Oiabetologia 9 Springer-Verlag 1984

Transcription

1 Diabetlgia (1984) 27: Oiabetlgia 9 Springer-Verlag 1984 Binding and mlecular weight prperties f the insulin receptr frm mental and subcutaneus adipcytes in human besity J. N. Livingstn 1, K. M. Lerea 1, J. Blinder 2, L. Kager 2, L. Backrnan 3 and P. Amer 2 1Department f Medicine, University f Rchester, Rchester, New Yrk, USA, and Departments f Medicine and Surgery, 2Huddinge and 3 Danderyd Hspitals, Karlinska Institute, Stckhlm, Sweden Summary. The insulin binding prperties and the mlecular weights f the insulin receptr and its insulin binding subunit were studied in mental and subcutaneus adipcytes prepared frm bese- and nrmal-weight subjects. Insulin binding by such adipcytes was decreased in besity when the binding activity was expressed per unit f cell surface area. N significant difference frm the lean cntrls was evident, hwever, when binding was calculated n a per cell basis, indicating that the ttal receptr cntent f the cells frm the bese subjects was nt altered. In additin, the nrmal difference in the receptr binding affinities previusly reprted between mental and subcutaneus cells frm lean individuals was unaffected by the bese cnditin. Studies f the mlecular weight f the nn-reduced insulin receptr in fat cell membranes prepared frm pieces f mental and subcutaneus fat demnstrated a majr receptr species f K Mr. In cntrast, adipcytes islated by cllagenase treatment f the fat had hetergenus nn-reduced receptr species f Mr 355K, 285K and small amunts f 427K and 182K. Althugh different nn-reduced receptr species were evident depending n the adipcyte receptr preparatin (e. g. islated adipcytes r fat cell membranes), n differences were fund between bese and lean cntrls r between subcutaneus and mental receptrs when the apprpriate cmparisns were made. Upn sulphydryl reductin, all receptr preparatins had a majr binding subunit f 125K M~. In cnclusin, besity is characterized by a dilutin f the insulin receptr ver the adipcyte cell surface in the absence f a change in ttal cellular cntent f receptrs. The difference in insulin binding affinities between mental and subcutaneus adipcytes culd nt be explained by an alteratin in receptr mlecular weight. Key wrds: Insulin receptr, besity, adipcyte, subcutaneus fat, mental fat. Obesity is a well recgnized insulin resistant state in man [1]. A large amunt f wrk has fcussed n the inciting factr(s) that induces this resistance and n the alteratin(s) in target cells respnsible fr the change [2]. One such area invlves the insulin receptr and the pssibility that a decrease in insulin binding cntributes t the diminished insulin actin reprted in human adipcytes. Results frm several f these studies have indicated a decrease in the ttal number f insulin receptrs per adipcyte in besity, which suggested that a change in receptr regulatin might cntribute t the hrmnal resistance [3-6]. Other studies, hwever, have demnstrated a change nly in the receptr density n the cell surface [7-9], a feature assciated with increased size f the adipcytes. All previus insulin binding studies in besity have used adipcytes taken frm subcutaneus fat even thugh mental adipse tissue frms a substantial part f the verall fat depsits [10]. In the present study, we have examined the insulin binding prperties f mental adipcytes frm bese- and nrmal-weight subjects and re-examined insulin binding by the subcutaneus cells. Since little is knwn f the structure f the human adipcyte receptr, affinity labelling prcedures and s- dium ddecyl sulphate electrphresis have been used t evaluate the mlecular weight f the receptrs in these tissues. This investigatin als prvided an pprtunity t determine whether besity alters the nrmal difference fund in insulin-binding affinities f mental and subcutaneus receptrs previusly described in lean individuals [11]. Subjects and methds Human subjects Nine nn-bese cntrl subjects and seven bese subjects were studied. Their clinical data are given in Table 1. The cntrl subjects had uncmplicated gallstne disease, but were therwise healthy and were admitted fr elective chlecystectmy. They had nt been n any special diets t help cntrl gallstne frmatin. The bese subjects were therwise healthy and were admitted fr gastric peratins fr besity. All subjects had maintained cnstant bdy weight during the 3 mnths preceding admittance, and nne was n any specific treatment. All subjects were examined as in-patients but remained active at their usual exercise level. They were put n an iscalric weight-maintaining diet (45% carbhydrate, 20% prtein, and 35% fat) fr 3 days. After an vernight fast, general anaesthesia was induced with a shrtacting barbiturate and maintained by phentanyl and a nitrus xidexygen mixture. The specimens f adipse tissue (abut 10 g) were

