Insulin-dependent protein trafficking in skeletal muscle cells

Min Zhou1, Lidia Sevilla2, Gino Vallega1, Peng Chen1, Manuel Palacin2, Antonio Zorzano2, Paul F. Pilch1, and Konstantin V. Kandror1

1 Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118; and 2 Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We have established a simple procedure for the separation of intracellular pool(s) of glucose transporter isoform GLUT-4-containing vesicles from the surface sarcolemma and T tubule membranes of rat skeletal myocytes. This procedure enabled us to immunopurify intracellular GLUT-4-containing vesicles and to demonstrate that 20-30% of the receptors for insulin-like growth factor II/mannose 6-phosphate and transferrin are colocalized with GLUT-4 in the same vesicles. Using our new fractionation procedure as well as cell surface biotinylation, we have shown that these receptors are translocated from their intracellular compartment(s) to the cell surface along with GLUT-4 after insulin stimulation in vivo. Denervation causes a considerable downregulation of GLUT-4 protein in skeletal muscle but does not affect the level of expression of other known component proteins of the corresponding vesicles. Moreover, the sedimentation coefficient of these vesicles remains unchanged by denervation. We suggest that the normal level of GLUT-4 expression is not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment.

rats; glucose transporters; hindlimb denervation; translocation

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

THE REGULATION of postprandial blood glucose levels by insulin is achieved mainly by increased glucose transport into skeletal and cardiac muscle and fat (9, 12, 23, 38). These are the only tissues that express a specific isoform of the glucose transporter protein, GLUT-4, which mediates the hormonal effect. It has been shown that in these tissues, under normal conditions, GLUT-4 is localized in intracellular membrane structures ("GLUT-4-containing vesicles") (7, 19, 24, 29, 49, 58) and is translocated to the plasma membrane in response to insulin (21, 22, 26, 37, 51-53). Because total glucose uptake in insulin-sensitive tissues is, in general, proportional to the amount of GLUT-4 molecules at the cell surface, this translocation process is the major if not the exclusive mechanism of insulin action on glucose transport (3, 20, 25, 31, 32, 45, 54).

In fat cells, identification of major protein components in GLUT-4-containing vesicles, i.e., those that are readily detectable by protein staining, has been largely completed. In addition to GLUT-4, these proteins include the insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor (33), the transferrin receptor (34), an insulin-responsive aminopeptidase (IRAP) (30, 35, 36), and a novel protein, sortilin (41). So, several physiologically important proteins in adipocytes are colocalized in a recyclable compartment or compartments, and their cell surface presentation appears to be coordinately regulated by insulin on a posttranslational level.

However, the expression and function of GLUT-4 in fat tissue account for a relatively small part of the total insulin-stimulated glucose uptake in the body, whereas the predominant site for insulin-stimulated glucose disposal is skeletal muscle (9, 12, 23, 38). When the relative mass of skeletal muscle and fat tissues in normal individuals is considered, this estimation is most probably true for the other proteins that are colocalized with GLUT-4 in the same compartment. However, unlike adipose cells, which represent a convenient, well-characterized experimental model for subcellular fractionation, skeletal muscle is a technically difficult tissue for this type of experiment, and, therefore, it has been studied to a lesser degree. Also, because there are substantial differences in the physiological functions of fat and skeletal muscle cells, it is not yet clear to what extent we can apply the results obtained for vesicle trafficking in adipocytes to this event in skeletal myocytes. The substantial progress that has been made in adipose cells can be explained, at least partially, by the early invention of a reliable protocol for their subcellular fractionation that allows the separation of the plasma membrane from intracellular microsomes and thus the ability to analyze the translocation process (50). A number of attempts have been made to establish an equivalent protocol for skeletal muscle tissue (14, 15, 19, 37). All such protocols, however, are long and complicated. Besides, different protocols yield slightly different results in terms of the recovery of subcellular fractions and their relative protein composition. Nevertheless, on the basis of these and other similar studies (43, 46) as well as immunocytochemical data (10, 13, 49), it is clear that in skeletal muscle tissue, just like in fat, insulin causes translocation of GLUT-4 to the cell surface, which in muscle consists of the T tubules as well as sarcolemma. However, the protein composition of the GLUT-4-containing recycling intracellular compartment in skeletal muscle cells has been studied in much less detail than in adipocytes. It remains unclear, therefore, whether skeletal myocytes can translocate any other proteins besides GLUT-4 to the cell surface in an insulin-dependent fashion.

