Effect of Cytochalasin B and D on Groups of Insulin

Receptors and on Insulin Action in Rat Adipocytes

POSSIBLE EVIDENCEFORA STRUCTURALRELATIONSHIP OF THE

INSULIN RECEPTORTO THE GLUCOSETRANSPORTSYSTEM

LEONARDJARETT and ROBERT N1. SMITH, Divisiotn of Laboratory Mledicinie,Departments of Pathology and Medicine, Washington UniversitySchool of Medicine, and Barntes Hospital, St. Louis, Missouri 63110

A B S T R A C T The possible physiological imiiportaineeof the groups of insulin receptors on rat adipocytesand the relationship of these groups to insulin actionwere investigated. The effect of cytochalasin B aind Don l)iological actions of insulin was measured aind coimi-pared with the effect of these agents on the ultra-structural distribution of groups of insulin receptors.Cytochalasin B had no effect on epinephrine-stimulatedlipolysis, insulin inhibition of epinephrine-stimulatedlipolysis, or insulin stimulation of protein synthesis.Cytochalasin B, over a concentration rainge of 50 nMto 5 ,uM, progressively inhibited the basal glucosetransport system, as measured by glucose oxidation,2-deoxyglucose transport, and 3-O-methylglucosetransport. Insulin was capable of fully stimulating re-maining basal transport at submaximal concentrationsof cytochalasin B. Insulin pretreatment of adipocytespartially protected the glucose transport system frominhibition by cytochalasin B. Cytochalasin B markedlyaltered the distribution pattern of insulin receptors,which caused an increase in the number of single re-ceptor molecules by decreasing the number of largergroups. A significant correlation (r = 0.964; P < 0.001)was found between the percent increase in single re-ceptors and the percent decrease in glucose transport.Ferritin-insulin pretreatment of adipocytes preventeddisruption of the groups of insulin receptors by cyto-chalasin B. Cytochalasin D had no effect on the bio-logical actions of insulin or on the groups of insulinreceptors. These data suggest that the ability of insulin

This study was presented in part at the Annual Meetingof the American Society for Cell Biology in San Diego,Calif., November 1977, and at the Endocrine Society inChicago, Ill., June 1977.

Received for publication 15 August 1978 and in revisedform 4 December 1978.

to affect adlipoeyte metabolism is independent of thehormonie occupying adjacent, groupedl receptor sites.The marked contrast in effects of cytochalasin B andD on groups of insulini receptors ancd glucose transportsuggests that the microfilaimenit system is not involvedin insulin actioni or in holding the groups of insulinreceptors together, as both agenits are known dis-rupters of microfilamenits and inhibitors of actin gela-tion. The correlation betweeni the effects of cytochalasiniB on insulin receptor distribution and glucose trans-port leads to the speculation that the glycoprotein mole-cules containiing the insulin receptor are funcetionallylinked with the glucose transport systemii.

INTRODUCTION

It is generally accepted that insulini initially interactswith specific receptors on the plasma membrane ofcells. This has been demonstrated by both biochemical(1, 2) and morphological techniques (3). With ferritin-insulin we have shown that the insulin receptors onrat adipocytes are distributed on the surface of the cellassociated with the glycocalyx region of the plasmamembrane (4-6). The receptors were visualized eitheras single receptors or in groups of two to six. The groupswere shown to occur before, and independent of, thebinding of the hormone to the receptor.

This study attempted to determine the possiblephysiological importance of these groups of insulin re-ceptors and to determine if they were essential to in-sulin action. The experimental design was to attemptto disrupt the groups of receptors by a chemical agentand relate this to alterations in the biological responsesto insulin. Cytochalasin B was chosen as the pharma-cological tool for disrupting the groups of insulin re-ceptors for several reasons. Cytochalasin B is known

J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. 0021-9738/79104/0571109 $1.00 571Volume 63 April 1979 571-579

to disrupt clusters of aggregated receptors (7) and intra-membranous particles (8), possibly through its actionson microfilaments and actin (9). This agent has beenused to inhibit basal and insulin-stimulated glucosetransport (10), but has been shown not to affect nu-merous other insulin-sensitive processes (11), includingboth membrane-localized and intracellular responses.The unaffected membrane processes include a-aminoisobutyrate transport and a specific fructosetransport system, whereas the unaffected intracellularinsulin-sensitive action was the antilipolytic effect ofthe hormone. Data from this report demonstrate thatdisruption of groups of insulin receptors by cytochalasinB does not prevent insulin from exerting its action.However, disruption of the groups of insulin receptorscorrelated with the inhibition of the basal glucosetransport system, which suggests that some of theinsulin receptors are on the same glycoproteinswhich form the glucose transport system.