2 448 J. N. Livingstn et al.: Insulin receptr in besity Table 1. Clinical and metablic characteristics f the study grups Sex Age Height (F: M) (years) (cm) Bdy Fasting bld Fasting serum Fat ceil vlume weight glucse level insulin level (ram ) (kg) (retl/l) (mu/1) Omental Subcutaneus Cntrl subjects (n=9) 4:5 37_+4 173:t:3 69_+3 4.7_ _ _ _+ 66 Obese subjects (n = 7) 4: _ _ b _+ 8.1 a b t a The values are expressed as mean SEM. Significance between grups using Student's unpaired t-test: ~p < 0.01; b p< taken immediately after the abdminal wall had been pened. Subcutaneus fat first was remved frm the surgical incisin in the epigastric regin f the abdminal wall and mental fat was taken frm the majr mentum. The time perid frm the start f the first t the end f the secnd bipsy was always less than 2 min. The study was apprved by the Ethical Cmmittee f the Karlinska Institute. Each subject was given a detailed descriptin f the study and infrmed cnsent was btained. Fat cell determinatin and islatin f fat cells The fat cell size was measured in frzen-cut and frmaldehyde-fixed adipse tissue, accrding t the methd f Sj6strm et al. [t2]. In individual experiments, the diameters f 100 cells were determined with a calibrating phtmicrscpe (Zeiss, New Yrk, USA). Mean fat cell vlume, fat cell number and mean fat cell surface area were calculated by frmulae described previusly [13, 14]. Islated fat cells were prepared using the methd f Rdbell [15]. In brief, minced adipse tissue was shaken fr 60 rain at 37 ~ in Krebs-Hensdeit bicarbnate buffer (ph 7.4), cntaining cllagenase (0.5 mg/ml) and dialyzed bvine serum albumin (20 mg/ml). After the islatin prcedure, the fat cells were washed three times with cllagenase-free buffer. Preparatin f adipse plasma membranes Plasma membranes were prepared at 0-4 ~ as described by Belsham et al. [t6]. A mixture f sucrse (2 tl/l), Tris-HC1 (80 retl/l) and EGTA (8 retl/l) was prepared and adjusted t ph 7.4 (buffer 1). Apprximately 4 g f adipse tissue was hmgenized fr 10 s with a Plytrn hmgenizer, Brinkmann Instruments, Westbury, New Yrk) in 10 ml f sucrse (0.25 tl/l), Tris-HC1 (10 retl/l), EGTA (2 retl/l), ph 7.4 (buffer 2). After centrifugatin fr 30 s at 1000 g, the interphase between the fat plug and the pellet was aspirated and centrifuged fr 30 min at 30,000 g. The pellet was resuspended in 8 ml f Percll: buffer I: buffer 2 (7:1:32) and centrifuged fr 15min at 10,000g. One ml f the supernatant cntaining the plasma membranes was aspirated and diluted with buffer 2 (5 ml) and centrifuged fr 2rain at 10,000 g. The pellet was suspended in 0.5ml f buffer 2 and stred at - 70 ~ When islated fat cells were used fr the preparatin f plasma membranes, 2 ml f packed adipcytes were hmgenized in 10ml f buffer 2 and prcessed as described abve. Insulin binding t islated fat cells Islated tat cells were incubated in triplicate at the final cell cncentratin f 8% (v/v) fr 60 min at 24 ~ in Krebs-Henseleit bicarbnate buffer (ph 7.4), cntaining dialyzed bvine serum albumin (40 rag/ ml), glucse (1 mg/ml), mn125i-(tyr A14) insulin (0.05 pml/ml) and the indicated cncentratin f unlabelled insulin. In these studies, the final insulin cncentratins ranged frm 0.05 t 50pml/ml. The binding reactin was terminated by adding NaC1 (0.154mml/1, 10 ml) and the cells were subjected t centrifugatin thrugh 1.2 ml f silicne il [17]. Cell-bund radiactivity was determined in the cells suspended abve the il phase. Nn-specific binding was apprximately 4% f the ttal binding as determined by the additin f unlabelled insulin (20 l.tml/1). Specific insulin binding was based n the fat cell number r the fat cell surface area. Binding data were presented bth as a plt f the percentage f the specifically bund insulin versus the ttal insulin cncentratin (cmpetitin-inhibitin curve) and accrding t Scatchard [18]. Affinity labelling f intact fat cells with lesl-insutin Packed