Title: Role of Semicarbazide-sensitive Amine Oxidase on Glucose Transport and GLUT4 Recruitment to the Cell Surface in Adipose Cells
Abstract: The previous characterization of an abundant population of non-adrenergic imidazoline-I2binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle.Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells. The previous characterization of an abundant population of non-adrenergic imidazoline-I2binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle. Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells. Insulin stimulates glucose transport in adipose tissue and cardiac and skeletal muscle by promoting glucose transporter translocation from an intracellular locus to the cell surface (1Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (654) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (955) Google Scholar, 3Kandror K.V. Pilch P.F. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar). Muscle and fat express two isoforms of glucose transporters named GLUT4 and GLUT1. The latter is found mainly in the plasma membrane but also in the interior of the cell, and insulin causes its redistribution to the plasma membrane in adipocytes and cardiac myocytes (1Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (654) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (955) Google Scholar, 3Kandror K.V. Pilch P.F. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar, 4Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G. Kozka I.J. Palacı́n M. Testar X. Kammermeier H. Zorzano A. J. Biol. Chem. 1997; 272: 7085-7092Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In contrast, GLUT4 is excluded from the plasma membrane and instead localizes to an intracellular storage pool in the basal state, and insulin causes a recruitment of GLUT4-containing vesicles to the cell surface (1Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (654) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (955) Google Scholar, 3Kandror K.V. Pilch P.F. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar, 4Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G. Kozka I.J. Palacı́n M. Testar X. Kammermeier H. Zorzano A. J. Biol. Chem. 1997; 272: 7085-7092Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Since the translocation of GLUT4 is greater than GLUT1, GLUT4 accounts for most of the insulin-stimulated glucose transport in adipose and muscle cells (1Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (654) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (955) Google Scholar, 3Kandror K.V. Pilch P.F. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar, 4Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G. Kozka I.J. Palacı́n M. Testar X. Kammermeier H. Zorzano A. J. Biol. Chem. 1997; 272: 7085-7092Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In the absence of insulin, GLUT4 is concentrated in membrane vesicles, which can be separated from cellular microsomes by velocity gradient centrifugation (5James D.E. Lederman L. Pilch P.F. J. Biol. Chem. 1987; 262: 11817-11824Abstract Full Text PDF PubMed Google Scholar). To delineate the intracellular trafficking pathway of GLUT4, it is important to identify the proteins that colocalize with GLUT4 in the same vesicles. The mannose 6-phosphate/insulin-like growth factor II receptor and secretory carrier membrane proteins (SCAMPs) 1The abbreviations used are: SCAMP, secretory carrier membrane proteins; SSAO, semicarbazide-sensitive amine oxidase; MAO, monoamine oxidase. have been detected in GLUT4 vesicles obtained from rat adipocytes, cardiomyocytes, and skeletal muscle (4Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G. Kozka I.J. Palacı́n M. Testar X. Kammermeier H. Zorzano A. J. Biol. 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Ther. 1995; 272: 681-688PubMed Google Scholar, 29Tesson F. Limon-Boulez I. Urban P. Puype M. Vandekerckhove J. Coupry I. Pompon D. Parini A. J. Biol. Chem. 1995; 270: 9856-9861Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 30Parini A. Gargalidis-Moudanos C. Pizzinat N. Lanier S.M. Trends Pharmacol. Sci. 1996; 17: 13-16Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 31Ozaita A. Olmos G. Boronat M.A. Lizcano J.M. Unzeta M. Garcı́a-Sevilla J.A. Br. J. Pharmacol. 