METHODSPreparation of isolated adipocytes. Adipocytes were pre-

pared as described by Rodbell (12). Minced epididymal fatpads from 120-g male Wistar rats were incubated in Krebs-Ringer phosphate buffer containing one-half the normalcalcium concentration, 3% bovine serum albumin (BSA),'and 0.2% dextrose with 1 mg crude collagenase per milliliterat pH 7.4. The pads were digested for 40-45 min at 37°C,after which the contents of the digestion flasks were filteredthrough a silk screen. The cells in the filtrate were cen-trifuged at 800 g for 30 s, and the infranatant was aspirated.The cells were washed three times by resuspension andcentrifugation in Krebs-Ringer phosphate buffer with BSAand dextrose as above. Dextrose was omitted from the washingbuffer of cells to be used for glucose oxidation or transportstudies. After the final wash, the cells were resuspended inthe wash buffer to a concentration of = 106 cells/ml and allowedto equilibrate at 37°C for 15 min before beginning any assay.Protein ineasurements of whole-cell suspensions were de-termined by performing cell counts as previously described (13).

Biochemical studies. Adipocyte lipolysis was determinedby measuring the amount of glycerol released during a 30-min incubation at 37°C (13). Triplicate vials were preparedwhich contained (a) epinephrine (0.125 /ig/ml; (b) cyto-chalasin B (1 ,uM); (c) insulin (50 uU/ml) in the presence ofepinephrine; or (d) insulin, epinephrine, and cytochalasin B.A set of control vials had no additions. Equilibrated adipocyteswere added to bring the incubation volume to 1 ml. The incu-bations were ended after 30 min by the addition of 0.3 ml2 N perchloric acid. The extract was transferred to centrifugetubes, and the extracted protein was sedimented. The in-franatant was removed and neutralized before assay forglycerol as described by Lowry et al. (14).

The incorporation of [14C]histidine into protein was meas-ured as previously described (13). Equilibrated adipocyteswere added to vials which contained 0.5 ,uCi [14C]histidineand the various agents being tested, to bring the final volumeto 1 ml. After 30 min of incubation at 37°C, aliquots were

I Abbreviations used in this paper: BSA, bovine serum al-bumin; Fm-I, monomeric ferritin-insulin; PBS-BSA, phos-phate-buffered saline containing 0.1% BSA.

removed from the vials to 2.54-cm squares of Whatman filterpaper (Whatman, Inc., Clifton, N. J.). The protein was pre-cipitated by immersing the filter papers in cold, 10% TCAfollowing the procedure of Mans and Novelli (15) as modifiedfor adipocytes (13). The filters were air dried, then placedin liquid scintillation vials containing 15 ml toluene-Omni-fluor (New England Nuclear, Boston, Mass.) and counted in aliquid scintillation counter.

The effect of cytochalasin B on insulin binding was de-termined by incubating 105 cells/ml for 15 min at 37°C with0-20 ,M cytochalasin B. 1251-labeled insulin (50 ,tU/ml, 0.6ACi) was then added to the cells, and the incubation con-tinued for 15 min at 37°C. The cell suspensions were dilutedwith 5 ml cold 0.1 M phosphate-buffered saline containing0.1% BSA (PBS-BSA) and decanted onto Whatman GF/Cglass fiber filters (Whatman, Inc.). The filters were washedunder moderate vacuum with 10 ml of the PBS-BSA buffer.Nonspecific binding of the labeled ligand was determinedin the presence of 5 ,g/ml of porcine insulin, andl the vatluesreported are the corrected or specific binding.

The effect of cytochalasin B or D on basal andl insulin-stimulated glucose oxidation was determined by adding5 x 104 cells to vials that contained various concentrations ofthe cytochalasin from 0 to 20 AM in a final volume of 2 ml.After 15 min at 37°C, 50 or 500 ,uIJ insulin/ml was added tothe appropriate vials and D-[1-14C]glucose (0.55 mM) wasadded to all vials. The vials were immediately capped. Atthe end of 60 min at 37°C, '4CO2 was determined as describedby Gliemann (16). For protective effect studies, cells wereincubated for 15 min at 370C with 250 ,uU insulin/ml beforecytochalasin B and D-[1-_4C]glucose addition.

