Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Bioehimica et Biophysica Acta, 763 (1983) 393 407

393

Elsevier BBA 11230

I N S U L I N - S T I M U L A T E D T R A N S L O C A T I O N OF G L U C O S E T R A N S P O R T E R S IN T H E I S O L A T E D RAT A D I P O S E CELLS: C H A R A C T E R I Z A T I O N OF S U B C E L L U L A R F R A C T I O N S IAN A. SIMPSON, DENA R. YVER, PAUL J. HISSIN *, LAWRENCEJ. WARDZALA. EDDY KARNIELI **, LESTER B. SALANS and SAMUEL W. CUSHMAN Cellular Metabolism and Obesity Section, National Institute of Arthritis, Diabetes, and Digestic, e and Kidm~v Diseases, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)

(Received April 18th, 1983) (Revised manuscript received August 15th, 1983)

Key' words: Glucose transporter distribution," Subcellular fractionation," Insulin," (Rat adipose tissue)

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific D-glucose-inhibitable [3HIcytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4-5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7 • 106/cell). Insulin does not influence the K d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15 • 103 mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (I) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.

* Present address: Department of Chemistry, Mount Sinai Hospital, New York, NY 10029, U.S.A. ** Present address: Metabolic Unit, Endocrine Institute, Rambam Medical Center, Haifa, Israel. Abbreviations: galactosyltransferase, UDP-galactose : Nacetylglucosamine galactosyltransferase; sialyhransferase, CMP-N-acetylneuraminate:asialofetuin N-acetylneuraminyltransferase; SDS, sodium dodecylsulfate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 0167-4889/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Introduction

A primary action of insulin on adipose and muscle cell function is its stimulation of glucose transport. This stimulation is characterized by an increase in the maximum rate (Vmax) of transport and is not associated with changes in affinity ( K o) for either substrate or inhibitors [1].

394

Recent reports from this laboratory have proposed that this insulin-induced increase in the V,..... for glucose transport in the rat adipose cell results from a rapid, reversible, and insulin concentration-dependent translocation of glucose transporters from a low-density intracellular membrane pool to the plasma membrane [2-4]. This concept is based on an assessment of the distribution of glucose transporter systems among various subcellular fractions prepared by differential ultracentrifugation. The numbers of glucose transporters were determined using an equilibrium binding assay for cytochalasin B, a potent competitive inhibitor of glucose transport in a wide variety of cell types. Parallel studies by Suzuki and Kono [5] and Kono et al. [6,7], using a different subfractionation procedure and reconstitution of the glucose transport activity into liposomes, have suggested essentially the same mechanism for insulin's stimulatory action on glucose transport in the rat adipose cell. These investigators [5,6] have further identified the intracellular pool of glucose transport activity with a m e m b r a n e fraction enriched in galactosyltransferase activity, a marker enzyme characteristic of membranes of the Golgi apparatus. Kono et al. [6] have also demonstrated that this translocation process is energy-dependent, but proteinsynthesis independent. More recently, Wardzala and Jeanrenaud [8,9] have indicated that insulin appears to stimulate glucose transport in rat diaphragm by a similar mechanism. The present report defines in more detail the subcellular fractions with which these glucose transporters are associated in the rat adipose cell, with a view towards further understanding the molecular events which occur in response to insulin. Materials and Methods

Preparation of isolated adipose cells and subcellular membrane fractions. Isolated adipose cells were prepared from the whole epididymal fat pads of ad libitum-fed (Purina Rat Chow, Ralston Purina Co.), 180-200 g male Sprague Dawley rats (CD strain, Charles River Breeding Laboratories) by the method of Rodbell [10], as modified by Cushman [11]. Briefly, the whole epididymal fat pads (6-7 g) of 8 rats were removed, minced and di-

gested at 37°C with 20 mg crude collagenase (Worthington Biochemicals) in 6 ml Krebs-Ringer bicarbonate (10 m M ) / H e p e s (30 mM) buffer, pH 7.4, containing 10 m g / m l untreated bovine serum albumin (Bovine Serum Albumin Powder, Fraction V, Reheis Chemical Co.). Following 30 40 min of digestion, the liberated cells were washed five times by centrifugation (1 min at 600 × g ..... ) and resuspended in the same buffer. Finally, the cells from 12 g tissue (approx. 80- 106 cells) were then resuspended in 36 ml buffer in the presence or absence of 7.0 nM (1000/~U/ml) insulin (crystalline zinc insulin, courtesy of Dr. Ronald E. Chance, Eli Lilly and Company) in 950 ml polypropylene jars and incubated for 15 min at 37°C with gentle shaking. Following incubation, the cells were washed twice in a 20 mM Tris-HC1/1 mM E D T A / 2 5 5 mM sucrose buffer, pH 7.4, approx. 20°C, resuspended in 20 ml of this same buffer, and homogenized with 10 strokes in a 55 ml Teflon pestle homogenizer (Arthur H. Thomas and Co.). Subcellular fractions of the homogenized cells were then prepared by a modification of the method originally described by McKeel and Jarett [12]. Homogenization buffer was used throughout the fractionation procedure, and all centrifugation and handling of samples were carried out at 4°C. The original homogenate was centrifuged at 16000 × g ..... for 15 min, the solidified fat cake was carefully removed, and the supernatant was saved for preparation of the microsomal membrane fractions. The initial pellet was resuspended and recentrifuged once before being resuspended in 5 ml buffer, applied to a 1.12 M sucrose cushion containing 20 mM Tris-HC1/1 mM EDTA, and centrifuged at 101000 × g .... for 70 min. The mitochondria, nuclei and broken cell debris were collected as a pellet. This pellet was washed once, repelleted at 16000 × g ..... for 15 min, and finally resuspended to approx. 6 mg p r o t e i n / m l . The plasma membranes, collected at the interface, were resuspended in 50 ml buffer and centrifuged at 48000 N gmax for 45 min. The pellet was resuspended in 10 ml buffer, repelleted and finally resuspended to approx. 3 mg p r o t e i n / m l . The initial supernatant was centrifuged at 48 000 × g .... for 20 min yielding a pellet of highdensity microsomal membranes. The supernatant

395

was then recentrifuged at 212000 X g m a x for 70 min yielding a second pellet of low-density microsomal membranes. Both pellets were resuspended in 10 ml buffer and repelleted before final resuspension to approx. 2 mg protein/ml. All membrane fractions were stored in liquid nitrogen until use.