Thus we describe a novel and simple protocol for separating the intracellular pools of recycling proteins from the cell surface domains of rat skeletal muscle cells. Using this procedure, we were able not only to confirm the vesicle-mediated insulin-dependent recruitment of GLUT-4 to the plasma membrane but also to demonstrate, for the first time, that transferrin and IGF-II/Man-6-P receptors in skeletal muscle undergo an analogous translocation process. Furthermore, we immunopurified intracellular GLUT-4-containing vesicles from this tissue and showed that they contain the receptors for IGF-II/Man-6-P and transferrin. Therefore, the redistribution of all these proteins from their intracellular pool to the plasma membrane and T tubules in response to insulin is likely to be explained, at least in part, by their colocalization in the same hormone-sensitive recycling compartments.

As was demonstrated earlier, denervation of skeletal muscle causes considerable loss of GLUT-4 protein (4, 6, 8, 11, 17, 18). We show here that the expression of other component proteins of the corresponding vesicles does not change after 3 days of denervation, and the sedimentation coefficient of vesicles from denervated muscle remains unchanged. This suggests that the presence of normal amounts of GLUT-4 in its cognate vesicles is not necessary for their insulin-sensitive translocation. We intend to use this model in our future studies to explore the role of GLUT-4 protein in insulin-regulated vesicular traffic.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animals. Male Sprague-Dawley rats (150-175 g) were purchased from Taconic Breeding Laboratory. The animals were fasted overnight and then injected with insulin (1.5 units/animal) or with the buffer alone via the portal vein 8 min before being killed, as described earlier (7). All rats were anesthetized with pentobarbital sodium (60 mg/kg body wt) by intraperitoneal injection. For denervation experiments, extensor digitorum longus (EDL) muscles were denervated by sciatic nerve section 3 days before muscle isolation and fractionation. For the sham operation, the sciatic nerve was visualized but not touched.

Antibodies. In this study, we used monoclonal anti-GLUT-4 antibody 1F8 (22), monoclonal anti-secretory carrier-associated membrane protein (SCAMP) antibodies (56), monoclonal anti-transferrin receptor antibody (Zymed Laboratory), monoclonal anti-dihydropyridine receptor antibody (a kind gift of Dr. Kevin Campbell, Univ. of Iowa), monoclonal anti-vesicle-associated membrane protein (VAMP)-2 antibody (a kind gift of Dr. R. Jahn, Yale Univ. School of Medicine), DEAE-cellulose-purified anti-IGF-II/Man-6-P receptor polyclonal antibody (a kind gift of Dr. M. Czech, Univ. of Massachusetts Medical School), polyclonal anti-GLUT-1 antibody (a kind gift of Dr. C. Carter-Su, Univ. of Michigan), polyclonal anti-beta 1 integrin antibodies (a kind gift of Dr. Carles Enrich, Universitat de Barcelona), and polyclonal anti-IRAP antibody (30).

Preparation of membranes from skeletal muscles. Red and white gastrocnemius and soleus muscles from rat hindlimb were removed and trimmed of connective tissue, fat, and nerves. The muscles were then minced and homogenized on ice three times (10 s each) using a Polytron homogenizer set at 13,500 rpm in a buffer containing 20 mM HEPES, 250 mM sucrose, 1 mM EDTA, 5 mM benzamidine, 1 µM aprotinin A, 1 µM pepstatin, 1 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4. The homogenate was centrifuged at 2,000 g for 10 min. The pellet, which contained mainly unhomogenized pieces of tissue, was discarded, and the supernatant was centrifuged at 9,000 g for 20 min. The 9,000-g pellet (P1) was resuspended in PBS with the standard cocktail of protease inhibitors listed above and analyzed for the presence of marker proteins by Western blotting. The supernatant was centrifuged at 180,000 g for 90 min. The 180,000-g pellet was resuspended in PBS with protease inhibitors, loaded on a 10-30% (wt/wt) continuous sucrose gradient (3-4 mg protein/5 ml gradient), and centrifuged at 48,000 rpm for 55 min in a SW-50.1 rotor. Gradients were separated into 25-30 fractions, starting from the bottom of the tube. The pellet of the sucrose-gradient centrifugation (P2) was resuspended in PBS and analyzed together with the gradient fractions. All centrifugations were performed at 4°C.

