 5

I I I

5 IO If MINUTES

FIG. 6. (left) o-Glucose uptake by fat cell membranes in the ab sence (O-O) and presence of 1 mM (O--O) and 4 mM (O--O) phloretin. Fat cell membranes (104 rg of protein) were suspended in 0.1 ml of Krebs-Ringer-bicarbonate buffer contain- ing 3% bovine albumin and phloretin. After 10 min at room tem- perature, 3H-n-glucose (1 m&r) was added and the suspensions were incubated at 25” for varying times. Phloretin was dissolved in absolute ethanol; the ethanol concentration of both the control and the phloretin samples was 0.1% (v/v).

FIG. 7. (right) The effect of insulin on the rate of uptake of n- glucose by fat cell membranes. The membranes (196 rg of pro- tein) were suspended in 0.1 ml of Krebs-Ringer-HCOS-Alb (3yo) in the presence (0-O) or absence (O-O) of insulin (6 mil- liunits per ml of incubation medium). After 15 min at room temperature, 3H-n-glucose (1 mM) was added and the incubations were allowed to proceed at 25” for varying times.

The transport of n-glucose into fat cell membranes is inhibited strongly by a-o-methyl glucose (Fig. 4A), a nonmetabolizable sugar that appears to share the same transport mechanism as

n-glucose in several other systems characterized by stereospecific, facilitated diffusion characteristics. No significant inhibition

of n-glucose is observed with L-glucose (Fig. 4B). The inhibition

of n-glucose transport by S-O-methyl glucose is clearly com- petitive (Fig. 5). The apparent dissociation constant (KJ for

this sugar is 16.3 mM.

Low concentrations of phloretin, another relatively selective

inhibitor of glucose transport in a number of other transport systems (26), effectively decreased the rate of n-glucose uptake

by fat cell membranes (Fig. 6). This occurred even in the presence of a high concentration (3y(J of albumin in the medium. The latter has been shown to antagonize the inhibitory effects of phloretin on glucose metabolism or transport in adipose tissue

(2). The inhibition of n-glucose uptake by phloretin was not of a strictly competitive type, and no effect on the rate of I,-glucose

transport by this drug could be shown. The rate of n-glucose uptake by the fat cell membranes was

increased by insulin (Fig. 7). No insulin effect could be shown on the uptake of L-glucose. Not all membrane preparations

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TaBLE 1

Zdentijication of 14C accumulated in fat cell membranes incubated with ‘*C-glucose

Fat cell membranes (190rg of protein) were incubated for vary- ing times with 1 mM W-glucose (specific activity, 90 pCi per pmole) as described in the text. The Millipore filters containing the washed membranes (Fig. 1) were placed in vials and extracted three times by shaking in 1.5 ml of distilled water at room tem- perature for 15 min. The radioactivity remaining on the filters after extraction was determined directly by counting the filters as described in the text. The pooled eluates were lyophilized, and the residue was suspended in 60 ~1 of distilled water and centrifuged,. Thesupernatant was analyzed by thinlayer chroma- tography as described in the text.

I / Incubation period / Radioactivity extracted /Free’aC-glucose in extracts

min % % 1 7% >95 2 67 >95 4 66 >90

10 43 *

20 48 b

0 This represents nearly the maximal recovery of radioactivity by the procedures used since the extractable radioactivity of a sample kept at 4” and immediately extracted without incubation (zero time) was about 75yo.

b Small amounts of radioactive material migrating differently from glucose and glucose-6-phosphate on thin layer chromatog- raphy were detected. Only traces of these materials were de- tected, and these appeared only after 4 min of incubation.