Electron microscopy and gel electrophoresis. For electron microscopy, all membrane fractions were pelleted as described above, washed once in 0.1 M phosphate buffer by resuspension and pelleting, resuspended for 24 h in a 2% glutaraldehyde fixative in 0.1 M phosphate buffer, and finally repelleted at 5000 × gma×" Post-fixation was carried out for 1 h in a 1% osmium tetroxide solution in the same phosphate buffer, and the samples were dehydrated in acetone, stained with 0.5% potassium permanganate and embedded in Epon. Thin sections were examined and photographed in a Phillips 300 electron microscope. SDS-polyacrylamide gel electrophoresis was carried out by the method described by Laemmli [13], using Coomassie blue to stain for protein. Protein was determined as described by Bradford [14] and modified by Simpson and Sonne [15] using crystalline bovine serum albumin (Sigma Chemical Company) as the standard. Membrane marker enzyme assays. 5'-Nucleotidase activity was assayed as described by Avruch and Hoelzl-Wallach [16] in the presence of 0.05% Triton X-100. 5 mM 2',3'-AMP was included in the assay medium in order to inhibit nonspecific phosphatase activity. Isoproterenol-stimulated adenylate cyclase activity was measured by the method of Salomon et al. [17] in the presence of guanyl-5'-imido-phosphate ( G p p N H p ) (1 • 1 0 -.4 M), a guanine nucleotide analogue, in order to achieve maximal stimulation. Rotenone-insensitive NADH-cytochrome c reductase activity was determined by the method of Dallner et al. [18]. Glucose-6-phosphate phosphatase activity was assayed by the method of Nordlie and Jorgenson [19] in the presence of 25 mM sodium flglycerophosphate in order to minimize nonspecific phosphatase activity. Galactosyltransferase activity was assayed as described by Fleischer [20] using a 2-(N-morpholino)ethanesulfonic acid buffer, pH 6.5. Citrate synthase was assayed by the method of Srere et al. [21].

Cytochalasin B binding. The number of glucose transporters in each subcellular fraction was quantitated by specific D-glucose-inhibitable equilibrium [3H]cytochalasin B binding [2,3]. In order to accommodate the additional membrane fractions investigated in this study, the following centrifugation conditions were employed: (1) for the plasma membrane, mitochondrial/nuclear, and high-density microsomal membrane fractions, the binding assay was performed in small glass tubes and the membranes were pelleted at 48000 × g .... for 20 min at 4°C; (2) for the low-density microsomal membrane fraction, the assay was performed in cellulose proprionate tubes and the membranes were pelleted at 220 000 × g .... for 70 min at 4°C in a Beckman 42.2 Ti rotor. D-Glucose-transport. D-Glucose transport was measured in the plasma membrane fraction by the method of Baldwin et al. [22]. Briefly, 60 /~1 of a solution containing 0.9 mM L-[1-3H]glucose (155 /~Ci/mmol) and 0.9 mM D-[U-14C]glucose (77.5 /~Ci/mmol) in homogenization buffer were added to 60 /21 of plasma membranes (1 m g / m l ) suspended in the same buffer. The resultant suspension was then incubated at 22°C for variable periods of time before stopping specific transport by the addition of 120/~1 buffer containing 0.45 mM unlabeled L- and D-glucose and 2 . 1 0 5 M cytochalasin B. For zero time samples, the stop solution was added first. All samples were stored on ice following incubation. Two 100-~1 aliquots of each sample were then applied to 1 ml columns of Sephadex G-50 which were prepared in 1 ml plastic syringes fitted with appropriate filters and equilibrated with the 'stop' buffer. Immediately prior to the addition of the samples, the columns were centrifuged at 600 × gmax for 3 min. Following addition of the samples, this procedure was repeated and the effluent collected directly in mini-scintillation vials. Protein recovery was monitored using nonradioactive samples; diffusion and the nonspecific trapping of radioactivity were measured using L-glucose. The results are expressed as nmol D-glucose transported/mg membrane protein per min. Results

Characterization of subcellular fractions Typical electron micrographs and SDS-poly-

396

Fig. 1. Electron micrographs of subcellular fractions from basal (Panels A and C) and insulin-treated (Panels B and D) isolated rat ad'ipose cells. Panel A: intact cells, Panel B: plasma membranes, Panel C: high-density microsomes, and Panel D: low-density microsomes. No effects of insulin were observed. Bar = 0.5 nm.