Immunoadsorption of GLUT-4-containing vesicles. Protein A-purified 1F8 antibodies and nonspecific mouse IgG (Sigma) were coupled to acrylic beads (Reacti-gel GF 2000, Pierce) at a concentration of 0.4 and 0.6 mg antibodies/ml of resin, respectively, according to the manufacturer's instruction. Before usage, the beads were saturated with 2% BSA in PBS for 1 h and washed with PBS. The GLUT-4-containing fractions from the sucrose gradient were pooled, diluted 1:1 with PBS containing 1% BSA, and incubated with 1F8-coupled beads and nonspecific IgG-coupled beads overnight at 4°C. The beads were washed four times with PBS and then with 10 mM Tris, pH 7.4, and the adsorbed material was subsequently eluted with 1% Triton X-100 in PBS and with Laemmli sample buffer (39) without 2-mercaptoethanol.

5'-Nucleotidase assay. This was carried out as described (2). Briefly, membrane samples (20 µl) were suspended in 1 ml of buffer containing 50 µmol Tris · HCl, pH 8.5, 0.18 µmol Mg2+, 0.04 µmol AMP, 5 µmol adenosine 2',3'-monophosphate, 1% Triton X-100, and 10 µl of 1:3 diluted [14C(U)]AMP (NEN NEC347) and incubated at 37°C for 30 min. The reaction was stopped with 0.2 ml of 0.3 M ZnSO4 and 0.2 ml of 0.3 M Ba(OH)2. Tubes were centrifuged for 10 min, and the supernatant was counted in a standard scintillation cocktail (Ecolume, ICN).

Muscle biotinylation and immunoprecipitation of transferrin and IGF-II/Man-6-P receptors. Male Sprague-Dawley rats (50-100 g) were fasted overnight, and EDL muscles were rapidly dissected and attached by their tendons to stainless steel clips to keep the muscle in the stretched state as described by Maizels et al. (42). All incubations were carried out in medium saturated with 95% O2-5% CO2. Isolated muscles were placed in KRP buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.6 mM Na2HPO4, 0.4 mM NaH2PO4, 2.5 mM D-glucose, pH 7.4) for 20 min at 37°C. Muscles were incubated with or without insulin (60 nM) for 30 min, and sulfonated N-hydroxysuccinimide-biotin (sulfo-NHS-biotin; Pierce) was added to final concentration of 0.5 mg/ml. Biotinylation was performed for 5 and 20 min at 37°C, and muscles were washed with 50 mM Tris, pH 7.4, three times. Muscles were then homogenized in HEPES-EDTA-sucrose buffer with protease inhibitors as described above in a Polytron homogenizer. The homogenate was centrifuged at 2,000 g for 10 min, and Triton X-100 was added to the supernatant to a final concentration of 1% for 3 h at 4°C. After centrifugation at 13,000 rpm for 5 min in an Eppendorf microcentrifuge, an aliquot of supernatant (1 mg of total protein) was supplied with 5 µl of lyophilized anti-IGF-II/Man-6-P receptor antibody reconstituted to the original serum volume or with 3 µg of anti-transferrin receptor antibody together with 40 µl of 50% protein A-trisacryl suspension (Pierce). After overnight incubation at 4°C with rotating, beads were washed three times with 1% Triton X-100 in PBS and three times with 10 mM Tris, pH 7.4, and eluted with Laemmli sample buffer without 2-mercaptoethanol.