Transport of [2-14C]deoxyglucose and [3-O-3H]methylglu-cose was determined under similar conditions except that thefinal incubation volume was 0.25 ml, the sugar concentrationwas 0.1 mM, and the incubation time after addition of thera(lioactive sugar was either 10 min for 2-deoxyglucose or10 s for 3-O-methylglucose. The incubation was terminatedby diluting the cell suspension with 3 ml PBS-BSA and de-canting the cells onto a Whatman GF/C filter. The filter waswashed once with 10 ml PBS-BSA, air dried, and countedfor "4C or 3H in a liquid scintillation counter.

Morphological studies. Monomeric ferritin-insulin (Fm-I)wats prepared and characterized as previously described (6).The monomeric peak of ferritin was purified on a Bio-GelA 1.5 MI chromatographic columiin (Bio-Racd Laboratories,Richmond, Calif.). The monomeric ferritin was conjugatedto insulin with dilute glutaraldehyde that specifically reactswith lysine residues and does not cross-link ferritin (4-6),which makes the B-29 lysine of insulin the point of cross-linking. The resulting conjugate was repurified on Bio-GelA 1.5 M, revealing only Fm-I, which confirmied the lack ofcross-linking of ferritin by glutaraldehyde. The Fm-I peakwas concentrated by centrifugation at 150,000 g for 2 h, andthe pellet was resuspended in 0.1 M NaPO4 buffer (pH 7.4)and stored at 4°C. The biological and immunological activityof the conjugate were assessed by comnparison with the originalporcine insulin. These activities were equal to each other andrevealed that only about 2% of the ferritin contained insulillas previously reported (4-6), which indicated that only oneinsulin would be present on one ferritin. Thus, any ferritincore bound to the cells would represent an insulin receptor.The actual Fm-I used in this study was the same as thatpreviously used (6).

Adipocytes were incubated with Fm-I under conditions es-sentially identical to the biochemical studies. About 106 cellswere incubated with various concenitrations of cytochalasini Bor D for 15 min at 37°C before the addition of 500 ,LU Fmn-I/ml. For protective effect studies, the cells were incubated

572 L. Jarett and R. M. Smith

for 15 min at 37°C with 500 ,AU Fm-I/imil before add-ing cytochalasin B or D. The cells were then incu-bated for 30 min at 37°C. The suspension was dilutedwith cold PBS-BSA and centrifuged at 800g, and the in-franatant was aspirated. The cells were prepared for electronmicroscopy as previously described in detail (5). Only areas ofcells showing ferritin cores were photographed. As pre-viously reported, the adipocyte has -10() ferritin-insulinireceptors per square micron of plasma membrane (3). It wvasnot feasible, therefore, to photograph entirely at random theentire cell surface in any given section. The validity of thequalitative and quantitative results presented below was as-sured by careful and strict adherence to several principlesand methodological approaches. First, the sectionied speci-mens were numerically coded in such a manner that themicroscopist was unaware of the experimental condition be-ing observed. The entire cell surface of all cells in a givensection was careftillv observed, and all ferritin cores werephotographed. A minimium of 100 micrographs (x33,000)were taken of each experimental condition at a uniformmagnification. The negatives were printed to yield a finalmagnification of 90,000. The distribution of the ferritin-inisulinreceptors into groups of variousi sizes were characterized bythe proximity of the ferritin cores to each other (Fig. 7 [6]).This was determined independenitlv by at least two personiswho were also unaware of the experimental condition beingainalyzed. The data was tabulated, and the results presentedrepresent the mnean of all determinationis for a given experi-mental condition in up to five experiments.

Controls for electron microscopy were performed as pre-viously described (4-6) and includled (a) Fm-I plus 5 Ag/mlof porcine insulin and (b) the same amount of ferritin as pres-ent in the Fm-I. In neither case wvas more than a few cell-associated ferritin cores seen in multiple sections from mul-tiple blocks as previously reported (4-6), which indicatedthe almost complete absence of nonspecific binding. Thereare several reasons for this extremely low nonspecific levelcoompared with the nonspecific binding reported in Fig. 1.The biochemical nonspecific binding included trapped fluid.The extensive washing processes of the fixed cells formorphological studies removes all trapped Fm-I or ferritin.Also, only cell-associated ferritin cores vere considered forspecific or nonspecific binding.