397

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(2)

PM

HDM

Std

LDM

M/N

Fig. 2. SDS-polyacrylamide gel electrophoresis of subcellular fractions from basal (1) and insulin-treated (2) isolated rat adipose cells. 50 /~g of each subcellular fraction were applied: plasma membranes (PM), high-density microsomes (HDM), low-density microsomes (LDM), and mitochondria/nuclei (M/N). Molecular weight standards are phosphorylase (92000), bovine serum albumin (68000), ovalbumin (43000) and cytochrome c (18000).

they are readily distinguishable by vesicle size with the plasma membrane fraction containing the largest vesicles and the low-density microsomal membrane fraction containing the smallest vesicles (Fig. 1). However, with the exception of a few mitochondria in the plasma and high-density microsomal membrane fractions, and a small number of Golgi saccule-like structures in the high-density microsomal membrane fraction, the membrane species comprising these fractions cannot be identified. The distinct nature of all four subcellular fractions is further reflected by their characteristic protein compositions (Fig. 2). Nevertheless, exposure of intact cells to a maximally-stimulating concentration of insulin does not detectably influence either the morphology or protein composition of any of the four fractions prepared here. Table I demonstrates the recovery of protein in

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acrylamide gels of the subcellular fractions prepared from isolated rat adipose cells using the present differential ultracentrifugation procedure are illustrated in Figs. 1 and 2, respectively. Fig. 1 also illustrates the ultrastructure of the intact cell for comparison. Morphologically, while all three membrane fractions consist primarily of vesicles,

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PROTEIN RECOVERY IN SUBCELLULAR MEMBRANE FRACTIONS FROM ISOLATED RAT ADIPOSE CELLS Results are the means_+S.E.M, of the individual values obtained in the number of experiments given in parentheses. Cells Basal (rag/rat) Homogenate Plasma membranes High-density microsomes Low-density microsomes Mitochondria/nuclei

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TABLE 1

Fraction

A III B ]Ii ri ri

Insulin-stimulated (mg/rat)

3.04_+0.33(9) 3.10_+0.31(8) 0.20_+ 0.03(9) 0.22_+0.03(8) 0.084-0.03(9) 0.10_+0.02(8) 0.10_+0.02(8) 0.10_+0.02(8) 0.45_+0.07(7) 0.484- 0.08(7)

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Fig. 3. Enzyme distributions among subcellular fractions from basal (open bars) and insulin-treated (closed bars) isolated rat adipose cells. Panel A: 5'-nucleotidase, Panel B: isoproterenolstimulated adenylate cyclase, Panel C: rotenone-insensitive NADH-cytochrome c reductase, Panel D: glucose-6-phosphate phosphatase, Panel E: UDP-galactose: N-acetylglucosarnine galactosyltransferase, and Panel F: citrate synthase. Homogenate (HOM), plasma membranes (PM), high-density microsomes (HDM), low-density microsomes (LDM), and mitochondria/nuclei (MN). Results are the means_+ S.E.M. of the individual values obtained in 7-10 separate experiments.

398 each fraction and the original homogenates, and Fig. 3 illustrates the specific activities of various marker enzymes characteristic of different subcellular organelles. Again, neither of these two fractionation parameters is affected by incubation of the intact cells with insulin. However, examination of the distribution of marker enzyme activities permits identification of the primary m e m b r a n e species comprising each fraction. A comparison of the distributions of the two classic plasma membrane marker enzyme activities, 5'-nucleotidase (Fig. 3A) and adenylate cyclase (Fig. 3B), reveals that the latter is disproportionately low in the homogenate. This is probably due to the presence of an inhibitory c o m p o n e n t [23] which can be eliminated by a 1 : 10- to 1 : 20-fold dilution (data not shown). Conversely, the apparent 5'-nucleotidase activity observed in the m i t o c h o n d r i a l / nuclear fraction is anomalously high c o m p a r e d to that of adenylate cyclase. This fraction contains both a nonspecific phosphatase which can be inhibited by 2',3'-AMP, and an indigenous (non-inhibitable) phosphatase activity which, after correction for plasma m e m b r a n e contamination based on adenylate cyclase activity, corresponds in specific activity to 17% of that in the plasma m e m b r a n e fraction. A p a r t from these anomalies, the distributions of 5'-nucleotidase and adenylate cyclase activities parallel each other very closely

a m o n g all four of the fractions prepared here, and demonstrate the enrichment of the plasma membrane fraction with plasma membranes. Rotenone-insensitive N A D H - c y t o c h r o m e c reductase (Fig. 3C) and glucose-6-phosphate phosphatase (Fig. 3D), marker enzyme activities characteristic of membranes of the endoplasmic reticulum in other cell types, are most enriched in the high-density microsomal m e m b r a n e fraction. Their parallel enrichments in this fraction and distributions a m o n g all four fractions indicate that they serve as adequate markers for monitoring the distribution of endoplasmic reticulum. Galactosyltransferase (Fig. 3E), a marker enzyme characteristic of membranes of the Golgi apparatus in other cell types, is most enriched in the low-density microsomal m e m b r a n e fraction. Preliminary data indicate a similar d i s t r i b u t i o n for sialyltransferase a m o n g the various fractions, although the activity of this Golgi marker enzyme is relatively low and its distribution cannot, therefore, be determined with precision. Citrate synthase (Fig. 3F), the only mitochondrial marker enzyme activity examined, is most enriched in the m i t o c h o n d r i a l / n u c l e a r fraction and its distribution correlates well with the morphological data presented in Fig. 1. Tables II and III demonstrate the use of 4 × 4 matrix to calculate the relative organelle composi-

TABLE ll ORGANELLE COMPOSITION OF SUBCELLULAR MEMBRANE FRACTIONS FROM ISOLATED RAT ADIPOSE CELLS Values in Table A were calculated from the 5'-nucleotidase, glucose-6-phosphate phosphatase, galactosyltransferase and citrate synthase specific activity data in Fig. 3 assuming an indigenous mitochondrial 5'-nucleotidase activity of 17% of that found in plasma membranes. Values in Table B were calculated as for Table A assuming an additional indigenous high-density microsomal galactosyltransferase activity of 30% of that found in low-density microsomes. Similar values were obtained using each and every other possible combination of marker enzyme specific activities. Organelle

A. Plasma membranes Endoplasmic reticulum Golgi Mitochondria B. Plasma membranes Endoplasmic reticulum Golgi Mitochondria