Gel electrophoreses and immunoblotting. Protein samples were electrophoresed according to Laemmli (39) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). After transfer, membranes were blocked with 10% nonfat dry milk in PBS for 1 h at room temperature and incubated with specific antibodies. Secondary antibodies (Sigma) were either conjugated to horseradish peroxidase or labeled with 125I. In the former case, blots were developed using an enhanced chemiluminescence detection system (Du Pont NEN), and films were scanned using a computing densitometer (Molecular Dynamics) for quantitative analysis. When radioactive secondary antibodies were used, blots were analyzed in a phosphorimager (Molecular Dynamics) or radioactive bands were cut and counted in a gamma counter. In some experiments, both horseradish peroxidase-conjugated and 125I-labeled secondary antibodies were used in parallel with virtually identical results. Biotinylated proteins were stained with streptavidin-alkaline phosphatase conjugate (Boehringer), and the membrane was analyzed by densitometry as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Muscle membrane fractionation. Homogenized muscles were processed as described in EXPERIMENTAL PROCEDURES and as summarized in Fig. 1. Briefly, after cell homogenization and removal of the nonhomogenized tissue by a brief low-speed centrifugation, we carried out another spin at 9,000 g to pellet most of muscle fibers, cell nuclei, mitochondria, large fragments of sarcoplasmic reticulum, and other heavy subcellular structures and organelles. We call this pellet P1. It is important to keep it for further analysis, since P1 also contains functionally significant domains of the surface membrane where GLUT-4 and other proteins are translocated (see below).


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Fig. 1.   Flowchart of skeletal muscle fractionation. P1, 9,000-g pellet; P2, pellet of sucrose-gradient centrifugation.

All membranes in the supernatant are pelleted by a high-speed centrifugation (180,000 g), resuspended in PBS with protease inhibitors, and fractionated in a continuous 10-30% sucrose gradient. This centrifugation also results in a significant membrane pellet (P2). The distribution of total protein between P1, P2, and the gradient fractions is shown in Table 1. Figure 2 demonstrates, by Western blot analysis and enzymatic assay, the distribution of various individual proteins in these fractions. P1 and P2 contain virtually all plasma membrane and T tubule marker proteins that have been tested (5'-nucleotidase, the dihydropyridine receptor, beta 1-integrin), none of which can be detected in the gradient fractions under standard exposure conditions. Because equal amounts of total protein were taken from P1 and P2 fractions for analysis, the dihydropyridine receptor and beta 1-integrin are not visible in P1. However, these proteins are readily detectable when more protein from P1 fraction is taken for electrophoresis (not shown). Practically all GLUT-1 is also recovered in P1 and P2 and thus should be localized mainly at the plasma membranes of skeletal myocytes, which is consistent with the results of previous immunocytochemical and fractionation studies (27, 44, 47, 57).

                              
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Table 1.   Distribution of total protein between P1, P2, and gradient fractions


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Fig. 2.   Representative pattern of relevant proteins assessed by protocol of Fig. 1. The 180,000-g pellet (3-5 mg of protein depending on experiment) was loaded on a 10-30% continuous sucrose gradient and centrifuged at 48,000 rpm for 55 min in an SW-50.1 rotor. Gradient was fractionated into 24 fractions starting from bottom of tube, and pellet (P2) was resuspended in 200 µl of PBS. A: total protein content (open circle ) and 5'-nucleotidase activity (bullet ) in fractions. B: P1 and P2 (each 80 µg of protein) and 30 µl of soluble gradient fractions were analyzed by Western blotting with different antibodies. A representative result of 7 independent experiments is shown. VAMP-2, vesicle-associated membrane protein-2; SCAMPs, secretory carrier-associated membrane proteins; AU, arbitrary units; cpm, counts/min.

The distribution of GLUT-4 under these conditions is drastically different from that of GLUT-1 and other plasma membrane markers. Although ~25% of GLUT-4 is present in P1 and P2, its major pool is recovered in the middle of the sucrose gradient (Fig. 2). Thus, as previously shown (29), a large population of GLUT-4 molecules is localized in rather homogeneous membrane vesicles that can be separated from other membranes and the sedimentational characteristics of which are similar to those of intracellular GLUT-4-containing vesicles from fat cells (29). The advantage of the present approach in comparison to our previous results is that now we can monitor the distribution of GLUT-4 between the surface domains and the intracellular vesicles with a high degree of accuracy, because these fractions are separated within the same centrifuge tube.