Materials. Cytochalasin B and D were purchased fromAldrich Chemical Co., Inie., MIilwvaukee, Wis., and stock solu-tions were prepared in absolute ethanol. The concentrationof ethanol in the inicubationsi (<0. 1%) was fouind to b)e withouteffect on the biochemical or morphologicial paraimetersstudied. Witistr rats vere from National Animiial Laboratories,St. Louis, NMo. Collageniase anid BSA were prodlucts obtainedlfrom Sigma Chemical Co., St. Louisi, MIo. Other reatgents aildprodlucts vere obtained from standcardl sources or as dletailedlin previotis reports.

RESULTS

Biochemnical studies. The effect of cvtochalasin Bwas tested on various metabolic pathways th<at areinsulin sensitive. Cvtochalasin B had Ino effect on basalor epinephrine- (0.125 /ug/mnl) stimulated lipolvsis ofthe adipocvte (data not showiv) as reported previouslybv Loten and jeainrenaud (11). Insulin (50 ,uU/ml) in-hibited epinephrine-stiimiulatedl lipolysis to the sameextent in the presence or absenice of cytochalasin B.The insulin concentration was chosen to give a sub-mlaximial inhibition of the lipolysis so that ainy minior

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FIGURE i The effect of cytochalasin B on 1251-labeled insulin(1251-INS) binding to adipocytes was determined by incubatingadipocytes (105 cells/mil) for 15 min with 0-20 t4M eytochalasinB (Cvto B) before the addition of 50 AU/ml 1251-insulin. After15 min, the cells were diluted and filtered as describedl inMethods. Specific bindinig was determined in triplicatesamples, and the effect of cvtochalasin B on '251-insulin bind-ing was plotted to show the meean percent difference (-SEM)from the control at each concentrattion of cytochalasin B. Non-specific binding was determined in the presence of 5 uginsulin/ml. Cytochalasin B had no effect on nonspecific bind-ing, which aecountedl for <20% of totall binding.

effect of cvtochalasin B would be found aind not pre-vente(l bvy a supermiiaximiial insulini coneenitrattionl.

Cvtochalasini B at 5 ANI had negligil)le effect on pro-tein svnthesis of the adipocyte (Table I). Insulini at 50and 100()tU/ml produced a 47 and 88% stimulation ofadipoeyte protein synthesis as meeasured 1yI [14C]his-tidine incorporation into protein. C tochalasin B had noeffect on the ability of insuliin to stimulate proteinsy,,nthesis.

The effect of cvtochalasin B on insulin biinding toa(lipocytes was determined because it had been re-porte(l that a 2-h incubation of INI-9 lymphocytes withhigh concenitrations (> 2 g.I) of cytochalasin B causedsignificaint jnhibition of subseqluenit insulini binding tothe cells (17). Fig. 1 shows that exposure of adipocytesto a range of cvtochalasin B conieentrationis (0.5-20ftM\I) for 15 min, followed by 1251-insulin (50 ,uU/ml)resulted in ac smiiall but progressive inierease in theaimounit of insulin bound with inereasinig cytochalasin

TABLE IEffect of.5 ,uM Cytochalasint B ont Basal atnd

Insulin-Stimtlulated Incorporationt of['4C]Histidine into Adipocyte

Protein

Siamiiple -Cvtochalasin B +C(vtochallaSsin B

Basal 1(0 103±4.050 AU insulin/mlii 147±3.1 160±0.2100 uU insuliri/ml 188±6.8 194±9.8

Relatiotiship of Insulini Receptors to the Glucose Transport System 573

B concentrations. A maximum increase of 28% wasfound at 20 ,uM cytochalasin B.