cells (1.5 ml) were suspended in Krebs-Henseleit bicarbnate buffer (ph 7.4, 7 ml) cntaining albumin (40 mg/ml), glucse (1 rag/ ml), 125I-insulin prepared by the chlramine-t methd (0.5 pml/ml) and unlabelled insulin (0 r 20 p~ml/1). The cells were incubated at 21 ~ fr 60 min. A slutin f the crss-linking reagent, disuceinimidyl suberate (0.1 ml) was then added (0.5 mg/ml, final cncentratin) and the incubatin was cntinued fr anther 15 min at 21 ~ [19]. The crss-linking reactin was terminated by the additin f 2 tl/1 Tris HC1 (ph 7.6, 0.1 ml) cntaining 0.2 tl/1 Na EDTA. After remval f the medium, the fat cells were washed twice in 15 ml f insulin-free incubatin buffer and the plasma membranes were prepared as described abve. Affinity labelling f adipcyte plasma membranes with 125 I-insulin Adipcyte membranes ( ~tg prtein) prepared frm fat tissue were incubated at 21 ~ with 1~sI-insulin (0.5 nml/1) in a Krebs Ringer phsphate buffer (ph 7.4), cntaining bvine albumin (40 mg/ml) and bacitracin (1 mg/ml). Nn-specific labelling was determined in a parallel experiment using 20 lxml/1 unlabelled insulin. After a 2-h incubatin, disuccinimidyl suberate (final cncentratin 1 mml/1) was added [19] and the incubatin cntinued fr 10 rain. The reactin was terminated by the additin f the Tris, EDTA slutin described abve. The membranes were islated by centrifugatin and disslved in the apprpriate sdium ddecyl sulphate (SDS) slutin fr gel electrphresis. SDS gel eleetrphresis The affinity labelled membranes were slubilized and subjected t SDS electrphresis by tw different methds. Nn-reduced affinity labelled receptrs were slubilized in the absence f reductant and subjected t electrphresis in a 3.3% SDS acrylamide gel as described by Weber et al. [20]. Fllwing electrphresis, the gels were fixed in 10% trichlracetic acid (w/v), stained with Cmassie blue R-250, destained and dried. The dried gels were expsed t X-matic film (S and W X-rays, Rchester, New Yrk) using X-matic intensifying screens (Eastman-Kdak, Rchester, New Yrk). The film was previusly sensitized by preflashing with a flash unit (Sunpak, Berkey C., Wdside, New Yrk). The subunit cmpsitin f the labelled receptrs were determined by slubilizing the membranes in an SDS buffer cntaining 13- mercaptethanl (100mml/1) as described by Laemmli [21]. The electrphresis was cnducted in a 5-12% acrylamide gradient gel [21]. Autradigraphs f the gels were prepared as described abve. Chemicals Crystalline prcine insulin was supplied by Vitrum, Stckhlm, Sweden, and Eli Lilly, Indianaplis, Indiana. Ctlagenase was btained frm Wrthingtn, Malverne, Pennsylvania, and Armur Pharmaceuticals, Kanakee, Illinis supplied bvine serum albumin. Disuccinimidyl suberate was purchased frm Pierce Chemical, Rckfrd, Illinis. Acrylamide, N, N' methylenebisacrylamide, and N,N,N',N'-tetramethylethylenediamine were supplied by Eastman Kdak, Rchester, New Yrk. High mlecular weight crss-linked standards (M r range f 336,000-56,000) were purchased frm British

3 J. N. Livingstn et al.: Insulin receptr in besity " A? x 9 84 % ~r " c: 6-.O C ~ 3- I c- O,,n 2" 0.;5@ il 5. Insulin cncentratin (pml/mt) 3 ; ; Bund? x E 12- * 6.. 5, B "O C a -5 if) c 3" i 0 u_ i "" 3~ f i 050, i, ;;0 00 C i' l Fig. 1A and B, Insulin binding t adipcytes islated frm A bese and B nn-bese subjects. Subcutaneus (0---0) and mental (O... O) fat cells were islated frm five bese and five nn-bese cntrl subjects matched fr age and sex. Specific insulin binding is expressed per unit cell surface area (mean_+ SEM). Left panels are cmpetitin-inhibitin curves; fight panels are the crrespnding Scatchard plts. *p < 0.05 Insulin cncentratin (pml/ml) Bund c- A Drug Huse Bichemicals, Drset, Ple, UK. Bi-Rad, Richmnd, Califrnia, supplied mid-range mlecular weight standards (M~ range f 200,000-43,000). Mn 125I-Tyr A14-insulin (209 Ci/g) used in the insulin binding studies was purchased frm Nv, Cpenhagen, Denmark. The 125I-insulin used in the affinity labelling prcedures was prepared by the chtramine-t methd as described [22], using 125I-Na purchased frm Amersham Internatinal, Amersham, Bucks, UK. Z s ~a -% c3- a~ J 5- c 2- c "T - ~0-0.b5 d, ; 50 Insulin cncentratin (pmllml) Insulin cncentratin (pmt / ml) Fig.2A and B. Scatchard analysis f the insulin binding data f Figure I expressed per cell. Data fr A mental and B subcutaneus fat