Fraction Plasma membranes (%)

High-density microsomes (%)

Low-density microsomes (%)

Mitochondria/ nuclei (%)

54 10 22 14 54 15 18 14

13 28 48 11 13 40 36 11

3 14 79 4 3 21 73 4

4 4 6 86 4 6 4 86

399 T A B L E III O R G A N E L L E RECOVERY F R O M INITIAL H O M O G E N A T E OF ISOLATED RAT ADIPOSE CELLS Values were calculated from the protein recovery data in Table I, the organelle composition data in Table liB, and the homogenate 5'-nucleotidase, glucose-6-phosphate phosphatase, galactosyltransferase and citrate synthetase specific activity data in Fig. 3. While similar values were obtained using the rotenone-insensitive NADH-cytochrome c reductase specific activities, organelle recoveries could not be calculated using the adenylate cyclase specific activities because of their anomalously low levels in the homogenates. Fraction

Organelle Plasma membranes

Endoplasmic reticulum

Golgi (%)

Mitochondria (%)

(%)

(%)

Plasma membranes High-density microsomes Low-density microsomes Mitochondria/nuclei

45 4 1 7

19 23 13 19

13 12 27 6

5 2 1 67

Total

55

74

58

75

specific activities. However, while similar values for the relative organelle compositions (Table II) were obtained using each and every other possible combination of marker enzymes, the recoveries of organelle protein (Table III) could not be calculated using adenylate cyclase as the plasma membrane marker enzyme because of its anomalously low specific activity in the original homogenate. Table IIA illustrates that 54% of the protein in the plasma membrane fraction is plasma membrane protein, 79% of the protein in the low-density microsomal membrane fraction is Golgi membrane protein, and 86% of the protein in the mitochondrial/nuclear fraction is mitochondrial protein. Because of the second assumption used in this calculation, mitochondrial protein must in-

tion of each of the four subcellular fractions prepared here and the corresponding recovery of organelle protein in each fraction from the original homogenate, respectively. The calculation assumes only that (1) each marker enzyme activity is specific for one of the four major organelles considered (plasma membranes, endoplasmic reticulum, Golgi apparatus and mitochondria), and (2) the four fractions are comprised only of combinations of these four organelles. The i n d i g e n o u s mitochondrial 5'-nucleotidase specific activity was set at 17% of that in the plasma membrane fraction as discussed above. The values illustrated represent those calculated using a combination of the 5'-nucleotidase, glucose-6-phosphate phosphatase, galactosyltransferase and citrate synthase

T A B L E IV C H A R A C T E R I S T I C S OF SPECIFIC o - G L U C O S E - I N H I B I T A B L E C Y T O C H A L A S I N B B I N D I N G SITES IN S U B C E L L U L A R M E M B R A N E F R A C T I O N S F R O M ISOLATED R A T ADIPOSE CELLS Results are the means_+ S.E.M. of the individual values obtained in 10 separate experiments. R o, number of sites; K a, dissociation constant; B.D., below level of detection. Fraction

Binding site parameters Basal cells

Plasma membranes High-density microsomes Low-density microsomes Mitochondria/nuclei

Insulin-stimulated cells

Ro ( p m o l / m g protein)

Kd (nm)

R0 ( p m o l / m g protein)

Kd (nM)

7± 1 8 _+ 1 81 _+6 B.D.

98+19 75+10 141 + 20 B.D.

32+2 14_+1 32 ± 2 B.D.

98+ 8 133_+20 93 -+ 15 B.D.

400

clude nuclear and cell debris protein by definition. However, while the high-density microsomal membrane fraction is enriched in endoplasmic reticulure relative to the other three fractions, both the plasma and high-density microsomal membrane fractions appear to contain more Golgi membranes than endoplasmic reticulum, despite the nominally greater density of the latter. This apparent anomaly might be due either to a galactosyltransferase activity indigenous to, or to distinct membrane subspecies of the Golgi apparatus with sedimentation characteristics similar to those of, the endoplasmic reticulum a n d / o r plasma membranes. Indeed, Bretz et al. [24] have reported separation of morphologically and functionally distinct Golgi subfractions each containing galactosyltransferase activity. In addition, Anderson and Erikson [25] have recently reported a UDP-galactose: asialomucin galactosyltransferase activity indigenous to rat liver endoplasmic reticuhim. Table liB illustrates an empirical recalculation of the relative composition of each of the four subcellular fractions assuming an indigenous endoplasmic reticulum galactosyltransferase activity. The specific activity of this marker enzyme was set at 30% of that in the Gotgi membranes since approx. 30% of the total activity appeared to cofractionate with the endoplasmic reticulum marker enzymes when a further separation of the high- and low-density microsomal membranes was attempted (data not shown). This recalculation raises the endoplasmic reticulum content and lowers the Golgi membrane content of all four fractions, but especially those of the high-density microsomal membrane fraction. Table III illustrates the calculated percent recoveries of organelle protein in each subcellular fraction, as well as the calculated total percent recoveries of these four organelles, from the original homogenate (intact cell), based on the fractional composition data shown in Table lIB. For these calculations only data for 5'-nucleotidase activity could be used due to the anomalously low adenylate cyclase activity found in the homogenate. Among the four fractions, 55-75% of the total quantity of each organelle in the original homogenate is ultimately recovered. Qualitatively similar results are obtained using the fractional composition data shown in Table IIA (data not shown).