1997; 121: 901-912Crossref PubMed Scopus (82) Google Scholar). These results together with the observation that GLUT4 vesicles contain several proteins as yet unidentified (3Kandror K.V. Pilch P.F. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar, 9Sevilla L. Tomàs E. Muñoz P. Gumà A. Fischer Y. Thomas J. Ruiz-Montasell B. Testar X. Palacı́n M. Blasi J. Zorzano A. Endocrinology. 1997; 138: 3006-3015Crossref PubMed Google Scholar) compelled us to characterize the amine oxidases present in adipocytes. As a result, we have identified a semicarbazide-sensitive amine oxidase (SSAO, E.C. 1.4.3.6) as another protein found in GLUT4 vesicles from rat adipocytes, 3T3-L1 adipocytes, and rat skeletal muscle, and we demonstrate a stimulatory role of SSAO on glucose transport and GLUT4 translocation to the cell surface in adipose cells. The identification of SSAO as a protein found in GLUT4 vesicles is in keeping with a recent report by Morris et al. (32Morris N.J. Ducret A. Aebersold R. Ross S.A. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 9388-9392Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) 125I-Protein A was purchased from ICN (Irvine, CA). ECL and [14C]benzylamine (59 Ci/mmol) and [3H]idazoxan were from Amersham Pharmacia Biotech. [14C]Tyramine (45 Ci/mmol) and 2-d-[1,2-3H]deoxyglucose (26Ci/mmol) came from NEN Life Science Products. Immobilon polyvinylidene difluoride was obtained from Millipore (Bedford, MA). γ-globulin, goat anti-mouse IgG, semicarbazide hydrochloride, pargyline, clorgyline, tyramine, benzylamine, and most commonly used chemicals were from Sigma. Purified porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN). All electrophoresis reagents and molecular weight markers were obtained from Bio-Rad. Anti-GLUT4 antibody (OSCRX) was produced from rabbit after immunization with a peptide corresponding to the last 15 amino acids of the carboxyl terminus (33Gumà A. Mora C. Santalucı́a T. Viñals F. Testar X. Palacı́n M. Zorzano A. FEBS Lett. 1992; 310: 51-54Crossref PubMed Scopus (23) Google Scholar). Anti-SSAO antibody was produced from rabbit after immunization with the membrane-associated SSAO purified from bovine lung. Rabbit polyclonal antibodies against rat β1-integrin were kindly given by Dr. C. Enrich (Universitat de Barcelona). Adipocytes were isolated from the epididymal fat pads of male Wistar rats (180–220 g) by digestion in Krebs-Ringer buffer containing 15 mm sodium bicarbonate, 10 mm Hepes, 2 mm sodium pyruvate, bovine serum albumin (3.5% w/v), and 1.5 mg/ml collagenase. After digestion for 35–45 min at 37 °C under shaking, isolated fat cells were filtered and washed three times in the same buffer without collagenase (KRBH buffer). After a preincubation period of 45 min at 37 °C, each vial containing 400 μl of cell suspension in KRBH containing the tested drugs (added in 4 μl of suitable dilutions to obtain the final concentrations) received an isotopic dilution of 2-deoxy-d-[3H]glucose, giving a final concentration of 0.1 mm equivalent to approximately 1,300,000 dpm/vial. Assays were further incubated for 10 min and then stopped with 100 μl of 100 μm cytochalasin B. 200-μl aliquots of the cell suspension were centrifuged as described by Olefsky (34Olefsky J.M. Biochem. J. 1978; 172: 137-145Crossref PubMed Scopus (171) Google Scholar) in microtubes containing dinonyl phthalate. After centrifugation, the cells (upper part of the tubes) were placed in scintillation vials, and the incorporated radioactivity was counted. The extracellular 2-deoxyglucose present in the cell fraction was determined with adipocytes previously stopped with cytochalasin B; it did not exceed 1% of the maximum 2-deoxyglucose transport in the presence of insulin, as previously reported (35Carpéné C. Chalaux E. Lizarbe M. Estrada A. Mora C. Palacı́n M. Zorzano A. Lafontan M. Testar X. Biochem. J. 1993; 296: 99-105Crossref PubMed Scopus (32) Google Scholar). Fat cell suspensions were incubated in KRBH buffer in the absence or presence of 100 nm insulin for 30 min. Cells were homogenized with a Potter-Elvejheim Teflon pestle, and subcellular membrane fractions were prepared as described previously (36Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar). 