The distribution of SCAMPs under these conditions is close to GLUT-4, which is consistent with the earlier observations about enrichment of SCAMPs in GLUT-4-containing and other cell surface recycling vesicles (5, 40, 56). VAMP-2-containing membranes, however, have a broader distribution than SCAMPs and GLUT-4. VAMP-2 partially overlaps with GLUT-4-containing vesicles; a significant amount of this protein is also recovered in P1 and P2 (plasma membrane and/or T tubules).

Immunoadsorption of GLUT-4-containing vesicles from skeletal muscle. On the basis of the results shown in Fig. 2, we felt confident that we separated the intracellular pool(s) of GLUT-4-containing vesicles from plasma membrane and T tubule domains. This provides an opportunity to immunoisolate pure GLUT-4-containing vesicles without risk of contaminating the preparation with GLUT-4-containing sheets of the plasma membrane. This is an ongoing concern in the case of skeletal muscle tissue, because a certain amount of GLUT-4 is always present at the sarcolemma and T tubules because of the basal blood insulin levels in vivo.

The protein composition of intracellular GLUT-4-containing vesicles from skeletal muscle cells has not been extensively characterized. Although previous studies have detected IRAP in GLUT-4-containing vesicles (29, 55), the presence of the receptors for IGF-II/ Man-6-P and transferrin in this compartment is controversial (1, 48). To address this question, we immunoadsorbed intracellular sucrose gradient-purified GLUT-4-containing vesicles with the use of monoclonal anti-GLUT-4 antibody, 1F8. The result of a representative experiment is shown in Fig. 3. As is the case in adipocytes, GLUT-4-containing vesicles from skeletal muscle include IRAP and the receptors for IGF-II/Man-6-P and transferrin. These proteins can be quantitatively eluted from immunobeads after solubilization of the vesicle membrane with 1% Triton X-100, whereas elution of GLUT-4 itself requires SDS. Under conditions in which we immunoadsorb virtually all GLUT-4-containing vesicles (Fig. 3), we are able to bring down 84 ± 4.7% of the total IRAP population, 28 ± 5.2% of the total transferrin receptor populations, and 31 ± 3.1% of the total IGF-II/Man-6-P receptor population, which again is quantitatively consistent with the distribution of these proteins in fat cells (29, 33, 34).


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Fig. 3.   Protein composition of GLUT-4-containing vesicles from skeletal muscle. GLUT-4-containing fractions of sucrose gradient shown in Fig. 2 were pooled, diluted with equal volume of PBS containing protease inhibitors and 1% BSA, and immunoadsorbed with 100 µl of either 1F8 beads or nonspecific mouse IgG beads. Immunobeads were washed and subsequently eluted with 1% Triton X-100 in PBS and then with Laemmli sample buffer without 2-mercaptoethanol and analyzed by Western blotting. A representative result of 3 independent experiments is shown. IGF-II/Man-6-P receptor, insulin-like growth factor II/mannose 6-phosphate receptor; IRAP, insulin-responsive aminopeptidase.