C3'tochalasin B binding to subcellular fractions The cytochalasin B binding assay for determining the number of glucose transporters present in each subcellular fraction is based on the measurement of only those sites to which the binding of cytochalasin B is specifically inhibited by D-glucose [2,3]. In practice, binding isotherms are obtained in the presence and absence of 500 mM D-glucose and plotted as described by Scatchard [26]. Subtraction of the curves obtained in the presence of glucose from the corresponding curves obtained in the absence of glucose along radial axes of constant free cytochalasin B concentration then gives rise to linear 'derived" Scatchard plots. The latter permit determination of both the number of specific D-glucose-inhibitable binding sites and their dissociation constant (Kd) for cytochalasin B. Cytochalasin E (2/~M) is included in the binding assay because it markedly reduces the level of binding to other specific sites, without affecting the binding characteristics of the D-glucose-inhibitable site [2]. Table IV illustrates that the number of specific D-glucose-inhibitable cytochalasin B binding sites/mg protein among the four subcellular fractions prepared here from basal cells is greatest in the low-density microsomal membrane fraction. This distribution is markedly altered, however, when these same four fractions are prepared from cells exposed to a maximally stimulating concentration of insulin (7 nM). Insulin induces a 5-fold increase in the concentration of binding sites in the plasma membrane fraction, a 2-fold increase in the high-density microsomal membrane fraction, and a 60% reduction in the concentration of binding sites in the low-density microsomal membrane fraction. These changes in distribution are accompanied by a 34% decrease in the apparent dissociation constant ( K d) for cytochalasin B binding of those sites remaining in the low-density microsomal membrane fraction and a 77% increase in the K d in the high-density microsomal membrane fraction. In contrast, no change is observed in the K d for cytochalasin B binding in the plasma membrane fraction. The insulin-induced changes in the K d for cytochalasin B binding reported in previous studies from this laboratory [3] were not statistically significant due to the relatively small number of samples examined.

401 TABLE V CALCULATED SUBCELLULAR DISTRIBUTION AND TOTAL NUMBER OF SPECIFIC D-GLUCOSE-INHIBITABLE CYTOCHALASINB (CB) BINDING SITES 1N THE INTACT ISOLATEDRAT ADIPOSECELL Values were calculated from the cytochalasin B binding data in Table IV, the protein recoverydata in Table l (5.106 cells/rat), and the organelle recoverydata in Table III. Cells

Membranes

CB binding in membranes (observed) (pmol/mg)

Recovered protein (observed) (pg/cell)

Recovery from homogenate (calculated) (%)

CB binding in intact cells (calculated) (pmol/cell)

Basal

Plasma membranes Low-densitymicrosomes Total

7 81

40.2 20.2

45 27

0.65 6.06 6.71

Insulin

Plasma membranes Low-densitymicrosomes Total

32 32

43.6 20.0

45 27

3.24 2.37 5.61

A comparison of the data in Fig. 3 and Table IV clearly demonstrates that the distribution of D-glucose-inhibitable cytochalasin B binding sites among the subcellular membrane fractions examined here does not correlate in either the basal or insulin-stimulated states with the distribution of any of the marker enzyme activities used in this study. However, by assuming that (1) all of the D-glucose-inhibitable cytochalasin B binding sites observed in the low-density microsomal membrane fraction come from the intracellular pool of sites and are recovered from the original homogenate in parallel with the recovery of the Golgi-specific galactosyltransferase activity in this fraction, and (2) all of the binding sites in the plasma membrane fraction are associated with plasma membranes and are recovered from the original homogenate in parallel with the recovery of plasma membranespecific marker enzyme activities, then the numbers of glucose transporters per cell in the plasma membrane and intracellular pool, and their totals in the basal and insulin-stimulated states can be calculated from the data in Tables III and IV. The results of such a calculation are illustrated in Table V. In the basal state, approx. 10% of the cell's glucose transporters appear to reside in the plasma membrane while the remaining 90% are localized to the cell's intracellular pool. In the insulinstimulated state, the number of glucose transporters in the cell's plasma membrane is increased to greater than 50% while the calculated total

number of glucose transporters itself is not significantly influenced.

D-Glucose transport in plasma membrane vesicles The electron micrographs shown in Fig. 1 clearly indicate the vesicular nature of the membranes obtained in the plasma and microsomal membrane fractions, and the lack of any discernible morphological differences between these fractions prepared from basal and insulin-stimulated cells. The data presented in Fig. 4 confirm the vesicular character of the membranes in the plasma membrane fraction by demonstrating the ability of these membranes to specifically transport D-glucose under conditions where the leakage of L-glucose, a non-transportable hexose, is relatively low. However, despite the vesicular nature of the membranes in the low-density microsomal membrane fraction, specific D-glucose transport is not measurable in this fraction because of small vesicle size and leakiness. Transport activity in the high-density microsomal fraction correlates well with the contamination of this fraction with plasma membranes (data not shown). Since glucose transport is a simple exponential function, the rate of glucose transport can be calculated from the initial slope of the glucose uptake curve. For the plasma membrane fractions from basal and insulin-stimulated cells illustrated in Fig. 4, these rates are 1.8 and 10.8 n m o l / m i n per mg membrane protein, respectively, in the

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Fig. 4. Time course of D-glucose uptake into plasma membrane vesicles prepared from basal and insulin-treated isolated rat adipose cells. Experiments were performed at 22°C at an initial D-[UJ4C]glucose concentration of 0.45 mM. The data are plotted after correction for trapping and vesicle leakage (see Materials and Methods) using the non-transported sugar l,-[l3H]glucose. Transport rates are 1.8 and 10.8 nmol/min per mg membrane protein for the plasma membranes from basal and insulin-stimulated cells, respectively.