TABLE II

Accumulation ratio of 3H-D-glucose in fat cell membranes

Membrane suspensions, equivalent to 20 ~1 of total membrane volume and 200 rg of protein, were incubated in 0.16 or 0.82 mM 3H-n-glucose (specific activity, 40 fiCi per pmole, in a total volume of 0.33 ml). The samples were cooled in ice and centrifuged at 37,000 X g for 5 min. A sample (20 ~1) of the supernatant was counted. The pellets were drained and resuspended in water in a final volume of 0.50 ml. The radioactivity in lOOr of this suspen- sion was determined. Identical incubations were performed in the presence of carboxyl-14C-inulin to determine the interparticu- late space. This was used to correct for the amount of 3H-D- glucose trapped in the pellet. The true volume of the membranes (intramembranal space) was calculated as described in the text.

~-Glucose concentration

m‘w

0.16

0.82

Time of ID- incubation Glucose]in”

min CPm CPm

2 55,140 141,700 6 70,140 137,280

20 77,430 134 ( 940 4 351,860 702,416 6 380,030 678,054

20 464,410 612,326

[D-Glucose]in/ [D-glucose]out

0.37 0.51 0.57 0.50 0.56 0.75

a Corrected for 3H-n-glucose trapped in extramembranal fluid. b Radioactivity contained in a volume of supernatant equiva-

lent to the intramembranal space (i.e. total membrane volume of the pellet minus the inulin space).

showed sensitivity to insulin. In 13 out of 21 separate prepara- tions, a 2- to 3-fold enhancement of glucose transport was ob- served, whereas no effect was observed in the others. The lack of insulin responsiveness in the latter could not be shown even

TABLE III

Ability of various energy sources and of some metabolic inhibitors to support or inhibit, respectively, glucose uptake in fat cell

membranes

Membrane preparations, corresponding to 1 mg of protein per ml of medium, were suspended in 0.1 ml of Krebs-Ringer-bicar- bonate buffer, pH 7.4, containing 3% bovine albumin. After the addition of each of the components listed below, the samples were assayed for 3H-n-glucose (1 mM) uptake at 25” as described in the text.

Addition

None............... ATP-DPN-TPN n-Lactate. n-Lactate plus DPN L-Lactate Dinitrophenol Ouakain. Ouabain. Ouabain. Ouabain.

Concentration Relative rate of glucose uptake

3 rnM

3 rnM

3 rnM

3 rnM

20 rnM

lo-’ M

lo-’ M

lo+ M

lo- M

100 100 100 ,100 100 106 116 108 106 107

after lowering the concentration of glucose in the medium to values as low as 0.1 mM. The reason for the lability of this

important functional response is not yet known and is currently under intensive investigation in this laboratory.

iLlore than 907, of the radioactivity accumulated by the fat cell membranes during the first 4 min of incubation with 14C-n- glucose was shown to be unmodified n-glucose (Table I). Signif- icant amounts of radioactivity which were not extractable with distilled water were observed in membranes incubated for longer than 5 min; the reason for this was not determined. Traces of water-soluble radioactive derivatives differing in their chroma- tographic behavior from n-glucose and glucose-6-phosphate were detected in suspensions incubated for longer than 4 min. The nature of these materials was not established. After a 4-min incubation period, the total amount of these derivatives was not more than 10% of the n-glucose present.

The fat cell membranes did not transport n-glucose against a concentration gradient for glucose (Table II). The intra- membranal concentration of radioactivity did not exceed the medium glucose concentration even after a 20-min incubation period.

No effect on the uptake of glucose into the membranes was detected by adding ATP, DPN, TPN, n-lactate, phosphenol- pyruvate, or creatine phosphate to the incubation medium (Table III). 2,4-Dinitrophenol, an inhibitor which blocks energy-linked uphill solute transport, did not impair glucose uptake by the fat cell membranes. Also, no inhibition of trans- port was observed by high concentrations (1 mM) of ouabain, which inhibits the sodium pump.