cells islated frm (0---0) nn-bese and ( bese subjects are shwn. The tw curves in each panel are nt statistically different Statistical analysis Reprted values represent the mean +_ SEM. Student's paired and unpaired t-tests were used fr statistical cmparisn f the data. Results All f the subjects had nrmal fasting bld glucse levels at the time f the study (Table 1). Hwever, the mean fasting serum insulin level was six times higher in the bese than in the cntrl grup (p<0.01), indicating that the bese subjects were insulin resistant. Table I als shws the increase in the vlumes f bth mental and subcutaneus fat cells frm the bese individuals. In bth grups subcutaneus cells were larger than the mental cells.

4 450 j.n. Livingstn et al.: Insulin receptr in besity Fig.3. Structure f the nn-reduced insulin receptr frm islated adipcytes and fat cell plasma membranes. Leftpanel: mentai (OM) and subcutaneus (SC) adipcytes were islated by cllagenase treatment and affinity labelled with 125I-insulin in the presence (+) r absence (-) f native insulin. The labelled receptrs were slubilized and subjected t SDS-PAGE and autradigraphy. Right panel: adipcyte plasma membranes prepared frm mental and subcutaneus fat pieces were affinity labelled with 125I-insulin and the structure f the receptr was examined as described abve Fig.4. The structure f the nn-reduced insulin receptr frm bese (O) and nrmal weight (N) subjects. Fat cell membranes were islated frm pieces f mental (OM) and subcutaneus (SC) tissue. Autradigrams f the dried gels were prepared after SDS electrphresis f the affinity labelled receptrs, The presence (+) and absence (-) f excess native insulin (INS) during the labelling prcedure is indicated Studies f insulin binding The results f insulin binding t islated fat cells expressed per cell surface area are illustrated in Figure I. Fr bth grups specific insulin binding was significantly higher in subcutaneus than mental cells at lw insulin cncentratins (p < 0.05). At higher insulin cncentratins (> 10 pml/ml), there was n difference in binding between the tw types f fat cells. Based n Scatchard analysis f the binding data, the difference in binding between mental and subcutaneus cells apparently was due t differences in verall affinity rather than differences in ttal binding sites. A difference in insulin binding between mental and subcutaneus fat cells was present als when binding was expressed per fat cell number, since subcutaneus cells are larger (Fig.2). Insulin binding t fat cells f bese and cntrl subjects can als be reliably cmpared, since the tw grups were matched fr age and sex. The cmpetitininhibitin curves shw that specific insulin binding t subcutaneus fat cells is, n average, 40% lwer in the bese than the cntrl grup (Fig. 1). The difference between the tw grups fr mental ceils is apprximately 30%. The Scatchard plts demnstrate a parallel shift t the left fr the bese grup in relatin t the cntrl grup fr bth mental and subcutaneus fat cells (Fig. 1). These data indicate that the decrease in insulin binding in the bese grup was largely due t a decrease in ttal binding sites per unit f cell surface. When insulin binding was expressed per cell (Fig. 2), n significant difference between the adipcytes frm bese subjects and cntrls was fund in the high affinity prtin f the Scatchard plts. In the lwer affinity prtin f the curve, e.g. > 5 nml/1 insulin, there was a slight but nt statistically significant decrease in the extent f binding by subcutaneus cells frm the bese grup. Characteristics f the mlecular weight f the adipcyte insulin receptr The mlecular weight f the receptr was examined by crss-linking tzsi-insulin t the insulin binding sites with disuccinimidyl suberate, fllwed by SDS plyacrylamide gel electrphresis. This methd was chsen because f its efficiency in cvalently attaching radilabelled insulin t the receptr [23]. Figure 3 (left panel) illustrates the nn-reduced insulin receptr species fund after crss-linking 125I-insulin t intact adipcytes islated frm subcutaneus and mental fat taken frm the same subject. There are several specific insulin binding species ranging in m-