3T3-L1 fibroblasts obtained from the American Type Culture Collection (Rockville, MD) were cultured in Dulbecco's modified Eagle's medium containing high glucose and l-glutamine and supplemented with 10% calf serum. Cells were maintained and passaged as preconfluent cultures at 37 °C in a 5% CO2-humidified incubator. Two days postconfluence (day 0), differentiation was induced with methylisobutylxanthine (0.5 mm), dexamethasone (0.25 μm), and insulin (5 μg/ml) in Dulbecco's modified Eagle's medium containing high glucose, l-glutamine, and 10% fetal bovine serum. After 2 days, the methylisobutylxanthine and dexamethasone were removed, and insulin was maintained for 2 additional days. On day 4, and thereafter, Dulbecco's modified Eagle's medium and 10% fetal bovine serum was replaced every 2 days. Before each experiment, cell monolayers were incubated in serum-free Dulbecco's modified Eagle's medium for 2 h. Cells were used for experimentation between days 8 and 14. Subcellular fractionation of 3T3-L1 membranes was performed as in rat adipocytes. Microsomes from rat skeletal muscle were prepared as described (37Muñoz P. Rosemblatt M.R. Testar X. Palacı́n M. Zorzano A. Biochem. J. 1995; 307: 273-280Crossref PubMed Scopus (33) Google Scholar). Crude extracts were prepared from white adipocytes or from rat liver homogenates as previously reported (25Carpéné C. Galitzky J. Larrouy D. Langin D. Lafontan M. Biochem. Pharmacol. 1990; 40: 437-445Crossref PubMed Scopus (20) Google Scholar, 28Carpéné C. Collon P. Remaury A. Cordi A. Hudson A. Nutt D. Lafontan M. J. Pharmacol. Exp. Ther. 1995; 272: 681-688PubMed Google Scholar) and assayed either for amine oxidase activity or [3H]idazoxan binding as described (28Carpéné C. Collon P. Remaury A. Cordi A. Hudson A. Nutt D. Lafontan M. J. Pharmacol. Exp. Ther. 1995; 272: 681-688PubMed Google Scholar). Protein A-purified 1F8 antibody was coupled to acrylamide beads (Reacti-gel GF 2000; Pierce) at a concentration of 1 mg of antibody/ml of resin according to the manufacturer's instructions. Before use, the beads were washed with phosphate-buffered saline (134 mm NaCl, 2.6 mmKCl, 6.4 mm Na2HPO4, 1.46 mm KH2PO4, pH 7.4) for at least 30 min at room temperature. Intracellular membranes were incubated with beads overnight at 4 °C (50 μg of membranes and 20 μl of beads). The beads were spun down, the supernatant was taken for later analysis, and the beads were washed five times in phosphate-buffered saline. The adsorbed material was used directly to determine amine oxidase activity; in some experiments, the adsorbed material was eluted with electrophoresis sample buffer (0.1 m Tris-HCl, 20% glycerol, 2% SDS, 5% β-mercaptoethanol, pH 6.8), incubated for 5 min at 95 °C, cooled, and microcentrifuged before subjecting the extracts to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed on membrane proteins following Laemmli (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Proteins were transferred to Immobilon in buffer consisting of 20% methanol, 200 mm glycine, 25 mm Tris, pH 8.3. After transfer, the filters were blocked with 4% fish gelatin, 0.02% sodium azide in phosphate-buffered saline for 1 h at 37 °C, and then incubated with antibodies in 1% nonfat dry milk, 0.02% sodium azide in phosphate-buffered saline. Transfer was confirmed by Coomassie Blue staining of the gel after the electroblot. Detection of the immune complex with the rabbit polyclonal antibodies was accomplished using 125I-protein A for 4 h at room temperature or using the ECL Western blot detection system (Amersham). The autoradiograms were quantified using scanning densitometry. Immunoblots were performed under conditions in which autoradiographic detection was in the linear response range. Amine oxidase activity was determined radiochemically following the procedure by Fowler and Tipton (39Fowler C.J. Tipton K.F. Biochem. Pharmacol. 