Insulin-dependent translocation of GLUT-4, the IGF-II/Man-6-P receptor, and the transferrin receptor as revealed by subcellular fractionation. Rats were injected with insulin or with buffer alone into the portal vein, and hindlimb muscles were isolated 8 min after injection and processed as described earlier (Fig. 1). Figure 4 shows the Western blot analysis of protein composition of P1, P2, and the gradient fractions. Under basal conditions, both pellets contain visible amounts of the receptors for IGF-II/Man-6-P and transferrin, whereas GLUT-4 was localized mainly in P2. The intracellular pools of these receptors present in the gradient fractions partially overlap with the distribution of GLUT-4-containing vesicles, which is consistent with the results of immunoadsorption experiments (Fig. 3). Insulin administration causes a profound decrease in GLUT-4 content in the fractions of the gradient and a corresponding enrichment of GLUT-4 in P1 and, to a lesser extent, P2. The transferrin receptor and the IGF-II/Man-6-P receptor show the same qualitative insulin response as GLUT-4. Quantitative analysis of the insulin-dependent protein translocation carried out with 125I-labeled secondary antibodies in a phosphorimager clearly and reproducibly showed an insulin-induced decrease in the intracellular content of all three proteins (gradient fractions) and a corresponding increase in the combined pellets (P1+P2), which contain cell surface domains (Fig. 4B). The insulin-dependent decrease in the intracellular pools of the receptors for transferrin and IGF-II/Man-6-P, however, is less pronounced than that of GLUT-4 and not statistically significant although highly reproducible. We presume this is because only one-third of the total population of these two receptors is present in the translocatable GLUT-4-containing vesicles (Fig. 3), whereas their major pools may not be sensitive to insulin, as is the case in adipocytes (33, 34).


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Fig. 4.   Translocation of GLUT-4, transferrin receptor, and IGF-II/Man-6-P receptor from intracellular compartment(s) to surface sarcolemma in response to insulin. Membrane samples were prepared from hindlimb muscles of insulin-stimulated and control rats as described in text, and equal amount of protein (3 mg) was loaded on 10-30% sucrose gradients. A: P1 and P2 (80 µg of each) and 30 µl of soluble gradient fractions were analyzed by Western blotting. A representative result of 5 independent experiments (2 with horseradish peroxidase-conjugated antibodies and 3 with 125I-labeled secondary antibodies) is shown. B: quantitation of results with 125I-labeled secondary antibodies. Results are expressed in relative percentage, with data from control muscle assigned a value of 100. Results are means ± SD of 3 separate experiments. * P < 0.05 and ** P < 0.01, significant difference from basal values according to Student's t-test for paired data.

Insulin-dependent translocation of the IGF-II/Man-6-P receptor and the transferrin receptor as revealed by cell surface biotinylation. We have used cell surface biotinylation with an impermeable amino group-specific reagent, sulfo-NHS-biotin, to study insulin-responsive protein traffic in adipocytes (28). We decided to use the same technique to detect the recyclable populations of the IGF-II/Man-6-P receptor and the transferrin receptor in skeletal muscle cells. Individual EDL muscles were incubated with sulfo-NHS-biotin in vitro in the absence and in the presence of insulin as described in EXPERIMENTAL PROCEDURES. After that, muscles were homogenized, and the receptors for IGF-II/Man-6-P and transferrin were immunoprecipitated with specific antibodies. Under these conditions, specific biotinylation of individual recycling proteins is roughly proportional to their residence time at the cell surface (28). As shown in Fig. 5, insulin treatment of EDL muscle increases the specific biotinylation of the IGF-II/Man-6-P and transferrin receptors by 28 ± 14.6 and 50 ± 8.1%, respectively. This effect is most likely explained by insulin-stimulated redistribution of the receptor proteins to the sulfo-NHS-biotin-accessible compartment, the surface of the muscle cells. In the case of isolated muscle, unlike primary culture of adipocytes (28), insulin has a rather modest but consistent effect on the specific biotinylation of the vesicle proteins. We explain this modest effect by the slow diffusion of both insulin and sulfo-NHS-biotin into the muscle in vitro, so that not every muscle fiber gets exposed to these reagents during the limited time of the experiment. Moreover and as previously noted, the presence of endogenous insulin raises the basal values. Nevertheless, the results of the biotinylation experiments also support the notion of insulin-dependent translocation of receptors for IGF-II/Man-6-P and transferrin in skeletal myocytes and confirm the data obtained by cell fractionation.