5 J. N. Livingstn et al.: Insulin receptr in besity 451 Fig. 5. Autradigram f the subunit cmpsitin f the adipcyte insulin receptr frm bese (O) and nrmal weight (N) subjects. Fat cell membranes were prepared frm mentat (OM) and subcutaneus (SC) fat pieces. The receptrs were affinity labelled in the absence (-) r presence (+) f native insulin (INS) and subjected t electrphresis after reductin with t00 retl/1/~-mercaptethanl lecular weight frm 427K t a small amunt f 182K. The majr species is a 285K cmpnent; smaller but prminent amunts f insulin binding species f 355K and 427K are als evident. The 182K is nt clearly shwn but is easily identified in the riginal autradigram. Bth mental and subcutaneus adipcytes cntained these species and their relative amunts were similar in bth tissues. Therefre, even thugh the insulin binding affinity was greater in subcutaneus cells, bth cell types cntained similar nn-reduced insulin binding cmplexes that have similar r identical mlecular weights. The insulin receptr is highly sensitive t prtease activity [24]. Since the cllagenase used in preparing the islated adipcytes cntains a number f different prteases, we als studied plasma membranes islated frm pieces f subcutaneus and mental fat. In these studies the plasma membranes were purified by a Percil gradient centrifugatin technique, which previusly was shwn t separate a membrane fractin crrespnding clsely t the plasma membrane f islated fat cells [16]. Figure 3 (right panel) shws the insulin binding species btained frm subcutaneus mental fat pieces. Clearly, there is a marked difference between these results and the findings btained frm islated fat cells shwn in the left panel. In the membranes islated frm the fat bipsies, a majr 400K insulin binding species is present with minr cmpnents f 122K and 60K M~. Only the 400K species binds insulin in a highly spe- cific manner, since the presence f a large amunt f native insulin des nt cmpletely inhibit the labelling f the tw minr bands with 12sI-insulin. Cnsequently, in membranes islated in the absence f cllagenase treatment, the majr nn-reduced insulin receptr has a mlecular weight in the 400K range. These results als shw that the insulin binding sites in membranes frm mental and subcutaneus tissue have similar mlecular weights. Therefre, as indicated by tw different receptr islatin prcedures, the changes in insulin binding characteristics cannt be explained by mlecular weight differences in the nn-reduced receptr species, at least as examined by the crss-linking technique and sdium ddecyl sulphate electrphresis. The Percll methd used t islate plasma membranes fr affinity labelling studies has an additinal advantage. It is mre efficient than the mre labrius technique f first islating the adipcytes, crss-linking radilabelled insulin t the insulin receptr, and then membrane islatin. This cnsideratin becmes imprtant in the studies f nn-bese subjects with limited amunts f available fat. Primarily fr this reasn, the mlecular weight cmparisns f nn-bese and bese adipcyte receptrs were carried ut using purified plasma membranes frm fat bipsies. Figure 4 shws the results frm tw bese and tw nn-bese subjects. The membranes frm bth grups have a high mlecular weight insulin binding species (429K - 390K) and small amunts f relatively nn-specific labelled material f 121K and 63K. There was n difference in the 125I-insulin labelling patterns between bese and nn-bese individuals, either in subcutaneus r mental fat. In these studies the amunt f plasma membrane used fr affinity labelling varied depending n the recvery frm the adipse tissue. It is nt pssible therefre t cmpare the actual cntent f the receptrs in bese and nrmal tissue by examining the autradigrams. The receptr subunit structure was examined in these subjects by subjecting the labelled receptrs t 100 mml/1/3-mercaptethanl (Fig. 5). A majr 125K subunit is present in all receptr preparatins alng with a minr 85K cmpnent. Again n difference is evident between bese and nn-bese r between subcutaneus and mental fat. Studies als were cnducted using 12sI-insulin crss-linked t islated adipcytes frm mental and subcutaneus fat (data nt shwn). In agreement with the results in Figure 5, the majr subunit in bth grups f cells had a mlecular weight f 125K. Discussin The basic alteratin(s) respnsible fr the insulin resistance f target cells in besity is unknwn. Current speculatin regarding the site f the alteratin has fcussed n either a change in the insulin receptr cntent r a change in pst-receptr (pst-binding) events [2].