The fat cell membrane preparations were also used for studies of the processes involved in the outward movement of n-glucose accumulated by the membranes (Fig. 8). Zero order kinetics prevailed during the first 5 min of efflux, and the initial velocities of D-glucose exit were proportional to the amount of membrane present. As observed for the processes of n-glucose uptake, the rate of efflux was temperature-dependent, stereospecific, and sensitive to the presence of insulin in the medium (Fig. 8). The

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MINUTES

FIG. 8. Rates of efflux of 3H-n-ghmose (0, 0, 0) and of W-L- glucose (4) from preloaded fat cell membranes. Fat cell mem- branes (118 pg of protein) were previously incubated in 0.1 ml of the standard medium containing 1 mM 3H-D- or SH-n-glucose for 4 min at 25”. Three milliliters of Krebs-Ringer-HCOa buffer, pH 7.4, were then added and the suspensions were incubated for vary- ing times at 4” (D---O), at 25’ (O-O, m-m), and at 25” in the presence of 12 milliunits of insulin per ml of buffer (O-O). At the indicated times, the reactions were stopped by rapidly filtering on H-A Millipore filters followed by washing with 15 ml of ice-cold buffer. The radioactivity on the filters was deter- mined as described in the legend to Fig. 1. The results are ex- pressed as the percentage of counts remaining in membranes; the zero time value was obtained from samples which were diluted with ice-cold buffer after the 4-min prior incubation and immedi- ately filtered and washed.

rates of n-glucose exit were also decreased by the addition of phloretin to the medium bathing the membranes (Fig. 9) ; this effect could be shown with concentrations of phloretin as low (1 mM) as those that affect glucose influx.

In the efflux experiments, the movement of glucose occurs into a very large volume of external medium. The initial concen-

tration of glucose in the medium (usually about 1 mM) during the influx phase is below the KT for influx and it is diluted 30-fold during the measurements of the efflux. The external glucose concentration during this phase is therefore nearly negligible and certainly far below the KT. Thus, in contrast to the influx experiments described earlier, a true unidirectional glucose flux is being determined in the efflux experiments. This feature of the experimental transport system permitted the design of experiments which show the presence of accelerated exchange diffusion, which is probably the single most important experi- mental feature yet described to incriminate a mobile carrier in transport processes (26). Fat cell membranes incubated for 4 min with 1 mM %-n-glucose were diluted in a medium containing 20 IIIM 3-O-methyl glucose. A direct acceleration of glucose

I I I I

5 IO 15 20 MINUTES

FIG. 9. Time course of release of 3H-n-glucose from preloaded fat cell membranes in the absence (O-O) and presence of 3-O- methyl glucose (O-O) or phloretin (O-U). The mem- branes were incubated in 1 lll~ 3H-n-glucose for 4 min and then diluted with 3 ml of Krebs-Ringer-bicarbonate buffer containing 20 rnM 3-O-methyl glucose or 1 mM phloretin. The incubations were performed at 25’ as indicated in Fig. 8.

that the limiting step in the glucose transport process is the relatively slow movement of the free carrier from the outer to the inner surface of the membrane where it can combine with glucose for the exit process (26). The movement of carrier from the outer to the inner membrane surface is increased by forming a complex with 3-O-methyl glucose during inward transport of this sugar. This view is based on the supposition that under the experimental conditions during which accelerated exchange diffusion is observed, the mobile carrier is concentrated predomi- nantly at the outer portion of the membrane.

These concepts were examined further by studying the depend- ence of efflux rates on the initial intramembranal concentration of glucose (Fig. 10). The membrane ghosts were incubated in varying concentrations of glucose for 4 min, followed by dilution with large buffer volumes (30 times) to ensure that the outer glucose concentration was negligible. The effect of a-o-methyl glucose on the unidirectional efflux rates was then determined as a function of the initial glucose concentration inside the mem- brane. Although the accelerated exchange diffusion phenom- enon was observed at all concentrations of glucose tested, the effects were much less dramatic at high intramembranal glucose concentrations (Fig. 10). The distribution of carrier at the inner phases of the membrane may be altered by increasing the concentration of free sugar on that side of the membrane, or the over-all turnover of carrier is increased in such a way that the relative increase in the inward movement of the carrier caused by 3-O-methyl glucose becomes less critical to the exit process. However, if the Lineweaver-Burk plot for the efflux in the pres- ence of a-o-methyl glucose (Fig. 10) can be validly compared

efflux was observed (Fig. 9). This is interpreted as evidence to that in the absence of this sugar, the data suggest that the

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0.8

0.6

“Vi

0.4

0.2 7

1

/

-A

0.