The effect of cytochalasin B was measured on basaland insulin-stimulated glucose uptake as measured byglucose oxidation. Data in Table II show that cyto-chalasin B inhibited basal glucose transport in a pro-gressive fashion reaching maximal at 5 uM. Additionof cytochalasin B before the addition of 50 ,uU insulin/ml resulted in an identical pattern of inhibition ofinsulin-stimulated glucose oxidation. Examinationof these data by comparing the insulin-stimulatedlevel of glucose transport in the presence of cyto-chalasin B with the level at each comparable cyto-chalasin B concentration revealed that insulin stim-ulated the glucose transport by the same percentregardless of the presence or absence of cytochalasin B(Table II). The slightly greater stimulation at the 5-uM cytochalasin B concentration reflects the dif-

ficulty in accurately measuring the low level of basalglucose oxidation. These data suggested that cyto-chalasin B acted only Qn the basal glucose transportsystem and did not interfere with the ability of insulinto stimulate the remaining uninhibited basal activity.

Fig. 2 demonstrates the ability of insulin to partiallyprotect against the cytochalasin B inhibition of glucoseoxidation. If cells were preincubated with 50 nM-5,uM of cytochalasin B, a progressive inhibition wasfound of the insulin- (250 ,uU/ml) stimulated glucoseoxidation. Pretreatment of adipocytes with insulin fol-lowed by the addition of cytochalasin B partially pro-tected the glucose oxidation system from inhibition.Over a log greater concentration of cytochalasin Bwas needed to demonstrate inhibition of the insulin-stimulated glucose oxidation if insulin was added firstcompared with cytochalasin B. This insulin effect wasfound to be dose dependent, with lower concentra-tions of insulin less effective (data not shown). Separatestudies were performed measuring the effect of 1 ,uM

TABLE IIEffect of Cytochalasin B on Basal and Insulin-Stimulated

Glucose Oxidation

Cyto- Percentage ofchalasin B Basal Insulin* stimulation

nmol "4CO, produced/mg %protein/h

0 8.9+0.3 134.0+ 1.0 1,50250 nM 8.6±0.7 128.6±0.7 1,5000. 1,uM 8.2±0.2 i24.1±0.9 1,5110.25 uM 6.4±0.3 96.8± 1.4 1,5030.5 ,M 4.5±0.3 67.3±0.7 1,5061 aM 2.5±0.3 37.5± 1.0 1,5015,M 0.3+0.2 4.8±0.3 1,865

* Insulin concentration was 50 juU/ml, and cytochalasin Bwas added to cells 15 min before insulin addition. All datarepresent means of triplicate determinations±SEM.

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FIGuRE 2 The protective effect of insulin on cytochalasin B(Cyto B) inhibition of adipocyte glucose oxidation was de-termined. One aliquot of isolated adipocytes was incubatedwith 250 ,uU insulin/ml for 15 min at 37°C before being addedto vials containing various concentrations of cytochalasin Bas indicated (U). Another aliquot of cells was incubated withthe cytochalasin B concentrations before being added to vialscontaining 250 ,uU insulin/ml (0). The cells were then in-cubated for 60 min at 37°C, and the conversion of D_[1-14C]glu-cose to G4CO2 was determined as described in Methods. Thevalues determined in the experiment, but not illustrated, were(in nanomoles 14CO2 per milligram protein per hour): basal,11.8±0.8; 250 ,uU insulin/ml, 225.1±1.6. Cytochalasin B in-hibited basal glucose oxidation by 3.2-95.7%. The values pre-sented represent the mean of triplicate samples from arepresentative experiment. The SEMswere too small (<1%)to be clearly illustrated.

of cytochalasin B on basal and insulin-stimulated glu-cose transport as determined by [2-14C]deoxyglucoseand [3-O-3H]methylglucose transport. Similar to find-ings in the glucose oxidation studies, cytochalasin Binhibited the basal but not the insulin stimulation ofthe remaining basal transport, and preincubation withinsulin partially protected against the cytochalasin Beffects (data not shown). This suggests that the re-sults from the glucose oxidation studies reflect theeffects of cytochalasin B on glucose transport. Cyto-chalasin D, as would be expected, had no effect oneither basal or insulin-stimulated glucose transport(data not shown).