presence of 0.45 m M D-glucose. Th e rate in the m e m b r a n e s from the insulin-stimulated cells represents a 6-fold increase over that in the m e m b r a n e s from the basal cells, c o r r e s p o n d i n g closely to the o b s e r v e d increase in the n u m b e r of cytochalasin B b i n d i n g sites (Table IV). By assuming that each glucose transporter contains one cytochalasin B b i n d i n g site as has been d e m o n s t r a t e d for the h u m a n erythrocyte [22], the data presented in Tab l e V can be used to calculate the t u r n o v e r n u m b e r of the glucose transporters in b o t h p l a s m a m e m b r a n e vesicles and intact cells. Since glucose transport in the plasma m e m b r a n e vesicles is m e a s u r e d here at 22°C using glucose as the substrate at a c o n c e n t r a t i o n of 0.45 mM, whereas glucose transport activity in the intact cells is routinely measured at 37°C using 3 - 0 m et h y l g l u co se as the substrate at a c o n c e n t r a t i o n of 0.1 raM, calculation of the respective turnover n u m b e r s requires two additional factors as follows: (1) the M i c h a e l i s - M e n t e n constants (K,, 1 values) for glucose and 3-O-methylglucose transport have been d e t e r m i n e d in several laboratories to be 10 and 5 mM, respectively, and (2) activation energy d e t e r m i n a t i o n s by Ludvigsen and Jarett [27] have d e m o n s t r a t e d that the rates of D-glucose transport in p l a s m a m e m b r a n e vesicles from both

TABLE VI TURNOVER NUMBER OF GLUCOSE TRANSPORTERS IN INTACT ISOLATED RAT ADIPOSE CELLS AND PLASMA MEMBRANES VESICLES 3-O-MG, 3-O-methylglucose; PM, plasma membranes. V,.... values were calculated assuming K,1 values for 3-O-methylglucose and glucose transport of 5 mM and 10 mM, respectively. Intact cells Cells

Basal Insulin

Transport rate (37°C, 0.1 mM 3-O-MG) (fmol/cell per min) 0.2 3.6

Transport V,,,x (37°C, 3-O-MG) (fmol/cell per min) 10.2 183.6

Cytochalasin B binding in PM a (amol/cell) 0.65 3.24

Turnover number (mol/min per site) 15.6.103 56.1.10 3

Plasma membranes Cells

Transport rate (22°C, 0.45 mM glucose) (mol/min per mg)

Transport Vm,~ h (37°C, glucose) (nmol/min per mg)

Cytochalasin B binding (pmol/mg)

Turnover number (mol/min per site)

Basal Insulin

1.8 10.8

83.6 501.6

7 32

11.9.10 3 15.7.103

" Data from Table V. b Calculated at 37°C based on activation energy determined in plasma membranes by Ludvigsen and Jarett [27].

403 basal and insulin-stimulated cells at 37°C are 2fold greater than those measured at 22°C. Similar temperature effects on 3-O-methylglucose transport in intact cells have been observed by Vinten et al. [1] and in this laboratory (unpublished data). The results of these calculations are illustrated in Table VI. In the plasma membrane vesicles, the turnover number of the glucose transporter in the insulin-stimulated state is only about 31% greater than that observed in the basal state. In the intact cells, on the other hand, while the turnover number of the glucose transporter in the basal state is similar to that observed in the plasma membrane vesicles from both basal and insulin-stimulated cells, that in the insulin-stimulated state is increased roughly 4-fold. Discussion

Electron microscopy and SDS-polyacrylamide gel electrophoresis (Figs. 1 and 2, respectively) clearly indicate the distinct nature of the four subcellular fractions prepared here from the isolated rat adipose cell with respect especially to their content of mitochondria, the size of their constituent membrane vesicles and their protein composition. None of these characteristics is detectably influenced by exposure of the intact cell to insulin. The marker enzyme composition of each fraction (Fig. 3) reveals, however, that all four fractions contain at least some of each of the various subcellular organelles identified. In order to measure the extent of cross-contamination and the yield of each membrane species, two marker enzymes have been used wherever possible for each organelle. This approach is particularly appropriate with regard to the plasma membrane markers, 5'-nucleotidase and isoproterenol-stimulated adenylate cyclase, since significant discrepancies are observed when their distributions are compared. This comparison and a similar comparison between the distributions of glucose-6phosphate phosphatase and rotenone-insensitive NADH-cytochrome c reductase, both marker enzymes of the endoplasmic reticulum, also reveal the importance of including nonspecific phosphatase inhibitors in enzyme assays designed to measure specific phosphatase activities. A quantitative comparison between the distributions of

galactosyltransferase and sialyltransferase, both marker enzymes of the Golgi apparatus, has not been possible because of the latter's low activity in the rat adipose cell. The membrane compositions of the four subcellular fractions prepared here have been computed using these marker enzyme activities (Table II) and compared with the distribution of glucose transporters over these same four fractions (Table IV). Among the subcellular fractions prepared from basal cells, glucose transporters are localized primarily to a membrane species which is selectively enriched in the low-density microsomal membrane fraction. This same fraction is also enriched in galactosyltransferase activity, probably representing one or more of the several components of the Golgi apparatus, particularly after a portion of this activity is attributed to an enzyme indigenous to the endoplasmic reticulum or to a membrane subspecies of the Golgi apparatus which cofractionates with the endoplasmic reticulum in the high-density microsomal membrane fraction. Nevertheless, the distribution of glucose transporters clearly does not parallel the distribution of this marker enzyme activity over all fractions, even after making such an adjustment for the distribution of galactosyltransferase. Furthermore, if an assumption were made that intracellular glucose transporters are recovered in parallel with the galactosyltransferase activity, then a recovery of 22 and 49 pmol glucose transporters/mg membrane protein would be expected in the plasma and high-density microsomal membrane fractions, respectively, from basal cells. These calculated values markedly contrast with the actual measured recoveries of 7 and 7 p m o l / m g membrane protein, respectively (Table IV). Thus, the membranes containing the intracellular glucose transporters appear to represent either a highly specialized subfraction of the Golgi apparatus or a unique membrane species. A similar conclusion was drawn by Kono et al. [6,7] from studies in which comparable membrane fractions were prepared on sucrose density gradients. The calculated cross-contamination and marker enzyme specific activities can be used, however, to estimate a corrected concentration of glucose transporter systems in the plasma membranes. On this basis, the plasma membrane fraction prepared