6 452 J. N. Livingstn et al.: Insulin receptr in besity There is evidence, summarized belw, that supprts bth pssibilities. Several studies have described an verall lss f insulin receptrs in adipcytes taken frm bese subjects [3-6]. This finding argues fr the first pssibility, i. e. the presence f a receptr alteratin in besity. Other wrk, hwever, has fund nly a change in the cell surface cncentratin f the adipcyte receptr withut a change in ttal receptr cntent [7-9]. The results f the present study supprt these latter data, which questin whether a receptr defect exists in the affected cells. We investigated bth mental adipcytes as well as the usually studied subcutaneus fat cells. Fr bth cell types, the insulin binding characteristics expressed per cell did nt differ significantly in adipcytes islated frm the lean cntrl subjects. The nly change detected in besity was a decrease in the number f receptrs per unit f cell surface area, i.e. a dilutin in the cncentratin f surface receptrs in the absence f a change in ttal receptr cntent. Clearly, if the ttal receptr cntent f the adipcytes is less than nrmal, a strng argument can be made fr an alteratin at the level f the receptr. In cntrast, a simple dilutin f the insulin receptr ver the cell surface is mre difficult t evaluate in terms f its rle in insulin resistance. The adipcytes enlarge during the develpment f the bese cnditin and this factr alne can accunt fr a change in the cell surface cncentratin f the receptr. Since very little infrmatin is available regarding the cupling f the insulin receptr t the cellular effectr systems, it is nt pssible t argue with any cnfidence that this dilutin effect gives rise t defective receptr functin. The insulin-mediated respnses f adipcytes frm verweight subjects have been measured in attempts at characterizing the changes present in besity. Alteratins in the maximum respnses t insulin have been reprted fr glucse metablism [9] and glucse transprt [8, 25]. Als a change (e. g. right-ward shift) in the insulin-dse respnse curve has been described fr these prcesses [8, 25]. The change in the maximum respnse is cmpatible with a pst-binding alteratin [2]. The right-ward shift in the dse-respnse curve is mre difficult t interpret since it may invlve the ill-defined prcesses that link the receptr t the effectr systems. Whether dilutin f the receptrs ver the cell surface cntributes t any uncupling between these structures and the effectr systems is impssible t judge at this time. Althugh changes in insulin actin ccurs in glucse metablism, anther system, antiliplysis, retains its nrmal respnse t insulin in adipcytes frm bese subjects [26]. This finding, plus the ther evidence fr pst-receptr alteratins in glucse metablism and transprt utlined abve, suggest that the majr change(s) in affected cells ccur, at selected sites beynd the receptr. Since insulin binding studies examine nly ne aspect f the insulin receptr, we als undertk an investigatin f the mlecular weight f the receptr, us- ing an affinity labelling prcedure [19]. Althugh this methd has a labelling efficiency f nly 20% fr the nn-reduced receptr [23], it is the mst efficient methd available t examine the mlecular weights f the nn-reduced receptr and its insulin binding subunit. Again, in agreement with the similarities in their respective insulin binding prperties and with the cncept f a pst-binding alteratin, n change in the mlecular weight f the receptr was detected in besity. Thus, dilutin f cell surface receptrs remains the nly change detected in mental and subcutaneus adipcytes frm bese subjects. Hwever, it cannt be excluded that a subppulatin f receptrs exists which is nt affinity labelled and which may differ in besity. The present study als prvided the pprtunity t examine the interesting difference nted previusly between the receptr affinities f mental and subcutaneus adipcytes frm lean individuals [11]. The mlecular weight studies shwed that membranes frm bth tissues cntained ne majr nn-reduced insulin binding species f K Mr. Sulphydryl reductin generated a majr insulin binding subunit f 125K and a minr 85K cmpnent frm bth types f tissues. It was interesting that affinity labelling the islated fat cells rather than the purified plasma membrane fractin frm fat pieces prduced different nn-reduced receptr