I I I I

:,:I::::::::I I I I

.2 2 4

‘4

FIG. 10. Relationship between the n-glucose concentration inside fat cell membranes and initial e&x velocities in the ab- sence (O---O) and presence of 20 mM 3-O-methyl glucose (O---O ). Membranes were previously incubated (as described in Fig. 8) at 25” for 4 min in various concentrations of 3H-n-gIu- case. The membranes were then incubated for 4 min (25’) in 3 ml of Krebs-Ringer-HCOS buffer which in some cases (O---O) contained 3-O-methyl glucose. The initial intramembranal con- centration of glucose was determined by the procedures described in Table II and in the text. The rates of efflux are expressed as initial velocities (Vi), expressed as nanomoles of n-glucose ner min per mg of protein; zero order kinetics prevails during the first 4 min of efflux (Fig. 8). The substrate (S) concentration is ex- pressed as mill&oh&. The lowest concentration indicated on the abscissa corresponds to 0.16 mM glucose.

outer binding, or the inward movement, of 3-O-methyl glucose causes a dramatic increase in the affinity of D-ghCOSe for the exit process and little change in the maximal efflux rate.

In the absence of a-o-methyl glucose, the apparent KT and V,,, for efflux is 0.2 mu and 3.8 (Fig. lo), respectively, whereas tha,t derived from the influx data is 3.3 mM and 12.3, respectively.

DISCUSSION

Much progress has been made during the past few years in elucidating the kinetic and chemical properties of the processes involved in sugar translocation across bacterial cell membranes (27-34). In these systems the availability of simple or rudi- mentary membrane preparations retaining transport functions has permitted study of transport by direct means, uncomplicated by the normal metabolic processes of the cell (27). Until now

it has not been possible to study directly the transport of glucose in hormone-sensitive mammalian tissues. In adipose tissue, the transport of glucose, and the effects of insulin on this process, have been examined indirectly by studying glucose oxidation or other aspects of it,s metabolism. Some of the problems inherent to the study of glucose transport by such means in adipose tissue or in isolated fat cells have been detailed previously (12, 13, 22, 23).

Rodbell described many properties of membrane preparations (ghosts) obtained by osmotic shock of isolated rat epididymal fat cells (20). Although all the fat and a major portion of the intra- cellular cytoplasmic components were expelled from the cell by

these procedures, sufficient residual glucose oxidation and metab- olism were retained to show the presence of glucose transport and its enhancement by insulin (21). These membrane prepara- tions have been used to measure directly the uptake of potas- sium (35) and amino acids (36), although the kinetics of these processes is examined over protracted time periods. Microsomal particles from isolated adipose tissue cells have recently been prepared which appear to be very promising for the study of glucose transport in metabolically inert organelles (37, 38). These preparations, however, do not display saturation kinetics for glucose transport, and they are not responsive to insulin added to the bathing medium.

In the present report, fat cell membrane preparations pre- pared essentially by the procedures described by Rodbell (20) are used successfully to measure directly the transport of glucose. Kinetics adequate for measurement of glucose transport exists only during the first 5 min of incubation since a steady state is achieved very quickly (Fig. 1). In addition, significant metabc olism of glucose probably occurs by 10 min. The basic prop- erties of the system satisfy some important technical parameters required for a reliable and sensitive assay system. Observa- tions can be made during periods in which zero order kinet,ics prevails and, therefore, allow initial velocity expressions. The substrate is not significantly altered metabolically during the period of study. The activity measured is directly proportional to the amount of membrane present over a wide range of con- centration. It is possible to determine reasonably well the processes of influx and efflux separately. Furthermore, bhe effects of insulin on these processes can be detected and measured.