404 using the present procedure comprises approx. 54% plasma membranes, representing 43% of the plasma membranes present in the original homogenate, regardless of the basal or insulin-stimulated state of the intact cells prior to homogenization. Purified plasma membranes thus contain approx. 13 and 59 pmol glucose t r a n s p o r t e r s / m g membrane protein when prepared from basal and insulinstimulated cells, respectively. The glucose transporters observed in the highdensity microsomal membrane fraction, the fraction enriched in marker enzyme activities of the endoplasmic reticulum, most likely represent contamination of this fraction with both plasma membranes and the specialized membrane species containing the intracellular pool. Plasma membranes contaminate this fraction to the extent of approx. 13% and thus account for roughly 1 2 pmol glucose t r a n s p o r t e r s / m g of high-density microsomal membrane protein. The remaining 5 - 6 p m o l / m g membrane protein appear to represent a roughly 5% contamination with membranes from the intracellular pool. Using these estimated levels of contamination (13 and 5%, respectively), a value of 10 p m o l / m g of high-density microsomal membrane protein would be expected in this fraction prepared from insulin-stimulated cells, in close agreement with the 14 p m o l / m g of membrane protein actually observed. Exposure of intact adipose cells to insulin does not influence the K a of the glucose transporters in the plasma membrane fraction for cytochalasin B binding. The K d of the glucose transporters remaining in the low-density microsomal membrane fraction, on the other hand, is significantly less than that of the glucose transporters in this fraction in the basal state. While the differences in K d between the intracellular pool and plasma membranes may simply reflect differences in the lipid a n d / o r protein environment in the two membrane species, the changes observed within the intracellular pool suggest the existence of two classes of glucose transporters, only one of which undergoes translocation to the plasma membrane in response to insulin. If this were the case, then the K d observed in the low-density microsomal membrane fraction in the basal state represents a composite of two K d values, and those glucose transporters which are translocated in response to insulin must

be characterized by a still higher K~ for cytochalasin B binding. In either case, these alterations in K~ suggest that intracellular glucose transporters undergo some as yet unidentified conformational change during their translocation to the plasma membrane in response to insulin. Nevertheless, in studies with streptozotocin-diabetic rats [28] and rats fed diets containing different ratios of fat to carbohydrate [29], the reduction in the concentration of intracellular glucose transporters induced by insulin remains proportional to that observed in this study despite large changes in the total concentration of glucose transporters. A comparison of the number of glucose transporters in the plasma membrane ~f the adipose cell with the glucose transport activity actually observed in the intact cell, and an examination of the stoichiometry of the translocation process require an estimate of the recovery of plasma membrane and intracellular glucose transporters from the original homogenates. Because of the hydrophobic nature of the ligand and the large lipid content of the adipose cell however, the number of glucose transporters in the homogenate cannot be directly measured using the equilibrium cytochalasin B binding assay [30]. An indirect method must, therefore, be employed based upon the recoveries of marker enzyme activities. The number of glucose transporters in the plasma membrane of the intact cell is readily obtained from the recovery of plasma membrane marker enzyme activities. That in the intracellular pool, however, can only be obtained by assuming a recovery comparable to that of the galactosyltransferase activity specific to the subfraction of the Golgi apparatus enriched in the low-density microsomal membrane fraction. Thus, estimates of 6.71 and 5.61 pmol glucose transporters/cell are calculated for the basal and insulin-stimulated states, respectively. These values are not significantly different ( P < 0.05). Literature values for insulin's stimulatory effect on 3-O-methylglucose transport in intact adipose cells are generally on the order of 10 20-fold, whereas the degree of stimulation observed when glucose transport itself is measured in the isolated plasma membrane fractions is only 4 6-fold in this study (Table VI), or 2 3-fold as determined by Ludvigsen and Jarett [27]. Similarly, when glucose transport is measured following reconstitu-

405 tion of the plasma membrane fractions into artificial liposomes, values of 2-3- [5,6] and 5-8-fold [7,31] are observed. Thus, in no case does the stimulatory effect of insulin on transport activity measured following cell fractionation approach the magnitude of that observed directly in the intact cell. This disparity may reflect modification of the glucose transporter during the preparation of the membranes. To assess this possibility, the apparent turnover number for the glucose transporter under these differing conditions has been calculated. The turnover numbers of the glucose transporters in the plasma membrane fraction (Table VI) prepared from basal and insulin-stimulated cells differ by only 31%. This failure to observe all but a small difference in turnover numbers is consistent with a variety of evidence suggesting the similarity of the glucose transporters in the plasma membrane in the basal and insulin-stimulated states: (1) the comparable K d values for cytochalasin B binding reported here and K~ values for the inhibition of cytochalasin B binding by glucose reported elsewhere [9,30]; (2) the similar K m values for 3-O-methylglucose transport [32] and K. values for the inhibition of 3-O-methylglucose transport by glucose [33] in intact cells; (3) the comparable K.,, values for, and temperature-dependence of, glucose transport in plasma membrane vesicles and liposomes reconstituted from plasma membrane preparations [6,27]; and (4) the virtually identical pH dependencies of glucose transport in plasma membrane vesicles [27]. The slightly lower turnover number observed in the basal, compared to the insulin-stimulated, state may actually represent a small degree (1.7%) of cross-contamination of the plasma membrane fraction with glucose transporters from the intracellular pool. The latter are incapable of net glucose transport and would, therefore, increase the apparent number of glucose transporters in the plasma membrane fraction in the absence of a proportional increase in glucose transport. In the intact adipose cell, the turnover number calculated in the basal state is similar to the turnover number observed in the plasma membrane vesicles from both basal and insulin-stimulated cells, while that in the insulin-stimulated state is roughly 4-fold higher (Table VI). When the