species. With islated cells, a majr species f Mr 285K was evident with a prminent amunt f a 355K cmpnent and small amunts f 427K and 182K species. Althugh tiffs difference between islated fat cells and plasma membranes is striking, the results with the cells are quite similar t thse reprted fr islated rat adipcytes [27] and t the recent findings f Berhanu et al. [28], wh examined the receptrs f islated human adipcytes. It is pssible that the difference bserved between the fat cell membranes and islated adipcytes is caused by the cllagenase treatment used in the cell islatin prcedure. The cllagenase preparatin cntains several different prteases, any ne f which culd act n the insulin receptr since it is highly sensitive t prtease activity [24]. A secnd pssibility is that the membrane preparatin cntains a large number f insulin receptrs f 400K Mr derived frm ther cell types, i.e. frm cells ther than adipcytes. The varius prprtins between ther cells and adipcytes in human fat bipsies are nt knwn, but in rat fat the adipcytes represent 35% f the ttal cell number [29]. Hwever, because f the much greater size f the adipcytes, the amunt f fat cell plasma membrane prbably accunts fr the majrity f the plasma membrane cntent f adipse tissue. Since we chse a methd specifically designed t islate adipcyte plasma membranes frm fat pieces as well as frm islated adipcytes [16], the plasma membrane fractin used in the present studies is prbably mre representative f the adipcyte than f the strmal cells. Mrever, strmal ceils, such as fibrblasts [30], have fewer insulin receptrs than adipcytes. Based n these cnsideratins, it seems unlikely that the 400K species is derived frm a strmal cell type.

7 3. N. Livingstn et al.: Insulin receptr in besity 453 A third and mre intriguing pssibility t explain the difference fund between affinity labelled membranes and islated adipcytes is that the binding f insulin t the surface f the intact cells causes the generatin f sme f these species. In supprt f this pssibility Berhanu et al. [28] fund a time-dependent prductin f different mlecular weight species after phtaffinity labelling f the adipcyte cell surface receptr. Thus, cvalent attachment f insulin t the receptr may cause the adipcyte t "prcess" this cmplex and alter its mlecular weight. Regardless f the causes f the different mlecular weight species, mental and subcutaneus adipcytes islated by cllagenase treatment have the same species in the same relative prprtins. Therefre, the cause f the difference in binding affinity between these tw cell types prbably arises frm anther reasn, like differences in membrane lipid cmpsitin [3t] r in membrane mdulatrs f affinity [32]. In summary, besity des nt change the mlecular weight f the adipcyte insulin receptr, its insulin binding prperties, r its cntent in the enlarged adipcyte. These findings suggest that the majr alteratin respnsible fr the insulin resistance f adipcytes frm bese subjects invlves pst-receptr sites. Acknwledgements. The authrs thank Dr. J. Amatruda fr helpful review f the manuscript and Dr. R Berhanu fr making available unpublished data. The present study was supprted by Natinal Institutes f Health Grant AM 25116, and The Swedish Medical Assciatin, The Karlinska Institute, the Swedish Diabetes Assciatin and the Fundatins f Flksam, Osterman and Nrdic Insulin. J. N.L. is a recipient f the Natinal Institutes f Health Research Career Develpment Award AM References 1. Kreisberg R, Bshell B, DiPlacid J, Rddman R (1967) Insulin secretin in besity. N Engl J Meal 276: Otefsky J (1981) Insulin resistance and insulin actin. 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J Clin Invest 72: Rdbell, M (1964) Lcalizatin f lipprtein lipase in fat cells f rat adipse tissue. J Bil Chem 239: Mtt D, Hward B, Bennett P (1979) Stichimetric binding and regulatin f insulin receptrs n human diplid fibrblasts using physilgic insulin levels. J Bil Chem 254: Guld R, Ginsberg B, Spectr A (1982) Lipid effects n the binding prperties f a recnstituted insulin receptr. J Bil Chem 257: Matur J, Hllenberg M (1978) Insulin receptr: interactin with nn-receptr glycprtein frm liver cell membranes, Prc Natl Acad Sci (USA) 75: Received: 19 January 1984 and in final frm: 17 July 1984 Dr. R Arner Department f Medicine Huddinge Hspital S-14t 86 Huddinge Sweden