Morphological studies. Studies were carried outwith Fm-I and cytochalasin B under conditions similarto the biochemical studies for morphological quan-titation of the groups of insulin receptors. An experi-ment illustrated in Fig. 3 showed that an increase inconcentrations of cytochalasin B caused a progressiveincrease in the number of single receptors and a de-crease in the number of larger groups of insulin re-ceptors. The results of another set of experiments withadditional cytochalasin B concentrations are illustratedin Fig. 4. The most dramatic change is seen in theincreasing percentage of receptors found as singlemolecules. The singletons comprised -22% of thetotal receptors in the control and over 50% of the total

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FIGURE 3 The effect of cytochalasin B on the distributionof Fm-I receptors on adipocytes was determined by incubatingadipocytes in the absence or presence of the indicated con-centrations of cytochalasin B for 15 min at 37°C before theaddition of 500 ,uU/ml Fm-I. After 30 min at 37°C, the cellswere washed and prepared for electron microscopic observa-tion. The distribution of the Fm-I receptors was analyzedas detailed in Methods. The results depicted represent atypical experiment, showing the percentage of the total re-ceptors observed in each group size of from one to six receptorsites per group.

after treatment with 20 ,uM cytochalasin B. The de-crease in the number of groups with three or fourreceptors was obvious. The number of receptors ingroups of two appeared to be relatively consistent. Thisprobably does not indicate that groups of two were notdisrupted, because as the disruption of the larger groupoccurred, more groups with two receptor moleculeswould be formed. Thus, the most reliable index of dis-ruption of the groups would be the percent increasein single receptors.

The similarity in the concentrations of cytochalasinB required for inhibition of glucose transport (Table II)and for disruption of the groups of insulin receptors(Figs. 3 and 4) suggested a possible relationship be-tween these two phenomena. A separate set of studieswas performed in which morphological observations

were carried out with one portion of an adipocyte prep-aration with 500 ,uU Fm-I/ml, while the rest of thecells were used for the biochemical measurements ofglucose oxidation with 500 ,uU insulin/mil. These dataare presented in Fig. 5. A significant correlation (r= 0.964, P < 0.001) was found between the ability ofcytochalasin B to inhibit glucose transport (as de-termined by glucose oxidation) and to disrupt thegroups of insulin receptors (as determined by measure-ment of the percent increase in single receptorsabove control levels).

The ability of Fm-I to protect the groups of insulinreceptors against disruption by cytochalasin B wastested, as insulin preincubation of cells was found toprotect against the cytochalasin B inhibition of glucoseoxidation (Fig. 2). Addition of Fm-I before the addi-tion of cytochalasin B did partially protect the groupsof insulin receptors from disruption (Fig. 6), similar tothe biochemical studies.

Cytochalasin B is known to have at least two majoreffects on cells: inhibition of glucose transport (10)and alteration of cellular cytoskeletal elements, such

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Relationship of Insulin Receptors to the Glucose Transport System

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FIGURE 5 The effect of cytochalasin B on glucose oxidationand the distribution of Fm-I receptors was determined whena single preparation of adipocytes was divided into two sets ofcells. One set was used to determine the effectiveness ofcytochalasin B in inhibiting glucose oxidation in the presenceof 500 ,U insulin/ml; the other set was used to determinethe distribution of insulin receptors after cytochalasin B treat-ment. Cytochalasin B concentrations were 0.1-20 ,uM, and thedetails of incubation and analytical procedures are describedin Methods. The data was plotted to illustrate the percentchange between cells incubated with 500 ,SU insulin orFm-I per milliliter in the absence of cytochalasin B and cellsincubated with each concentration of cytochalasin B for bothanalyzed parameters.

as microfilaments and actin (18). Cytochalasin D doesnot inhibit glucose transport (19) but does alter micro-filaments at even lower concentrations than cyto-chalasin B (20). Thus, the effects of cytochalasin Don the groups of insulin receptors was tested. Fig. 7shows that cytochalasin D had no effect on the dis-tribution of the groups of insulin receptors on theadipocyte surface, just as it had no effect on glucoseoxidation by the adipocyte.

DISCUSSION

Previous ultrastructural studies have shown that in-sulin receptors are present in randomly distributedgroups associated with the glycocalyx coating of theadipocyte plasma membrane (3-6). Data in one of thesereports suggested that the groups are present before,and independent of, occupancy by the ligand (6), whichindicated that the receptors are not randomly distrib-

uted. Similar groups of insulin receptors have beenfound on microvilli of human placental syncytial tro-phoblasts (21). Naturally occurring groups of melano-

* cyte-stimulating hormone receptors have also been re-ported (22). The questions to be addressed in this studyconcern whether the groups of insulin receptors were

/ necessary for insulin action, and what physiologicalfunction these groups might have.