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Fig. 5.   Demonstration of insulin-sensitive recycling of transferrin receptor and IGF-II/Man-6-P receptor by cell surface biotinylation. Isolated rat extensor digitorum longus (EDL) muscles were biotinylated in presence and in absence of insulin (INS) for 20 min as described in EXPERIMENTAL PROCEDURES. After that, muscles were homogenized and centrifuged at 2,000 g for 10 min. Triton X-100 was added to supernatant (1 mg of total protein) to final concentration of 1%, and transferrin receptor and IGF-II/Man-6-P receptor were immunoprecipitated. After electrophoresis and transfer, membrane was stained with streptavidin-alkaline phosphatase conjugate, and results were analyzed by computer densitometry. A: Western blot analysis. B: quantitation of results. Results are expressed in relative percentage, with data from control muscle assigned a value of 100. Results are means ± SD of 3 separate experiments. * P < 0.05 and ** P < 0.01, significant difference from basal values according to Student's t-test for paired data.

Effect of denervation on the component proteins and sedimentation coefficient of GLUT-4-containing vesicles. Denervation of EDL muscle causes a dramatic decrease in GLUT-4 content after 3 days (Fig. 6; see also Refs. 4, 11, 17, 18). However, other known component proteins of GLUT-4-containing vesicles are not sensitive to denervation (Fig. 6). In addition, denervation does not change the sedimentational distribution of the reduced amount of intracellular GLUT-4 (~20% of control) that is still present in denervated muscle nor does it affect the sedimentation of transferrin and IGF-II/Man-6-P receptors (Fig. 7). This suggests that these proteins are still compartmentalized in membrane vesicles that survive the loss of 80% of one of their major protein components, GLUT-4.


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Fig. 6.   GLUT-4 is the only protein from cognate vesicles that is affected by denervation. Rat limbs were denervated or sham operated 3 days before experiment. EDL muscles were extracted, homogenized, and centrifuged at 2,000 g for 10 min. Individual vesicular proteins were analyzed in supernatant by SDS-polyacrylamide gel electrophoresis and immunoblotting. Results are representative of 3 independent experiments.


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Fig. 7.   Distribution of GLUT-4 (A), transferrin receptor (B), and IGF-II/Man-6-P receptor (C) in sucrose gradients is not altered by denervation. EDL muscles from denervated or sham-operated rats were homogenized, membrane proteins were prepared as described in text, and equal amounts of protein (3 mg) were loaded on a 10-30% sucrose gradient. Soluble gradient fractions (30 µl) were analyzed by Western blotting. A representative result of 5 independent experiments is shown.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this paper, we describe a new and relatively easy technique for the subcellular fractionation of skeletal muscle cells that allows us to analyze the insulin-dependent translocation of three physiologically important proteins, GLUT-4, the IGF-II/Man-6-P receptor, and the transferrin receptor, from their intracellular pool(s) to the cell surface. A brief comparison of our procedure with the existing procedures for muscle fractionation is noteworthy. Our protocol includes four short centrifugation steps (total centrifugation time: 3 h) and takes less than 5 h until samples are ready for electrophoresis, whereas published techniques require ~2 full days of experimentation (14, 15, 19, 37). Another important feature of our protocol is that, with the exception of the very first low-speed-centrifugation pellet, which contains mainly nonhomogenized chunks of tissue (with a very low specific content of GLUT-4), we do not discard any membrane fraction and monitor the proteins of interest practically with no "leftovers." The advantages of our approach derive from the fact that it is based on fractionation of membranes according to their sedimentation coefficients, whereas previously published techniques are based on separation by buoyant densities. The rationale for our technique originated from earlier observations that GLUT-4-containing vesicles from both fat and skeletal muscle cells have a unique sedimentation coefficient of ~100-120 S and are very well separated from the bulk of membrane protein in a continuous sucrose velocity gradient (7, 29).