31% correction described above for the plasma membrane vesicles is applied to the basal intact cells, the calculated turnover number is increased from 15.6.103 to 20.5.103 m o l / m i n per site. However, this corrected value is still significantly lower than the 56.1-103 m o l / m i n per site observed in the insulin-stimulated cells. Several potential explanations exist for this apparent discrepancy. (1) Glucose transport in the intact cell may be regulated by modulation of both the number of glucose transporters in the plasma membrane and the glucose transporter's intrinsic activity. While the former is retained during cell disruption and membrane preparation, the latter may be lost or modified. Preliminary evidence for a direct effect of glucose-6-phosphate [34] and a cAMPmediated effect of isoproterenol [35] on glucose transporter intrinsic activity has recently been presented. (2) The activity of the glucose transporters associated with the isolated plasma membranes may not be fully expressed in the intact cell giving rise, especially in the basal state, to lower calculated turnover numbers. In the model proposed by Karnieli et al. [4], the existence of non-functional glucose transporters in the plasma membrane was suggested by the delay between translocation and the onset of glucose transport activity during the time course of the response to insulin. More recently, incubation of cells either with Tris [36] or at reduced temperatures [37] has been shown to induce the translocation of glucose transporters in the basal state in the absence of a corresponding increase in glucose transport activity in the intact cell. (3) Finally, the number of glucose transporters in basal membranes may be increased as a result of mechanical stimulation of the cells during membrane preparation. Such mechanical agitation has previously been reported to substantially increase glucose transport activity in the intact cell [38]. Further studies will be necessary to distinguish among these various possibilities and ultimately define the specific site, or sites, of insulin action. Czech and coworkers [31,39] have suggested that insulin stimulates glucose transport in the isolated rat adipose cell through the activation of glucose transporters already present in the plasma membrane. In one report, they demonstrated an activating effect of fluidizing agents such as cis-

406

vaccenic acid on the transport activity of plasma membranes from basal cells in the absence of an effect of these agents on the transport activity of plasma membranes from insulin-stimulated cells [39]. They suggested, therefore, that insulin might exert its stimulatory effect by enhancing membrane fluidity. In another report, these investigators observed that the glucose transport activity reconstituted from a total microsomal membane preparation comprising both plasma membranes and the intracellular pool from insulin-stimulated cells was 2-fold greater than that reconstituted from the total microsomal membrane preparation from basal cells. In separate experiments, insulin did not influence the transport activity reconstituted from a membrane fraction reported to represent the intracellular pool, but increased that reconstituted from the plasma membrane fraction by roughly 5-fold. The following observations suggest that the disparity between these data and those presented here may very well reflect marked differences in the methods employed. First, the glucose transport activities observed by Pilch et al. [39] in plasma membrane vesicles from basal and insulin-stimulated cells are substantially lower than those observed either by Ludvigsen and Jarett [27] or reported here. Furthermore, in striking contrast to Carter-Su et al. [31], Kono and colleagues [5-7] consistently report a reduction in glucose transport activity reconstituted from the intracellular pool of insulin-stimulated cells similar in magnitude to that reported here despite parallel increases in the plasma membranes. Thus, glucose transporters associated with the plasma membranes and intracellular pool may not reconstitute with equal efficiencies. In addition, glucose transport activity reconstituted from a total microsomal membrane preparation is highly dependent on the relative recoveries of plasma membrane and intracellular glucose transporters from the original homogenate. While the centrifugation procedure used by Carter-Su et al. [31] would appear to assure complete recovery of the plasma membrane glucose transporters (10 and 50% of the total in the basal and insulin-stimulated states, respectively), a low recovery of the intracellular glucose transporters would produce a much greater loss of glucose transporters from basal than from insulin-

stimulated cells, and an apparent activation of glucose transport activity by insulin in the total microsomal membrane preparation. Recent preliminary data from this laboratory indicate that the yield of intracellular glucose transporters can be increased relative to that reported here by collecting the low-density microsomal membrane fraction at 365 000 × gmax (unpublished data). Finally, Czech and coworkers themselves [40] have recently confirmed the present results both qualitatively and quantitatively using a subcellular fractionation procedure similar to that described here and a new technique for photoaffinity labeling the glucose transporter with cytochalasin B. The evidence presented here supports the hypothesis that insulin stimulates glucose transport in the isolated rat adipose cell by a rapid, reversible, and energy-dependent process which involves the translocation of membrane vesicles containing the glucose transporters from an intracellular site to the plasma membrane. Additional evidence from this laboratory further indicates that various states of insulin resistance in the rat including streptozotocin-induced diabetes [28], obesity [41] and high f a t / l o w carbohydrate feeding [29], are accompanied by a depletion of intracellular glucose transporters and a consequent impairment in this translocation process, apparently giving rise to some of the so-called 'post-receptor' defects in insulinstimulated glucose metabolism. The mechanisms regulating the level of intracellular glucose transporters and the factors determining their distribution between the plasma membrane and intracellular pool are presently under active investigation with a view to understanding this translocation process and its perturbation in these pathophysiological states.

Acknowledgments The authors wish to thank Mary Jane Zarnowski and Stephen Richards for their expert technical assistance, and Joy Glotfelty for typing the manuscript. We would also like to thank Dr. Donald Keefer and the University of Virginia Diabetes Research and Training EM Facility (P60AM-2-2125) for performing the electron microscopy. E.K. was a recipient of a Fogarty International Fellowship.

407

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