1981; 30: 3329-3332Crossref PubMed Scopus (128) Google Scholar). The reaction was performed in 200 μl of 0.2 m phosphate buffer at 37 °C in the presence of radioactive benzylamine (50 or 100 μm; 50 mCi/mmol) or tyramine (50 or 100 μm; 50 mCi/mmol) at pH 7.4 for 60 min unless otherwise stated. Reactions were carried out at 37 °C in a final volume of 225 μl of 50 mmpotassium phosphate buffer, pH 7.2. Reactions were stopped by the addition of 50 μl of 4n HCl, and the products were extracted into toluene/ethyl acetate 1:1 (v/v) containing 0.6% (w/v) 2,5-diphenyloxazole before liquid scintillation counting. Blank values were measured in assays stopped immediately after the addition of cellular extracts and represented less than 1% of total radioactivity added in each incubation mixture. Time course assays were performed to ensure that initial rates of the reaction were precisely determined; proportionality to enzyme concentration was established in each case. Protein concentrations were determined by the Bradford method (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) with γ-globulin as a standard. Preliminary studies indicated the existence in crude membranes from rat adipocytes of substantial levels of [3H]idazoxan binding to imidazoline-I2 sites, characterized by an apparent dissociation constant (Kd) of 12 ± 2 nm and a maximal binding capacity (Bmax) of 390 ± 112 fmol/mg of protein (n = 7). This is in agreement with previous reports showing a large number of imidazoline-I2binding sites in membranes from white adipocytes (25Carpéné C. Galitzky J. Larrouy D. Langin D. Lafontan M. Biochem. Pharmacol. 1990; 40: 437-445Crossref PubMed Scopus (20) Google Scholar, 26MacKinnon A.C. Brown C.M. Spedding M. Kilpatrick A.T. Br. J. Pharmacol. 1989; 98: 1143-1150Crossref PubMed Scopus (27) Google Scholar, 27Langin D. Paris H. Lafontan M. Mol. Pharmacol. 1990; 37: 876-885PubMed Google Scholar). Because amine oxidases have been reported to display imidazoline-I2binding sites (28Carpéné C. Collon P. Remaury A. Cordi A. Hudson A. Nutt D. Lafontan M. J. Pharmacol. Exp. Ther. 1995; 272: 681-688PubMed Google Scholar, 29Tesson F. Limon-Boulez I. Urban P. Puype M. Vandekerckhove J. Coupry I. Pompon D. Parini A. J. Biol. Chem. 1995; 270: 9856-9861Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 30Parini A. Gargalidis-Moudanos C. Pizzinat N. Lanier S.M. Trends Pharmacol. Sci. 1996; 17: 13-16Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 31Ozaita A. Olmos G. Boronat M.A. Lizcano J.M. Unzeta M. Garcı́a-Sevilla J.A. Br. J. Pharmacol. 1997; 121: 901-912Crossref PubMed Scopus (82) Google Scholar), we initially determined the type of amine oxidase present in crude extracts from rat adipocytes, and this was compared with crude membrane preparations derived from rat liver. This was done by assaying the oxidation of tyramine, a substrate than can be oxidized by monoamine oxidases A and B (41Yu P.H. Boulton A.A. Baker G.B. Yu P.H. Neuromethods: Five. Series I, Neurochemistry. Neurotransmitter Enzymes. V. Humana Press Inc., Totowa, NJ1986: 235-272Google Scholar) and by SSAO in the absence or presence of pargyline (a preferential irreversible inhibitor of MAOs) or semicarbazide (an inhibitor of SSAO). Results shown in Fig.1 clearly indicate that liver extracts express MAO activity since all tyramine oxidation is blocked by pargyline and does not express SSAO activity since no inhibition occurs in the presence of semicarbazide. On the other hand, adipocyte extracts contain both MAO and SSAO; complete inhibition of tyramine oxidation is only obtained by the addition of semicarbazide and pargyline (Fig. 1). Previous studies have also reported the activity of SSAO in crude extracts from adipose tissue (42Barrand M.A. Callingham B.A. Biochem. Pharmacol. 1982; 31: 2177-2184Crossref PubMed Scopus (70) Google Scholar, 43Raimondi L. Pirisino R. Ignesti G. Capecchi S. Banchelli G. Buffoni F. Biochem. Pharmacol. 1991; 41: 467-470Crossref PubMed Scopus (55) Google Scholar). Further studies were performed comparing the oxidation of tyramine to that of benzylamine (a preferential substrate of SSAO) but also oxidized by MAO-B (42Barrand M.A. Callingham B.A. Biochem. Pharmacol. 