We do not claim, however, that our procedure results in substantial purification or enrichment of surface membranes. It is well known that the surface sarcolemma of muscle fibers has several structurally different domains and subdomains, separation and purification of which require much more sophisticated biochemical and immunochemical techniques (47). Nevertheless, we believe that our experimental conditions readily separate the surface sarcolemma and T tubules from hormone-sensitive intracellular vesicles, thus allowing us to detect and measure the insulin-dependent protein translocation between these fractions in skeletal muscle cells. In addition, this new protocol provides an opportunity to isolate and study intracellular vesicles with the use of a conventional technique of immunoadsorption without contaminating the preparation with the plasma membrane proteins. We show here that these vesicles compartmentalize, in part, the receptors for IGF-II/Man-6-P and transferrin. By several independent approaches, we demonstrate that these receptors are translocated to the surface of skeletal muscle cells along with GLUT-4 in an insulin-dependent fashion. Thus translocation of GLUT-4-containing vesicles may be responsible, at least partially, for the cell surface recruitment of the receptors for IGF-II/Man-6-P and transferrin. In our earlier experiments (7, 29), we prepared intracellular microsomes from skeletal muscle cells by using a variation of the protocol of Klip et al. (37), and we showed that IRAP in skeletal muscle cells is localized in GLUT-4-containing vesicles (29) and that its intracellular content is decreased in response to insulin (7). Also, Sumitani et al. (55) have recently demonstrated colocalization of IRAP and GLUT-4 in the same vesicles and insulin-sensitive translocation of IRAP to the surface of skeletal myocytes. We show here that GLUT-4-containing vesicles purified by our new procedure also contain 84 ± 4.7% of the total IRAP population. These data are consistent with previous studies, so that GLUT-4-containing vesicles appear to maintain their compositional integrity regardless of the type of procedure used for subcellular fractionation.

An interesting feature of skeletal myocytes is that, unlike fat cells, they do not seem to have any significant amount of GLUT-1-containing vesicles, since virtually all GLUT-1 in these cells is found at the cell surface (16, 27, 57). Moreover, after insulin administration, GLUT-4 and GLUT-1 are likely to be localized in the different domains of the surface sarcolemma, since they are found predominantly in P1 and P2, correspondingly. We are now trying to determine the immediate biochemical environment for both transporters at the plasma membrane.

We have also begun to apply our technique to address the effect of denervation on the nature and translocation of GLUT-4-containing vesicles in skeletal muscle. After 3 days of denervation, GLUT-4 expression dramatically decreases (Figs. 6 and 7), whereas the levels of other known protein components of GLUT-4-containing vesicles do not change. Nor does the distribution of GLUT-4, the transferrin receptor, or the IGF-II/Man-6-P receptor in the sucrose velocity gradient change after denervation (Figs. 6 and 7), which suggests that all these proteins may still be compartmentalized in membrane vesicles of the same sedimentation coefficientas in untreated animals. We believe, therefore, that aside from the reduction in the GLUT-4 content, there are no obvious changes in composition and structure of these vesicles after denervation. This presents a convenient experimental model, which we intend to use to determine whether the presence of GLUT-4 in vesicles is required for their insulin-sensitive translocation. Moreover, our preliminary data suggest that denervation does not change insulin responsiveness of this compartment. We are now trying to accumulate more experimental data about insulin signal transduction in denervated cells to evaluate the possible direct role of GLUT-4 protein in the insulin response.

    ACKNOWLEDGEMENTS

We are grateful to T. G. Kurowski and Drs. S. J. Heydrick and D. J. Dean for methodological advice and assistance in muscle denervation. We thank Dr. Anthony Filippis for assistance with statistical analysis of the data and Amr El Jack for help during the experiments.

    FOOTNOTES

This work was supported by research grants from the Dirección General de Investigación Científica y Técnica (PB95/0971), from Generalitat de Catalunya (GRQ94-1040; 1995SGR 537), and from Fondo de Investigación Sanitaria (97/2101), Spain (to A. Zorzano); by North Atlantic Treaty Organization Collaborative Research Grant CRG 950873 (to A. Zorzano and P. F. Pilch); by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-49147 (to N. B. Ruderman), DK-30425 and DK-36424 (to P. F. Pilch), and DK-52057; by Research Grant 197029 from the Juvenile Diabetes Foundation; and by a research grant from the American Diabetes Association (to K. V. Kandror).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. V. Kandror or P. F. Pilch, Dept. of Biochemistry, Boston Univ. School of Medicine, 715 Albany St., Boston, MA 02118.

Received 21 January 1998; accepted in final form 17 April 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(2):E187-E196
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