The general properties of glucose transport in fat cell mern- branes agree with those previously proposed for adipose tissue, and they support the notion that transport occurs by a carrier- mediated facilitated diffusion mechanism (2). Glucose trans- location occurs through a bidirectional system which has similar, if not identical, properties in both directions. Influx and efflux are temperature-sensitive processes, stereospecific and saturable with respect to glucose, insensitive to ouabain, and not linked to metabolic energy-producing processes. -4s expected, a.ccumula- tion of glucose does not occur against a chem:cal gradient. Phloretin applied to the external membrane inhibits the unidirec- tional efflux of glucose. The transport of glucose is enhanced in either direction by the presence of insulin outside the membranes.

It is of interest that the apparent kinetic constants deter- mined for the efflux process are quite different from those ob- tained from the influx studies. The apparent K, (KY) for influx is 16 times larger than that for efflux, whereas the apparent v max for efflux is one-third that calculated for influx. It is tempting to speculate that these differences may reflect func- tional adaptations of the cell membrane to its environment. The normal cell surface is exposed to the relatively large concentm- tions of glucose in the circulating blood, whereas very little free glucose would ordinarily be expected to be present inside the cell. However, the absolute values of apparent kinetic constants ob- tained on complicated multistep biological processes, such as transport, are very difficult to interpret, and the differences may be more apparent than real. 41~0, it should be recalled that influx kinetics is obtained under conditions which measure net fluxes, whereas the efflux data reflect true unidirectional fluxes. Although all the data in the present study support the general hypothesis of transport by facilitated diffusion, the kinetic constants camrot be used in support of the current thesis that

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Issue of April 25, 1971 G. Illiano and P. Cuatrecasas 2479

predicts that the system is symmetrical and possesses identical substrate-carrier dissociat’ion constants on both sides of the membrane (26).

The general kinetic considerations which support the mobile carrier hypothesis of facilitated diffusion transport have recently been reviewed (26). Support for a mobile carrier mechanism for glucose transport in adipose tissue (2, 5) has come from the demonstration of competitive exchange diffusion (39) or counter- transport (40) phenomena. However, both of these processes, which involve the same mechanism and differ only in the experi- mental conditions during which they are observed (41), do not distinguish a mobile site mechanism (4244) from a system of unidirectional pores (26, 41). These processes are adequately explained by any model which consists of physically separate entry and exit pathways. Evidence favoring the existence of a mobile mechanism over fixed pores or channels comes principally from two sources. (a) Factors (phloretin, analogue inhibitors, and insulin) added to the external surface which affect the rate of entry also affect the rate of exit; it has not been possible to alter or modify one of these processes independently from the other. (6) The demonstration of acceleration of unidirectional flux by the transport, in the opposite direction, of a sugar which shares the same transport mechanism. This has been referred to as accelerative exchange diffusion (45-48). It is postulated that the carrier-substrate complex traverses the membrane faster than the carrier or substrate alone. The concentration of free carrier in the outer portions of the membrane is high when unidirectional transport occurs. The limiting step is the return of this carrier to the inner portions, a process which is facilitated by the presence in the external medium of a sugar which can form a complex with the carrier for inward transport. The enhancement of n-glucose efflux from the fat cell membranes by 3-O-methyl glucose (Fig. 9) is subject to these interpreta- tions, and the kinetics of this acceleration (Fig. 10) in general supports these views.

The demonstration that the glucose transport system presented here is sensitive to insulin indicates that it may serve as an excellent simplified model system for further, more detailed examination of the molecular basis of insulin action.’

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Gennaro Illiano and Pedro CuatrecasasGlucose Transport in Fat Cell Membranes

1971, 246:2472-2479.J. Biol. Chem. 

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