1982; 31: 2177-2184Crossref PubMed Scopus (70) Google Scholar) in light microsomes from isolated rat adipocytes; they indicated the presence of a high SSAO activity in light microsomes. This was substantiated by (a) tyramine oxidative activity largely inhibited (89% decrease) by 10−4m semicarbazide (Fig.2 A) and (b) benzylamine oxidative activity totally blocked by semicarbazide (Fig.2 B). In contrast, the tyramine oxidation measured in mitochondrial fractions from rat adipocytes was barely inhibited by semicarbazide (less than 9% inhibition at 10−3m inhibitor) but was totally blocked by the MAO inhibitor clorgyline (90% inhibition at 10−4m) (Fig.2 C). Of note, clorgyline blocked tyramine oxidation by only 10%, and it did not alter benzylamine oxidation in light microsomes from rat adipocytes (Fig. 2 A). This indicates that most of benzylamine and tyramine oxidation by light microsomes is not due to MAO activity. Furthermore, light microsomes from rat adipocytes contain SSAO, which can be assayed by using benzylamine as a specific substrate or by evaluating the fraction of tyramine oxidation that is semicarbazide-sensitive. Substantial semicarbazide-sensitive amine oxidase activity was also detected in light microsomes obtained from 3T3-L1 adipocytes or from intracellular membranes obtained from rat skeletal muscle (data not shown). Subcellular fractionation of membranes from isolated rat adipocytes revealed high semicarbazide-sensitive amine oxidase activity, assayed as the oxidation of benzylamine or the oxidation of tyramine sensitive to semicarbazide, in plasma membrane preparations (Fig. 3 A and data not shown). SSAO activity in plasma membrane was near 4-fold greater than in light microsomes (Fig. 3 A). Low monoamine oxidase activity was detected both in plasma membrane and in light microsomes (data not shown). The utilization of a polyclonal antibody raised against SSAO purified from bovine lung microsomes detected a single band in adipocyte membranes showing an electrophoretic mobility similar to that observed in bovine lung microsomes (Fig. 3 B). Analysis of immunoreactive SSAO protein indicated that it was more abundant in plasma membrane than in light microsomes (Fig.3 B). Incubation of adipocytes for 30 min in the presence of 100 nm insulin did not alter SSAO activity in light microsomes or in plasma membrane preparations (Fig. 3 A). No alteration in the abundance of immunoreactive SSAO protein was detected after incubation with insulin in plasma membranes or in light microsomes (Fig. 3 B). Based on the presence of SSAO activity in light microsomes from adipose cells and skeletal muscle, we next examined whether semicarbazide-sensitive amine oxidase activity colocalized with intracellular GLUT4-containing vesicles. In a first step, we fractionated intracellular membranes obtained from rat skeletal muscle in sucrose gradient. These membranes are highly enriched in intracellular markers such as GLUT4, SCAMPs, cellubrevin, or vesicle-associated membrane protein 2 and are free from plasma membrane markers (9Sevilla L. Tomàs E. Muñoz P. Gumà A. Fischer Y. Thomas J. Ruiz-Montasell B. Testar X. Palacı́n M. Blasi J. Zorzano A. Endocrinology. 1997; 138: 3006-3015Crossref PubMed Google Scholar, 37Muñoz P. Rosemblatt M.R. Testar X. Palacı́n M. Zorzano A. Biochem. J. 1995; 307: 273-280Crossref PubMed Scopus (33) Google Scholar, 44Muñoz P. Mora S. Sevilla L. Kaliman P. Tomàs E. Gumà A. Testar X. Palacı́n M. Zorzano A. J. Biol. Chem. 1996; 271: 8133-8139Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Analysis of fractions obtained from sucrose gradient centrifugation demonstrated a parallelism between the profile of GLUT4 abundance and the activity of SSAO present in the different membrane fractions (Fig. 4). More direct evidence was obtained in vesicle immunoisolation assays. Quantitative vesicle immunoisolation analysis using monoclonal antibody 1F8, specific against GLUT4, and coupled to acrylic beads was performed in light microsomes from rat adipocytes. This method of vesicle immunoisolation adsorbed 70–90% of total GLUT4 from the fractions in a specific manner (Fig. 5 A). Under these conditions, GLUT4 vesicles specifically contain immunoreactive SSAO protein, which accounted for by 18–24% of total SSAO present in light microsome membranes (Fig. 5 A). Furthermore, GLUT4 vesicles contain active SSAO. Thus, whereas the material nonspecifically bound to IgG beads contained very low levels of SSAO activity, 1F8 antibody brought a substantial level of SSAO activity (Fig. 5 B). Near 25%