* Cytochalasin B was capable of markedly altering thedistribution pattern of insulin receptors in groups ofvarious sizes. The most obvious effect of cytochalasin Bwas to increase the number of single insulin receptormolecules and decrease the groups of three to four(Figs. 2 and 3). This disruption or alteration in re-ceptor grouping occurred without affecting the actionof insulin on membrane-related or intracellular proc-esses. The biochemical data confirmed the findingsof Loten and Jeanrenaud (11) that cytochalasin B hadno effect on the antilipolytic effect of insulin. In addi-tion, cytochalasin B had no effect on insulin stimula-

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FIGURE 6 The protective effect of Fm-I on cytochalasin-B-induced redistribution of Fm-I receptors was determined.One aliquot of adipocytes served as the control and wasincubated with 500 ,uU Fm-I/ml for 30 min at 37°C (0). Asecond aliquot was incubated with 500 ,uU Fm-I/ml for 15min before the addition of 5 AM cytochalasin B (Cyto B).These cells were incubated an additional 15 min at 37°C(-). A third aliquot was incubated with 5 AMcytochalasin Bfor 15 min before adding 500 ,U Fm-I/ml and incubatingfor an additional 30 min at 37°C (A). All cells were preparedfor electron microscopy, and the receptor distribution wasquantitated as described in Methods. The data presented arethe results of a single experiment.

576 L. Jarett and R. M. Smith

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1 2 3 4 5 6RECEPTORSITES PER GROUP

FIGURE 7 The effect of cytochalasin D on the distributionof Fm-I receptors was determined by incubating adipocytesfor 15 min at 37°C with 10 ,uM cytochalasin D before theaddition of 500 ,uU/ml Fm-I. After a 30-min incubation at37°C the cells were prepared for electron microscopy andanalysis as described in Methods. The results depicted showthe percentage of the total insulin receptors observed foundin each group size from one to six receptor sites per groupin the control (0) or cytochalasin-D-treated (X) cells.

tion of protein synthesis. Cytochalasin B was capableof inhibiting the basal glucose transport system, andthereby prevented insulin from stimulating the dis-rupted basal system. However, any remaining un-inhibited basal glucose transport was responsive toinsulin to the same extent as in the absence of anycytochalasin B. This is consistent with the data of Czechet al. (10). Thus, it would appear that maximum dis-ruption of the groups of insulin receptors by cyto-chalasin B did not interfere with insulin action. At leastthree explanations exist for this. First, and most likely,the glycoproteins to which insulin binds contain themechanism for initiating insulin action and can func-tion whether part of a group or not. The second isthat not all of the groups are disrupted, and the re-maining ones can account for maintaining insulin actionthrough spare receptors (23, 24); however, concentra-tions of cytochalasin B that caused maximal disruptionof receptor groups did not alter the actions of insulinat submaximal concentrations of insulin (making thisexplanation unlikely). A third possibility is that thereare two functionally distinct types of groups of insulinreceptors: one group is sensitive to cytochalasin B andrelates to glucose transport as discussed below; andthe second type of group is insensitive to, and not dis-rupted by, cytochalasin B and may account for all otheractions of insulin.

The data in this study would suggest that the groups

of glycoproteins containing the insulin receptors, or atleast a subset of them, function as a structural part ofthe glucose transport system. A number of lines of evi-dence support this theory. First, several experimentalconditions showed a correlation of morphological andbiochemical observations with cytochalasins. Disrup-tion of the groups of insulin receptors occurred overthe same concentration range of cytochalasin B thatinhibited glucose transport. Cytochalasin D over thesame concentration range had no effect on glucosetransport or on the groups of insulin receptors. Pre-treatment of adipocytes with insulin protected glucosetransport from inhibition by cytochalasin B and other in-hibitors of glucose transport such as N-ethylmaleimide(25), and the pretreatment with insulin prevented disrup-tion of the groups of insulin receptors by cytochalasin B.Secondly, antibodies to the partially purified adipocytehexose transport system mimic insulin by stimulat-ing glucose transport (26) and inhibiting stimulatedlipolysis.2 Thirdly, there appears to be the same num-ber of cytochalasin B binding sites associated with theglucose transport system of adipocyte plasma mem-branes (27) as has been previously reported for insulinbinding sites based on biochemical and morphologicaldata (3).

Singer (28) and Guidotti (29) have recently pro-posed a structural model for transport based on integralproteins aggregating into groups of various sizes (twoto four) and forming an aqueous channel. A numberof transport proteins have been identified which fitthis model, such as the Na+-K+-ATPase (30), theerythrocyte band 3 anion transport protein (31), thebacteriorhodopsin in the purple membranes whichtransport H+ ions (32), rhodopsin (33), the Ca++-ATPase of sarcoplasmic reticulum (34), and theacetylcholine receptor (35). The data present in thisreport are consistent with such a model, which suggeststhat the groups of insulin receptors form the glucosetransport pore. This would explain why disruption ofthe groups by cytochalasin B inhibits the basal glucosetransport system, and would suggest that the groupingof insulin receptor glycoproteins are necessary forbasal transport to occur.

Another physiological function of the groups,besides forming the glucose transport pore, might be tofacilitate microredistribution of receptors after oc-cupancy by insulin. The data of Kahn et al. (36), fromstudies with antibodies to insulin receptor that mimicinsulin action, suggest that microredistribution of thereceptors might be important in initiating insulin ac-tion. Bivalency of the antibody or its fragments wasessential for the insulin-like effects. Ultrastructuralstudies have shown a slight difference between pre-

2 Pillion, D. J., J. R. Grantham, and M. P. Czech. 1979.J. Biol.Chem. In press.

Relationship of Insulin Receptors to the Glucose Transport System 577

fixed and 0-4°C-incubated membranes in the distribu-tion of the insulin receptors in groups of various sizes(6). This may be consistent with microredistribution ofthe receptors occurring. In conitrast, a number of argu-ments can be made against the microredistribution be-ing necessary for insulin action. First, a single insulinmolecule is normally considered to be monomeric, notbivalent as is the receptor antibody. Second, themarked disruption of the groups of insulin receptorsby cytochalasin B did not alter the biological effects ofinsulin, even though it would be more difficult formicroredistribution to occur after disruption of thegroups. Finally, it is not clear yet as to the mechanismby which the antibody actually mimics insulin. Anti-bodies to both the insulin receptor (36, 37) and theglucose transport system (26) mimic insulin as doesconcanavalin A (38). All of these multivalent ligandsmay cause major alterations in the membrane that differsfrom the actual mechanism of insulin action, but re-sult in activation of a common subsequent pathway.This is especially true in light of recent data (39), whichshows that anti-insulin receptor antibodies have asimilar magnitude of effect as insulin on 2-deoxyglucoseuptake and glycolysis, but a smaller effect on glycogensynthesis than insulin, all in skeletal muscle. Cautionmust be exercised at this time in the interpretationof the antibody data as to its implications to the mecha-nism of insulin action.

The actin or actin-myosin cytoskeletal system wouldnot appear to be involved in the mechanism of insulinaction or in holding the groups of insulin receptorstogether. Both cytochalasin B and D disrupt micro-filaments (18, 40) and interfere with actin gelation(9, 41), with cytochalasin D even more effective thancytochalasin B. The concentrations of cytochalasin Bused in this study were at the lower level of effectiveconcentrations that disrupt microfilaments or interferewith actin gelatin in other cell systems studied. In con-trast, the cytochalasin D levels were at the high endof the effective range. Cytochalasin B has a uniquebinding site not shared with cytochalasin D, whichhas been related to the glucose transport glycoproteinsystem (19). Cytochalasin B, but not cytochalasin D,inhibited glucose transport and disrupted insulin re-ceptor groups. Thus, the disruption of the groups ofinsulin receptors concomnitant with the inhibition ofglucose transport suggests that the disruption relatesto the mechanism by which cytochalasin B inhibitsglucose transport rather than any action on micro-filaments or actin gelation. The mechanism by whichcytochalasin B inhibits glucose transport is not known,but has been suggested (42) to be because of competi-tive binding with glucose on the glucose transportglycoprotein as a result of an identical spatial dis-tribution of four oxygen atoms to those found in theCl conformiation of 8-D-glucopyranose. These bio-

chemical and morphological observations should pro-vide a test system for further study into the mechanismby which insulin and cytochalasin B act on the glucosetransport system.

ACKNOWLEDGMENTS

This study was supported, in part, by U. S. Public HealthService grants AM-11892 and AM-20097, and a grant from theJuvenile Diabetes Foundation.

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